SEROLOGICAL STUDIES ON AVIAN VISCERAL LYMPHOMATOSIS USING THE COMPLEMENT FIXATION REACTION By Robert T. A Gentry THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR Department Year 07 of PHILOSOPHY Bacteriology 1953 SEROLOGICAL STUDIES ON AVIAN VISCERAL LYMPHOMA TO SIS USING THE COMPLEMENT FIXATION REACTION By Robert AN F. Gentry ABSTRACT Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Scienoe in partial fulfillment of the requirements for the degree of DOCTOR Department Year Approved OF of PHILOSOPHY Bacteriology 1953 ABSTRACT Mortality in ehiekens, resulting from avian visa oral lymphomatosis, is of major economic import ana a to the poultry industry. Isolation and genetic resistance influanaa this m ortality rata, but ara not: praatieal for tha widespread oontrol of the d i s ease. The development of a diag- nostie tost would ba a major tantribution in this field, and this work was dona in an attempt to develop sueh a teehnique by use of the indiroet aomplament fixation reaetion. Attempts were made to utilize the teehnique of Rice (19L&) and Mayer (I9I4.7 ), both of whom employed the slope of the hemolytic curve in the calculation of the amount of fixation. The slope value was found to vary greatly, depending on the reagent present, and a technique was developed whioh eliminated the use of suah a value. The amount of fixation was determined in terms of milliliters of undiluted oosplement necessary for 50 percent hemolysis by plotting the e le otrophotometer readings against the volume of undiluted complement present. From this the units cf com- plament fixed oould be oaloulated. Influenza virus was employed foe* the standardization of the test teohnique. Rabbits were first used as a source of complement fixing antibodies, but were found unsuitable due to the formati cn of hetero­ phils antibodies which reacted with heterophils antigen of the ohioken serum. The production of these antibodies had been stimulated by hetero­ phils antigen in the influenza virus suspension and had originated from the embryos used for its propagation. Guinea pigs were found not to produce this antibody and were used for subsequent inooulations. Test antigens were prepared by extracting normal ohloken tissue and tumors of RPL strain 12 visceral lymphomatosis (Burnsster et al., 19146). They were used for the production of anti s e r u m ahiokens, and ducks. in guinea pigs, Infeoted and normal chicken s e r u m were also used as antigens, both in tests and for inoculations. The first tests were designed to determine if the agent of the strain 12 tumor could be detected in the chicken serum. Infeoted chicken serum was used as an antigen and allowed to react witti guinea pig anti serums that had been produced by the inoculation of normal and infeoted chioken serum and normal tissue and tumor extracts. Some reaction took plaoe, but was due to the presence of normal tissue materials and not the result of tumor agent activity. A direct test, employing tumor antigen and guinea pig antitumor serum was standardised for use in testing chicken antitumor serums for their tumor agent antibocy content. A reaction, in excess at that due to the normal tissue materials was noted, apparently due to either tumor oel 1 components or the agent. Chioken anti tumor serums were tested by the indirect technique, but no agent stimulated reaction could be detected. The reaction taking plaoe was found to be due to normal tissue materials. Normal tissue antibodies were adsorbed from the guinea pig anti tumor serum with normal chicken red blood cells, but when the adsorbed antiserum was used in direct reactions for the indirect titration of ohioken antltumor serums, no difference was noted from the titers obtained with the untreated antiserum. The detection of the agent of visceral lymphomatosis or specific antibodies was not possible with the techniques used, and will depend upon the development of more highly purified agent suspensions and 'the elimination of the normal tissue materials. VITA Robert Francis Gentry candidate for the degree of Doctor of Philosophy Pinal examination, Dissertation: Serological Studies on Avian Visceral lymphomatosis using the Complement Fixation Reaction Outline of Studies Major subject: Bacteriology Minor subjects: Pathology, Biological Chemistry Biographical Items Born, May 31, 1921, Topeka, Kansas Undergraduate Studies, Kansas State College, 1940-1944, D. V. M. 1944 Graduate Studies, University of Missouri, 1944-1947, M. A. 1947 Michigan State College 1947-1953 Experience: Instructor, University of Missouri, 1944-1947; Graduate Fellow, Michigan State College, 1947-1950; Veterinarian, Regional Poultry Research Laboratory, 1950-1953 Member of American Veterinary Medical Association, Society of the Sigma XI, Poultry Science ACKNOWLEDGMENTS Hie author wishes to express his appreciation to Dr. H. J. Stafseth, under whose supervision this investigation was undertaken. Acknowledg­ ment is also due Mr. Berley Winton, Director of the Regional Poultry Research Laboratory, for his interest and permission to carry on the study and for the use of the Laboratory materials and equipment. Deepest thanks are given Dr. B. R. Burmester for his guidance and assistance in'the preparation of the materials, particularly the antigens, and for his help in the interpretation of the findings. The author also wishes to thank Drs. N. F. Waters and G. E. Cottral of the Regional Poultry Research Laboratory, and Drs. H. D. Anderson and M. Kurts of the Michigan Department of Health Laboratories for their suggestions and assistance in one way or another. The investigator deeply appreciates the financial support of the National Cancer Institute and is indebted to Michigan State College for the administration of these funds. TABLE 07 00NTENTS I. II. III. INTRODUCTION PAGE ............................................. 1 REVIEW 07 L I T E R A T U R E .................................... 5 MATERIALS AMD M E T H O D S .....................................16 a. C o m p l e m e n t .......... .16 Is. H e m o l y s i n .............. 17 c. Sheep red blood c e l l s ................. 17 d. D i l u e n t s ............ . • • • • • . • • • • • • • • 1 9 e. Electrophotometer.............................. .. f. Red blood cell concentration standardisation g. Selection of complement fiscation technique ... 36 • ... 30 h. Calculation of d a t a ..................... IV. 39 TITRATION 07 R E A G E N T S ...................................... 46 a. Complement t i t r a t i o n ....................... ... 46 b. Hemolysin t i t r a t i o n .......... V. 48 FBSPERATION 07 TEST R E A G E N T S .............................. 51 A« Antigen ............. a. Influensa rirus b. Normal tissue c. Tumor • • • • • • • • 5 1 .............................. antigen • . 51 ................• • • • 5 1 antigen . . . . . . . . . ................ .53 d. Tumor euatigen -LI • • • • ................. .. . . . e. Tumor antigen -L3 • .......... f. Tumor antigen - L 3 ......... 53 • • • • • • • • • • 5 4 55 g# Infected chicken s e r u m ............................. 56 h. Normal chicken serum ................... . • • 57 PAGE i. Tumor antigen -PT4 ............... • • • • • • • • 5 8 J. Tumor antigen -L4 k* Tumor antigen -L5 • • • • • • • • • • • • • • • • • 5 9 l v Tumor antigen -L7 • • • • • • • • • • • • • • • • *59 m« Tumor antigen -L9 • • • • • • • ...................* 6 0 n, Tumor antigen -Lyl ........................ • • • • 6 0 .......... • • • • • • • • • • B. Antibody Production 61 &• Bleeding P r o c e d u r e ..................................61 b. Inoculation Procedure • • • • • • • • • 1 « Babbita ••• ........... ........... 6? • • • • • • • • • • • 6 2 2. Guinea p i g s ...................................... 62 3. Chickens and ducks • • • • • • • • • • • • • • • c. Inoculations and Bleedings 1. Influenza antigen 2« Normal tissue antigen ........... 63 • • • • • • 6 3 .................... . . . . 63 • • • • • • • • • • • • • 6 4 a 3* Tumor antigens ..................... • • • • • • 6 6 4. Infected chicken serum ................. • • • • 6 9 VI. STANDARDIZATION 07 T £ C H N I < $ 7 3 ............................. 72 VII. BESULTS AND D I S C U S S I O N ................................... 81 a. Tests for the R.P.L. Strain 12 agent or specific antibodies in infected chicken serum by the direct reaction ............................. . . . 8 2 b. Standardization of a direct complement fixation test for use in testing for tumor agent antibodies in chicken serums . . . . . ............... • • • • 9 7 % PAOl c. Indirect complement fixation teste on chicken antiserums for the detection of tumor agent antibodies VIII. IX. SUMMARY • • • • • • • • . • • • ............... 104 ............................................119 ................................................ 125 B I B L I O G R A P H Y ............................................. 127 LI8T 07 TABLES PAGE The Effect of Calcium and Magnesium Cations on the Titer of C o m p l e m e n t .......................... .. 22 • • • • • • • • • • • • • • 33 Table of Proportionate 7alues ......... • • • • • • • • 40 Complement Titration ••• 47 ............. 49 A 8ample Slope Calculation • • • • • ................. Hemolysin Titration • • • • • • • . . • • Inoculations with Influenza Virus 65 Inoculations with Normal Tissue Antigen • • • • • • • • 65 Inoculations with Tumor A n t i g e n s ...............« . . . 67 Inoculations with Chicken Serums 71 • • • • • • • • • • • Indirect Tests on Chicken Anti-influenza Serum with Babbit and Guinea Pig Anti-influenza Serums in the Direct f a c t i o n s ................... ................. 77 Preliminary Tests with Pooled Guinea Pig Antinormal and Anti-infected Chicken Serum Serums Against Pooled Normal and Infected Chicken Serum Antigens . • • • • 85 Preliminary Tests of Pooled Normal and Infected Chicken Serum Antigens Against Individual Guinea Pig Anti­ normal and Anti-infected Chicken Serum Serums • • • • 88 Preliminary Test of Guinea Pig Antinormal and Anti­ infected Chicken Serum Serums Against TA -L2 and NT Antigens 90 PAGB XIII. Titration of Guinea Pig Antinormal and Anti-infected Chicken Serum Serums Against TA -L2 and NT Antigens • • 92 XIV. Titration of Individual Guinea Pig Antinormal and Anti-infected Chicken Serum Serums Against TA -L2 and N T Antigens XV. ................................ 93 Titration of Normal and Infected Chicken Serum Antigens with Pooled Guinea Pig Antinormal Tissue, Antitumor -LI and Antitumor —L2 Serums XVI. • • • • • • • • • • • • • • • 9 6 Titration of Pooled High Titered Guinea Pig Antinormal Tissue Serum with Normal Tissue Antigen XVII. .............. 99 Titration of Tumor Antigen -L3 Against Guinea Pig Antitumor -LI, Antitumor -L2 and Antinormal Tissue Serums XVIII. • • • • • • • ..................... 103 Indirect Titers of Chicken Antitumor Antigen -LI Serums when Tested with TA -L3 (Antigen) and TGPS -LI and L2 (Antiserums) in the Direct Reaction • .105 XIX* Indirect Titers of Chicken Antitumor Antigen -Ll Serums when Tested with NTA (Antigen) and TGPS -Ll and L2 (Antiserums) in the Direct Reaction XX. • • • • • .107 Indirect Titers of Chicken Antinormal Tissue Serums when Tested with Normal Tissue and Tumor Antigens and Guinea Pig Antinormal Tissue and Antitumor Serums in the Direct Reaction . . • • . . • • . • • . • • • • • .108 PAGZ XXI* XXII* Indirect Titere of Duck Antiserums when Tested with Normal Tissue and Tumor Antigens and Tumor Antiserum in the Direct R e a c t i o n ..................... Indirect Titers of Individual Infected Chicken Serums when Titrated with TA -L3 - TOPS -Ll Direct Reaction. XXIII* XXIV Indirect *111 *112 Titers of Various Chicken Antitumor Serums • • *114 Titers of Guinea Pig Antinormal Tissue and Anti tumor -Ll Serums Before and After Adsorption with Chicken Red Blood C e l l s .............................................. 116 XXV. Indirect Titers of Chicken Antiserume when Adsorbed Guinea Pig Anti tumor Serum Used in the Direct R e a c t i o n .......... 118 LIST or GRAPHS AND FIGURES GRAPH 1. PAGE The Relationship of the Percent Hemolysis to the Blectrophotometer Reading Using Pilters of Various Ware L e n g t h s ............................................ . 9 4 2. The Relationship of the Slectrophotometer Readings With a 525rau. Filter to the Percent Hemoglobin When 100 Percent Equals 3* a Reading of 7 0 ........................ 27 The Relationship of the Electrophotometer Readings to the Amount of Complement • . • • • • • • • . • • • • • • 3 7 4* Graphical Determination of Hemolysin Titer • • • • • • • • 4 9 FIGURE la. Diagram of the Indicator System of the Complement Fixation Reaction • • • • • • ......................... 6 lb* Diagram of a Positive Complement Fixation Reaction • . • • 6 lc. Diagram of a Negative Complement Fixation Reaction • • • • 6 2a* Diagram of a Positive Indirect Complement Fixation Reaction • • • • • . • . • • • • • • • • • • • • • • • • 2b. Diagram of a Negative Indirect Complement Fixation Reaction 3* 10 Chart used ..................... in Determining Proportionality V a l u e s ....... 38 10 I. INTRODUCTION Lymphomatosis, a naturally occurring malignant disease of chickens, is character! tod by the formation of lymphoid tumors in almost any of the tissues of the bird's body. The lymphoid cells infiltrate the tis­ sues, produoing a generalised enlargement of the area and giving it a characteristic grayish oolor. In some oases nodules are formed whiah are made up exclusively of the malignant lymphoid cells. The occurrence of lymphomatosis is widespread throughout the world. The economic loss in this country alone has been estimated to be over $60,000,000 annually (Tsnth Annual Report of the Regional Poultry Research Laboratory, 19U9)» This amount represents the monetary value of the birds that die from the disease. It is only a portion of the total loss involved, since the cost of feed and maintenance up to the time of death must also be taken into consideration. The neural form attacks primarily the younger birds, while the visceral form appears somewhat later, especially during the laying period of pullets, and deaths are continuous there after as long as the birds are maintained (Biester and Devries, 19W+) (Waters and Prickett, 19^.6) . The losses from this disease are felt in all branohes of the poultry industry. From what has been said, it is evident that the development of a method for the control of lymphomatosis would be of great economic importance. There are at present, two methods far limited control of the disease: breeding for genetic resistance, and isolation. In breed­ ing for genetic resistance, ohiokens are selected on the basis of the performance of their ancestors (Johnson and Wilson, 1937) (Waters, 19U5) (Hutt and Cole, 19U7). The seoond method of control is isolation. By segregating a group ef birds so that they hare no contact with other chickens at any time during their life, a marked reduction, and in some instanoes, the eonplete elimination ef disease manifestations, can be brought about. This procedure ooxuiists of selecting .eggs from certain hens, and incubating, hatching, and rearing the resultant; chickens in complete confinement and under rigid quarantine (Waters and Priokett, I9M 4) • This procedure, as well as the method ef selective breeding, would not be practical for widespread control of the disease. Transmission of the agent eausing lymphomatosis in its various forms appoars to eeour by the following methods: from parent to off­ spring through the egg (Cottral, 19^9)» hy direct bird to bird eontaet (Waters, 19U7) (Waters and Bywaters, 19U9)» and possibly by indlreot eontaet (Waters, 19^7) (Hutt and Cole, 19U7)» Indirect contact by means of feoes and other body exerations m a y play an important role (Biester and Devries, I9W 4) • Hie only ex­ cretions, however, which have been definitely shown to transmit the infection, are nasal and traoheal washings (Cottral, 1952). The methods of transmission mentioned previously all indicate the presenoe of an infective agent. Hie genetic resistance exhibited by certain inbred lines is apparently due to a faotor whi oh prevents the manifestation of infection in the form of tunors, but not one that pre­ vents the ent ranee of the agent into the chicken Ts body. The first isolation of an agent capable of producing lymphomatosis was achieved by Purth and Breedis (1933)* More reoently Burmester (19l4.7e), using high speed centrifugation, concentrated the agent of a lymphoid tumor He also demc 't designated as KPL* Strain 12 (Burmester et^ *1/* 19lj6) trat_ i filtrable agents of other lymphoid tumor strains designated as RPL 16, 16, 19, 20, and 21 (Burmester, 19U7b). The tumor material, usually liver, was minoed, suspended in saline and oentrlfvged to sediment the oellular components. If the supernatant fluid was lnooulated into young ohioks, usually at one day of age, no immediate response was noted. However, after a period of approximately 75 to 160 days,, tunors, indistinguishable From those occurring naturally, were produoed (Burmester et_ al., 19U6). Similar results were obtained follow ing inoculation of tumor filtrates whioh had been prepared by passage through a bacteria retaining filter (Burmester et al^., I9U 6 ). It was thus demonstrated that an agent, oapable of passing a baoteria retaining filter, was present, and that this agent would produce tumors indistin­ guishable, both grossly or microsoopically, from the naturally ooourring tumor of visceral lymphomatosis. With the establishment of the presence of a oausative agent which could be ccnoentrated by oentrifugation, its use in the development of a test was considered. It had been shown by Burmester (19U7o) that the lymphoid tumor cells stimulated the production of a fector considered to be an antibody, whioh had a strong oytotoxic notion toward subsequently inoculated tumor cells. This faotor was found in the serums of ohiokens which had survived a tumor oell inoculation or had been inoculated repeatedly with a suspension of non-viable tumor cells. Serum contain­ ing this faotor showed a toxio effect upon tumor cells both in vivo and * Regional Poultry Research Laboratory im vitro. Even though there was apparently am anti bo - Hemolysin, Complement (3) m Influensa Antigen (l) __ .. __ _ Non-Specific Ouinea Pig Antiserum (2) figure lc. Diagram of a Negative Complement fixation Reaction * ( ) is the order of addition of reagents portion remains aotive and la capable of reaoting with additional amounts of the same or a different antigen-antibody complex. In order to deter­ mine if any complement remains free after the ant igen-antibody oonplex being tested has had time to react, the red blood cel1-hemolysin mixture is added. If the test system has fixed all of the complement, no action on the red oells occurs and the test is oalled positive (Figure lb). It must be assumed that the amount of complement present in a test, such as the one shown in Figuie lb, had been adjusted so that all would be fixed b y the antig en-antibody complex of the test system. If this had not been done, the exoess complement would react with the indicator system, and the test system reactian could not be detected. If, an the other hand, the antigen and antibody of the test system are not specific or there is insufficient antibody present, no detectable effect on the oonp lament will take place. This will leave complement free to react with the red cell-hemolysin complex (indicator system) and hemolysis will be noted (Figure lc). In this case the test is oalled negative. Influenza virus and guinea pig anti-Influenza serum are used in the illustrations. This basic procedure for the deteotion of the fixation of complement was developed by Bordet and Gengou (1901), (Gengou, 1902) and is often referred to as the Bordet-Gengou phenomenon. Soon after Bordet an4 Gengou's experiments, Citron (1906) showed that bacterial extracts could be successfully substituted for baoterial suspensions in these reaotions. Wasserman, while working with Neisser (Wasserman et^ a l ., 1906a) and Sohuoht (Wasserman et al., 1906b), developed a method for the diagnosis of syphilis in man by oonp lament fixation, whioh is known as the Wasserman reaction. This was the first widely used diagnostic test employing the phenomenon of complement fixation. In complement fixation reactions whioh use 100 peroent hemolysis as the end point, as is the case with the Wasserman reaotion, the degree of hemolysis is determined by holding the tube containing the material front of a bright light and observing the turbidity. in The variations in turbidity are graded as 1|># 3^# 2*, 1*, ♦, and -, which correspond to approximately 0, 1+0, 80, 9 0 , 97, and 100 peroent hemolysis. Whan amounts of complement are plotted against the percentages of hemolysis produced by them, a sigmoid curve is formed. Larger amounts of complement are. required per unit change of hemolysis as the hsmolysis approached either 0 or 100 percent. It has been shown (Brooks, 1918) that the differences in the susceptibilities of the red oells to lysis by complement are responsible for the deviation from a straight line relationship and are due to various kinetic factors involving the con­ centrations of erythrocytes, antibody, and complement, and their respec­ tive speeds of interaction (Mayer srt a l ., 19i+8a), and the susceptibili­ ties in the red blood cell population to lyeis by complement (Waksman, 19U9). In the range between 20 and 80 percent hsmolysis, there is praoticnlly a straight line relationship between the amount of complement and the hemolysis produced, and within this range, where a small amount of complement produoes a large change in the degree of hemolysis, more accurate measurement of the amount of complement present can be made. A highly quantitative test was developed using $0 percent hemolysis as the end point (Wadsworth, 1939. Maltaner and Maltaner, 1935# Thompson •ad Malt an ar , 19U°» Wadsworth et a l ., 1931, 1938*, 1938b, «nd 19380), and utilization made of an instrument such as an els ot rephot emeter or a speotrophotometer (Friedewald, 19U3, Mayer et al^, 19U6b). This type of technique was used in the studies reported in this thesis. The complement fixation test, as it has been outlined, could not be used for the detection of antibodies in chicken serum beoause the reac­ tion of these antibodies with specific antigen does not form a coup lex capable of fixing, complement (Rice, 19i+8b). will combine with specifio reaction is However, chicken anti serum antigen, and the antigen involved in such a no longer freeto react with oth or antiserum. For example, if chicken anti-influenza serum is mixed wi th influenza antigen, they will combine but will have no effeot on the camplemerrt . If guinea pig anti-inf1 uenza serum is added, it can not combine with the antigen and no complex capable of fixing complement will be formed. If there were ne specific antibodies in the chicken serum, the guinea pig anti -infl ue nza serum weuld react with the antigen and the complement weuld be fixed as is shewn inFigure lb. The extent te which the antigen-guinea pig anti - serum complex is inhibited measures the antibody titer of the chickm antiseruii, and since this measurenont is made indirectly, the technique has been called the "Indirect Complement Fixation Test.” The actual mechanics of the reaotion may be more olearly understood with the aid of diagrams such as those shewn in Figures 2a and 2b. Figure 2a represents a positive indirect reaotion. The chicken anti- influenza serum contains antibodies which will combine with the influenza antigen; therefore, their symbols are enclosed in a solid line to indi­ cate this reaction. The guinea pig anti-inflnenza serun, when added, can Hemolysin (5) Sheep Red Blood Celle Complement (4) Guinea Pig , Anti-influenza Serum (3) I ^ f l j j e n p a _ A n tig e n (2 T ^ v Chicken Anti-Influenza Serum (1) Figure 2a. Diagram of a Positive Indirect Complement Fixation Reaction ^ '-Sheep Red Blood C e l l e d ------- (5) Hemolysin^ ' Complement (4) Guinea Pig Anti-influenaa Serum (3) Influenza Antlgen(3}' ^ \ 1 Hon-specific chicken Serum (l)* _ ____ ■ Figure 2h. Diagram of a Negative Indirect Complement Fixation Reaction • ( ) is the order of addition of reagents ■ot react, bodies. since all the amtigem has been bound by the ohloken anti­ The coop lemen t is thus not affected, and «h«i the lndioator system is added, the complement is free to not upon it and hemolysis occurs. In Figure 2 b , a negative indireot reaction is shown. The ohioken serum does not contain antibodies specific for the influenza antigen and no reaotion ooours. When the guinea pig anti-influenza scrum is added, the antigen reaots with it and the complex formed fixes the complement. With the addition of the indicator system, no complement is available and no hemolysis is seen. These illustrations assune that all or name of the various reagents are involved in the respeotive reaotions. In aotual practice, the proportionate part of each reagent whioh enters into the reaotion is quantitatively measured. The use of a serodiagnostic test for visceral lymphomatosis must be of a nature whioh will allow the detection of the agent oausing the disease, and not merely a demonstration of the presence of tumor tissue. In human cancer, the detection of the presence of tumor tissue would be of value. As has been pointed out by Maver (19UU), such a test could be used for the differentiation of cancer and tuberculosis or to indioate the absence of metastases or residual growths following surgery. Also, it would provide a method for detecting growths while they were still in the operable stage. develop such a test. Many procedures have been used in attempts to Reviews by Davidsohn (1936) and Maver (I9U+) are available whioh show that the tests for the various types of tumors in animals and humans may be divided into three groups: (1) those which measure the comparative concentrations of the constituents of the blood, or the ohemical or physical characteristics dependent upon these constituents, (2 ) those which depend upon a specific antlgen-antibody reaction, and (3) those which compare the proteolytic or lipolytic aotli vities of the serums* Only tests of the type mentioned in group 2 are of Interest here* These tests, however, are designed to allow the iden­ tification of a speoific material which is a component of the tumorous tissue, such as purified protein fraotions (Kidd, 19^+2) end not an agent* Many of the tumors of animals have been shown to be caused by an agent, presunably a virus. The first demonstration of an agent of this type was made by Rous (1911), while working with sarooma in fowls. Other animal tumors, such as the adenocarcinoma of frogs (Lucke*, 1938), benign papillomas of the skin of western cottontail rabbits (Shope, 1933), the mouse mannary oarcinoma (Bittner, 1936), and Leukosis of fowls (Ellerman and Bang, 190*8) are known to be produced by virus-like agents. Serodiagnostic methods have been attempted for all these various types of tumors and are discussed in a review by Mavcr (I9I1I+). Only the tests concerned with the various forms of the avian leukosis complex (Biester and Devries, 19M+) will be mentioned here. The classification "leukosis” includes all the various forms of the disease listed under the avian leukosis complex. made on the erythroblastic form. The early studies were In this disease, the immature forms of erythrooytes beoome the dominant blood cell. Thomsen et a l . (1933) used a ground suspension of these cells as an antigen for complement fixation studies. study. They reported fixation in about 20 percent of the serums under These serums were from chickens which had been inoculated with a suspension of the neoplastic cells. The fixation observed was not con­ stant on repetition and may have been due to faotors other than a specific ant1gen-antibody reaction, since antibodies formed in chickens are not of a type capable of reacting with conplement (Rice, 19^8b). Kabat and Furth (I9I+O) attempted precipitin and complement fixation studies on the sedimented agents of fowl erythroleukosis and sarcoma, but were unable to disclose any speoific antibody in immune serums pre­ pared from tumor extraot and leukotic serum. They attributed their failure to demonstrate difference by invnunological methods between the materials sedimented from tumorous and normal spleen extracts by hi^i speed oentrifugation, to the presence of large amounts of "normal heavy material” in the virus preparations. They concluded that if agents for these conditions were present, they were there either in small amounts or they were less effective antigens than the normal heavy substance. Kabat and Furth (19Ul) did demonstrate, however, that the agent of the Rous sarcoma produced neutralising antibodies in the rabbit. Similar sediments from normal chicken spleen produced no neutralizing antibodies, and the complement fixing antibodies produced by both materials were unrelated to the neutralizing antibodies. Similar results were obtained by Foulds (1957) and Lee (19U2), using the serums of ducks, turkeys and chickens which had been inoculated with lymphoid and myeloid tumor extraots. Pollard et a l . (19U3) obtained complement fixation reactions with one form of the avian leukosis oomplex, namely, hemocytoblastosis. The malignant blood cells were ground in a Ten Broeck grinder, centrifuged at low speed, and the supernatant fluid was used as the antigen. Serums from adult turkeys and guinea fowls which had been inoculated with whole blood from nortnvl chickens and from chickens showing lesions of various forms of lymphomatosis and leukemic conditions were tested against the antigen. No reaction was observed when the serums of the animals reoeiv- ii%; nprmal chioken blood were used, but all the serums of those receiv­ ing blood from infected chickens gave positive reactions. Since the antigen was an extract of malignant white blood cells or their precur­ sors, and the positive anti serums were produced by the inoculation of whole blood containing the same type of cells, the positive reactions were apparently not due to an agent responsible for the condition, but rather to components of the malignant cells and a specific antibody pro­ duced by the inoculation of an extract of the same type of cells. It indicated, however, that the tumor cells formed in these related condi­ tions were similar and contained common components distinguishable from normal blood cells. Kissling (I9i4?) described a rapid slide test using stained, washed, formal inised lymphocytes which agglutinated in the presence of serum from a lymphomatosis positive chicken. Darcel Investigations of this test by (1950) showed that whereas a high correlation was obtained with birds showing advanced oases, healthy birds in a flock also showed a high number of positive reactions. The lymphocytes were not found to be necessary in the reaction because the dye and serum would react similar to the reaction between horse serum and isamine blue, the phenomenon described by Dean (1937). The use of a test, particularly complement fixation, in efforts to detect the agent of visceral lymphomatosis, has not been reported. The segregation of the agent in sufficiently pure form to permit its use as an antigen in the production of antiserum, and the development of a test which would allow the identification of either the agent of visceral lymphomatosis, or specific antibodies in an infected chicken, were the aims of the wo r k reported in this thesis. III. a. MATERIALS AND METHODS Complainant i— It has baan shown by Wadsworth (19U?) that complement can be preserved for an indefinite period of time by freezing. Pooling of the serums of a large number of guinea pigs would afford a constant supply of complement and eliminate the necessity of collecting and pro­ cessing small batches at frequent intervals. Appraximetely $00 young guinea pigs, wei/tfiing 800 to 1000 grams each, were secured and bled by cardiac puncture. Eight to 10 milliliters of whole blood was taken from eaoh animal, placed in a small vial, and allowed to clot in an ioe water bath. The vials were then placed in a refrigerated oentrifqge and spun for five minutes at 1^00 g to constrict the clot. The samples showing hemolysis, due to the meohanioal breakdown of the red blood cells, were discarded. The serums of the remaining samples were then deeanted into a large flask which was immersed in an ice water bath and rotated continually to insure complete cooling and mixing. The resultant pool was transferred to small vials in 1, 3» and 5 milliliter amounts and sealed with rubber stoppers. Eaoh vial, imme­ diately after sealing, was placed in a wire basket in a mechanical freezer where it was frozen and stored at -35° C. The guinea pigs surviving the first bleeding (approximately 200) were held for seven days and bled a second time. The serums obtained from this second bleeding were handled in the same manner as outlined for the first group. Approximately 2000 milliliters of guinea pig serum was obtained from the two collections, and served as a common source of complement for all the tests. b. Hemolysin:--The deteotlon of a specific reaction between an antigen and an anti semam, by the complement fixation test, is dependent upon the demonstration of the fixing or inactivation of the complement. As was mentioned previously, the hemolysis of the sheep red blood cells is used as an indicator of this reaction; however, the presence of the red blood oells alone is not enough. The complement must have an antigen- antibody complex to act upon, and the sheep red blood cells act as the antigen for such a oomplex. When inooulated into rabbits, the sheep red blood cells stimulate the production of a specific called hemolysin (Kabat and Mayer, ant is arum whi oh is 19148). Commercial anti sheep hemolysin was used for the tests reported here. It had been preserved b y the addition of an equal volume of gly­ cerol, and when kept in a refrigerator at 2-1+° C, the titer remained constant for an indefinite period of time. e. Sheep red blood cells:— A special flask was prepared for the col­ lection of the sheep red blood oells. It consisted of a 500 milliliter Erlenmeyer flask fitted with a number 6, two-hole, rubber stopper. Through one hole was placed a three-inch piece of 6 millimeter glass tubing which was plugged loosely with cotton and acted as an air vent. The other hole was fitted with a six-inch piece of the glass tubing to which was attached a heavy walled rubber tube approximately 18 inches in length. The blunt end of a 16-gauge California type bleeding needle was inserted into the free end of the rubber tube. The beveled end of the needle, which protruded fran the tifce, was pushed through the center of a square pieee of gauze and inserted into a test tube which was of sufficient size to fit snugly over the end of the rvbber tube. The gauze prevented the rubber tubing from adhering to the glass wall of the test tube during sterilisation. A screw d a m p on the rubber tube completed the oolleotion flask. The preservative for the sheep red bleod cells was a modified Alsever's solution (Bukantz et a l ., I9U 6 ) consisting of: Dextrose ........... 2.05 grams Sodium citrate ............... 0.80 grams Sodium chloride .............. O.I42 grams Citric acid ................ Distilled water .............. . 0.055 grams 100 milliliters This amount was sufficient to preserve 100 milliliters of blood; it was placed in a collection flask and steam sterilized. The sterilization had to be limited to 15 minutes at 121* C (steam at 15 pounds pressure) to prevent oaramelization of the solution. Sheep were made available at the Michigan Department of Health laboratories, Lansing, Michigan for the collection of blood. An area approximately three inches square over the Jugular vein was clipped, lathered, shaved, washed with water, rubbed dry, and painted with iodine. After careful removal of the test tube and gauze, the needle was inserted into the Jugular vein and 100 milliliters of blood allowed to flow into the flask. The flask was agitated continually to insure complete mixing of the blood and ] reservative solution. TOien 100 milliliters of blood had been obtained (determined by a mark previously placed on the side of the flask) the screw clamp was tightened and the needle remeved. Before storage, the preserved cells were transferred to sterile glass bottles apd sealed with rubber stoppers. Sheep red blood cells, when collected and stored in this manner, remained in good oondition for six to eight weeks, after whioh time a portion of them started to break down and a new batoh had to be secured. d. Diluents:--The components of the complement fixation test vary in their activity and must be diluted so they will be present in optimal ooncentrations. Also, after the antigen, antiserum, and complement are present, the volume must be made constant so that w h e n the sensitised red blood cells are added, a uniform volume and red cell concentration are obtained. by Mayer The diluent seleoted for use in these tests was described et a l . (l9U6a). They had shown that the addition of cations, especially those of magnesium and calcium, and to a lesser extent, those of cobalt and nickel, had a marked enhancing action on the hemolytic activity of complement. Veronal buffer, containing magnesium and cal­ cium oations, was prepared as follows: Sodium c h l o r i d e ..... ............ ....... . 85*0 grams 5 ,5~diethyl -barbituric acid Na 5»5“di«thyl-barbiturate .......... . .......... . 5*75 grams 3»75 grams Mg Clg • 5^2® •••.......................... 1*02 grams Ca Clg • 2 H g O ............................. 0,22b grams Distilled w a t e r ........................... 2000 milliliters The acid was first dissolved in 500 milliliters of hot distilled water and the other components then added. The resulting solution was steri­ lised by steam at 121* C for 15 minutes. This constituted a stock solu­ tion whioh was diluted with four parts of distilled water before use. Due to the presence of the cations, the solution has been oalled "Ionic buffer” and will be referred to as such. The ionic buffer was uood as the vehicle for all of tha componsnts of tha tasts. This included the diluting of the antigens, anti serums and complement to the proper concentrations, addition to the test system far standardisation of the volume, dilution of the hemolysin, and the final suspension of the red blood cells. The only place where this diluent could not be used was in the washing of the red blood oells. The red oell preservative employed as an anticoagulant, sodium oitrate, which prevented clotting by its action on the calcium of the blood plasma (Dukes, I9U2 ). If the ionic buffer was used for washing the cells, an excess of oalcium cations was available which overcame the action of the sodlxsn citrate and clotting occurred. Therefore, a solution which did not contain an excess of these oations had to be used for the washing process. A phosphate buffer (Kent, I9U6 ) was selected for this purpose and prepared as follows: Sodium chloride ...................... KHg 170.0 grams 2»U grams NagH P O ^ .............................. 11*3 grams Distilled water ........................ 1000 milliliters The components were mixed and heated slightly to facilitate dissolving and the mixture sterilized by staam at 121° C for 15 minutes. This served as a stook solution and was diluted with 19 parts of distilled water before use. After several washings with this solution, the plasma components responsible for the clotting were sufficiently removed so that ionic buffer could be placed with the red cells for the preparation of the standard suspension. Titrations of complement were conducted to determine the extent ef the enhancing action of the oaloiun and magnesium ions. This was done by titrating complement with ionic buffer as the diluent and comparing the titer to titrations in whioh phosphate was employed. As will be seen in Table I, less than half as much complement was needed to produce 50 per­ cent hemolysis when the ionio buffer was used as when phosphate buffer was present, which agreed with the findings reported by Mayer et a l . (l9U6a). e. Bleotrophotometer:--It has been shown by Kent «rt al^. (19U6) and Mayer et_ al_. (19U6b) that the measurement of the amount of hemolysis by the spectrophotometric or electrophotometric teohnique is very accurate when 50 percent hemolysis is used as the end point. A Fisher model AC electrophotometer was used for these determinations. A straight line-should be obtained by plotting the log scale r e a d ­ ings from the drum dial of the eleotrophotometer for varying concentra­ tions of a given solution. Since the material measured is a solution of hemolized sheep red blood cells, a similar solution was prepared by add.ing 2.5 milliliters of washed sheep red blood cells to 122.5 milli— liters of distilled water. The resulting solution was then used for the preparation of a series of hemoglobin concentrations, considering the original solution as representative of 100 percent hemolysis. Three filters were available for use in the eleotrophotometer. They had their peak transmittance at different wave lengths, namely U2 5 * 525* and 590 millimicrons. To determine which of these was most applicable, the various solutions, ranging in hemoglobin concentration from 10 to 100 percent, were tested against each of the three filters. The values TABU I THE EFFECT 07 CALCIUM AND MAGNESIUM CATIONS ON THE TITER 07 COMPLEMENT Tube Number 1 2 3 4 5 6 7 8 9 Complement ml. (1 :1 0 0 ) Diluent ml. .30 •25 •30 •35 .40 .45 .50 .55 .60 .80 .75 .70 •65 •60 .55 •50. •45 •40 Sensitized cells ml. 1.0 1*0 1.0 1.0 1.0 1,0 1,0 1,0 1,0 1. Diluent Electrophotometer Phosphate Buffer (a) Ionic Buffer Readings 3 4 6 10 18 20 26 31 39 42 (b) 2 4 6 11 15 20 24 32 38 43 (a) 24 32 38 46 51 55 59 62 65 69 (b) 23 32 38 47 52 57 60 63 65 69 Milliliters of undiluted complement necessary for 50 percent hemolysis with: Phosphate Buffer 0,00575 Ionic Buffer 0,00275 for 'the eleotrophotometer readings are plotted in Graph 1_. The readings for the Ij25 millimicron filter did not give a straight line, the values for the lower concentrations dropping considerably below what would be expeoted. The readings for the 590 millimicron filter covered only 12 divisions on the log soale of the dial, which did not allow the accurate differentiation of readings for concentrations differing only a few per­ cent. The 525 millimicron filter gave a consistent increase in the scale readings with increased concentration and was selected for use in subsequent determinations. The plotting of these points, as shown in Graph 1, did not coincide exactly with a straight line; however, the variation was later found to be due to differences in the inside dia­ meters of the tubes containing the hemoglobin solutions. The variation in the tube site was first noticed when repeat deter­ minations on the various concentrations were attempted. To cheok the reproducability of the readings obtained with the 525 millimicron filter a hemoglobin solution was prepared by adding 10 milliliters of washed red oells to i+00 milliliters of distilled water. The reading fer this solution was then adjusted to 1+0 on the log soale by the addition of dis tilled water. Using this value to represent 100 peroent hemolysis, 25 milliliters of each of the other concentrations (10 to 100 percent) were prepared. Two milliliters of eaoh concentration was pipetted into 15 tubes, making 15 groups of the 15 concentrations. The eleotrophotometer readings for the tubes containing any given concentration varied consi­ derably. A few tubes were chosen at random, their inside diameters measured, and found to vary in size, with the smallest measuring 0*975 centimeters, and the largest 1.020 centimeters. g Hi 8 Si no* th* eleotr©photometric determinations are based on the amount of light transmittance, the differences in the diameters of th* tubes would alter th* amount of solution through which th* light would have t# pass* If the tube were large, the distance through the solution would be increased and the reading lowered, due to th* adsorption of more light. If the tube were small, the distance through solution would be decreased and the reading high. To obtain accurate readings with a large number of tubes, the inside diameters of all the tubes must be constant. Approximately 3600 tubes were available for us* in these tests. They measured three-eighths by three inches and were flanged at th* top. The tub* size was not th* same as the cuvette usually used with this model eleotrophotometer, and a special cuvette holder was made. The first step in th* standardization of the tube size was th* mea­ surement of their inside diameters with calipers. The calipers wore set at 0*995 centimeters, and the inside diameter of each tub* graded accord­ ing to th* manner in which it fitted over this size measure. The tubes wore found to fall into thro* general classes: small, into which th* measure oould not be forced; average, which fitted snugly over the mea­ sure} and large, which allowed considerable movement after it had been inserted into th* tub*. A second chock on th* uniformity of size was then mad* on th* tubes which had been classified as average. This in­ cluded approximately thro*-fourths (2J00) of the original group *f tubes. This second check consisted of preparing 200 milliliters of a hemoglobin solution giving a reading of 1+0 on the elect re photometer. The solution was pipetted into the tubes in the amount of two milliliters eaoh and the eleotrophotometer readings taken. Since the solution was standard for all tubas, those having tha same insida diameter should giva the same reading. A marked variation was noted in only about 10 percent of* tha tubas, and these ware discarded. Tha remaining tubas, all giving approx• imately the same reading, ware considered satisfactory and used in sub­ sequent determinations. f. Rad blood call.concentration standardisation;--A reading of 1+0 on the lag soale of the drum dial of tha electrephetometer was used as tha 100 percent hemolysis and point for tha determination of the uniformity of the tuba size. This value was chosen arbitrarily, since it was approximately in the middle of tha scale, where tha unit scale size was relatively large and allowed easy determination of slight variations in the readings. When selecting the 100 peroent hemolysis value to be used in the tests, however, a larger value was considered mere desirable. This allowed a greater range between 0 and 100 percent, thus making the unit interval on the scale representative of less difference in the amount of hemoglobin present in the solution being measured. The unit size on the scale was largest at the 0 end and became smaller as the values increased. soale units were marked off on the dial Individual from 0 to 75* above which the each. markings were at intervals of five units The value JO was chosen far 100 percent hemolysis, thus making 35 the reading representing 50 percent hemolysis. Concentrations of hemoglobin, varying from 10 to 100 percent, the same as were used in the testing of the filters, were prepared. The con centration of hemoglobin in this case, however, was adjusted to give a reading of 70 for the 100 percent value. When platted, the readings for these concentrations^gave approximately a straight line as shown in Iraph 2. 3 I 8 n I Th* preserved rod calls had to b* washed b*f*r* their concentration :*uld be adjusted. The washing procedure was conducted as follows: 10 milliliters of the preserved cells was placed in a graduated centrifuge tube and centrifuged for five minutes at 1000 g. Th* supernatant fluid, or preservative-bleod plasma mixture, was deoanted off and approximately five milliliters of phosphate buffer solution added. The tube was then * shaken vigorously until all of th* cells which had adhered to the bottom of the tube wore resuspended. The volume was then mad* up to 15 milli­ liters with the phosphate buffer, the tube shaken, and the suspension centrifuged in th* same manner as described above. This procedure was repeated until a total of three washings with th* phosphate buffer had been completed. Following the last washing, the volume of the packed red cells was measured, a small amount of ionic buffer added, and the cells resusponded by shaking. They were then transferred to a 500 milliliter graduate and ionic buffer added in an amount sufficient to make a 2 per­ cent suspension of the cells. A portion of the buffer was used to rinse th* centrifuge tub*. In th* tests th* rod colls wore broken down by th* action of the complement. For th* standardisation of the red cell concentration, h o w ­ ever, this breakdown had to be brought about in seme other way. addition of distilled water proved satisfactory for this purpose. The Hi* ratio of distilled water to the red cell suspension, however, had to be accurate, since the concentration ef th* resulting hemoglobin solution was critical. This ratio was dependent upon the volume of the red coll suspension and the total volume when all the components wore present in a given test, which had been set at two milliliters. Of this amount, one milliliter mas taken up by the antiserum, complement and diluent, the diluent (ionic buffer) being added in amounts sufficient to standard­ ize this volume. The other one milliliter was made up ef equal parts ef red cell suspension and hemolysin. Therefore, the standardized red cell suspension aooounted fer 0.5 milliliters of the total volume in any given test. By adding 1.5 milliliters of distilled water to 0.5 milli­ liters of the red cell suspension, a hemoglobin solution comparable to 10^ percent hemolysis in a test was obtained. The readings for hemeglo- bin solutions prepared in this manner from the 2 percent cell suspension, were uniformly higher than 7 0 , indicating that there was toe great a concentration of red cells. To correct for this, the formula Vg- was used (Kent et al_., I9U 6 ) . In this relation, 70 was the volume ef the suspension being measured, OD^ the reading obtained for this suspen­ sion, 70 the reading desired, and Vg the velume needed to give the read­ ing of 70. By inserting the proper values and calculating fer V2 , the amount of ionic buffer necessary to add to the suspension to give a reading of 70 was determined. If the reading for the 0D^ value was belew 70, the same formula was used to calculate the amount of diluent to be removed. This was accomplished by centrifuging a portion of the suspension and the calculated amount of diluent decanted off. The sedi­ mented cells were then resuspended and returned to the parent suspension. The reading was checked the same as before and, if necessary, additional adjustments made until a value of 70 * .5 was obtained. An example of the use of the above formula would be as follows. If the reading fer the original suspension or 0D^ was 78, tnd the total vol­ ume or was lLi.0 milliliters, these values would be inserted into the formula as: Vn z lhO x 78 2 ^ --V2 : 2 x 78 v2 = 156 Th* s*c*nd volume, V 2 , which should giv* a reading of 70, is 156 millilit*rs or an incrtase of 16 milliliters over th* original volume Vj. Therefor*, 16 millilitora of ionic buffer would bo added, the suspension a thoroughlv mixed, and th* reading again checked. g. Selection of complem ent fixation technique: In complement fixation tests conducted prior to those reported here, the techniques described by Mayer et a l . (191*7, 19U8b) and Rice (191*6, 19l*7a, b, 19l*8a) had been used. Both of these techniques utilise th* 50 percent hemolytic unit of complement. In the technique described by Mayer, an excess amount of complement was placed with the antigen-antibody system being tested. By titrating the amount of complement present before and after the entigenantibody complex had acted upon it, and subtracting the latter value from th* former, the amount of complement fixed was determined. In th* second technique, that used by Rice, a series of tubes, eaoh containing the same amounts of antigen and antibody, was prepared. Vary­ ing amounts of complement, usually 3# 6, 9, and 12 units, were added to those tubes. The degree of reaction was then calculated frem the amount of homolysis occurring in th* various tubes. Both of the techniques mentioned used th* slope of th* hemolyti* curve in the determination of th* units of complement which had been fixed in a given reaction. Th* slop* of th* line, represented by i, was the n slop* of the straight line obtained by plotting th* log x which was th* quantity af complement used, against tha lag _2L_ whera y aqualed tha 1-y percentage of hamelysis which had accurrad. By using this slapa value, tha units af complement responsible far any given ohanga in hamalysia cauld ba calculated. If thasa valuas were calculated aver the entire range af hamalysis obtained, a table cauld ba prepared which would give tha amount af complement (in units) responsible far a given amount af hemolysis. Those complement valuas changed, however, with a ohanga af tha slope valua. The actual determination of the slope was made by use of von Krogh *s alteration equation (von Krogh, 19l6)» x s K ( y ) 1/n “i V or in logarithmic form? log x = log K ♦ 1_ log y n 1-y In these relations, x represents the volume of complement used and y the corresponding percent of hemolysis. The constants l/n and K, respec­ tively, denote the slope of the hemolytic curve and the quantity of com­ plement giving 50 percent hemolysis (one unit). The direct calculation of l/n, however, was made by the use of the method of least squares (Kent et_ al., 191*6) in which the following formula was employed: T /„ - N .3 XY - (:-X x -Y) ' - N-.-Y2 ~ ~ W 7 P --In this relation X was the log x, and Y the log y , and N the number of i-y observations. The values X and Y had to be calculated for in a given titration. each of the tubes used To facilitate these calculations, tables were prepared which gave the values for X, Y, and Y2 over the range of values encountered. In the sample calculation shown in Table II, five values for x (the amount of the complement) were used, since they all gave read ings within the range of 20 to SO percent hemolysis. If the 50 percent end point had been near the smallest, or largest, of the x values, how­ ever, only a portion of them could have been used since those at the opposite extreme would have been out of the usable range. A minimum of three values was considered necessary for the calculation, with the 50 percent value falling between them. In other words, if there were only three values within the 20 to 80 percent range, but all were either above or below the 50 percent value, no calculation was made. The consistency of the slope value was questioned when, upon pre­ liminary calculations using complement titrations, a variation from 0.25 to 0.35 was obtained. A large group of complement titrations, the tech­ nique of which will be given later, was conducted, and the slopes calcu­ lated. The slope values ranged from 0.23 to 0.3^ for the 129 determina­ tions involved. The average for the group was 0.30, which was consider­ ably above the 0.20 given by Mayer et a l . (19l;6b) and Rice (19U2) as the optimal value. If the slope were considered to be 0.20 and conversion tables prepared (Rice, 19L-8a), considerable error would arise when suoh tables were used in calculating the amount of complement fixed in a test These slope determinations had been made with values obtained from complement titrations. It was obvious that they did not agree with the generally accepted slope value of 0.20. This discrepancy led to the consideration of the possible effect of either the antigen or antiservsn, or both, on this slope value, and tests were conducted to determine what effect they might have. TABLB II ▲ SAMPLX SLOPS CALCUIATIOK 0.30 0.35 0.40 0.45 0.50 1.477 1.544 1.602 1.653 1.699 (ml. of complement) X (log of x) Slectrophotometer readinc 15 22 36 43 48 •215 •315 •515 .615 •685 1.438 1.663 2.026 2.203 2.337 T2 2.068 2.766 4.105 4.853 5.462 XT 2.124 2. 568 3.246 3.642 3.970 y (percent hemolysis) T (log of j) Values needed in the calculation: >X 7.875 IT 9.667 2-T2 19.854 £XT 15.550 teY)2 93.451 (rX*2T) 77.094 Tozsala used in the calculation: l/n. «rU 5TY?)- (* Y ) 2 (6»18.650) - (77.094) (5 *19.254) - (93.451 77.750 - 77.094 96.270 - 93.451 -a.656 2.891 l/n - .23 * S is the number of values involved. The antigen selected for standardization of the technique had been the PR8 strain of human influenza virus Type A. The slope values fer titrations made -when 0.1 milliliter of the influenza antigen at a dilu­ tion of 1 : 2 0 .was present, differed from those obtained when only comple­ ment waa present, varying from 0.26 to 0.1+1 with an average of 0.33 f°r 123 determinations. The antiserum selected for these tests had been prepared by the inoculation of rabbits with the influenza virus as is described under the section on antiserum preparation. A series of titrations was made in which 0.1 milliliter of the antiserum was used at a dilution of 1:20, and the slopes calculated. The slope values varied from 0.26 to 0.1+3 with an average of 0.3U for 105 determinations, which was approximately the same as had been ^ound with antigen present. The most important determination in a complement fixation test is the titration of the speoific reaction between antigen and antiserun. To determine the variability of the slope value when this reaction took place, a series of titrations was made in which the amount of complement was adjusted so that there were sufficient electrophotometrlc readings in the desired range to allow the calculation of the slope values after a portion of the complement had been fixed by the reaction of influenza antigen and rabbit anti-influenza serum. The slope values in this oase ranged from 0.15 to 0.26 for 97 determinations, with an average of 0.21, which agreed clesely with the recommended 0.20 value mentioned previously. The variation between individual values, however, was considerably greater than had been desired. The determination of the amount of reaction occurring in a given titration, and the ameumt of complement necessary for the production ef 50 peroent hemolysis, when alone or in the presence of either antigen er antieerum, or both, is dependent upon the slope value in the techniques of both Rice and Mayer. If the slope were considered to be 0.20 for the canplement titration, but was actually 0.30 (the average of the slope values obtained for complement titrations), the ameunt of complement necessary for one unit could not be accurately calculated, and any deter­ minations employing the unit value calculated with the 0.20 slope value would be in error. The variability of the slope values, when the various reagents were present, and the possible errors which would occur unless separate slepe values were used in the preparation of conversion tables for each reac­ tion involved, led to the conclusion that a technique was needed in which the slepe calculation was not necessary. In the techniques mentioned previously, the amount of complement necessary for percent hemolysis, er one unit, was determined and a given number of units used in a reac­ tion. Since the amount of complement fixed b y a given reaction is used to determine the extent of the reaction, it was considered a more direct approach to determine the actual milliliters of complement involved, and then convert this to units, rather than to determine one unit and work in multiples ef this value. The determination of the exact ameunt of coirplement necessary te give 5^ percent hemolysis, either in a complement titration or when one or more roagents were present, was possible by platting the electrophoto­ meter readings against the milliliters of complement vised. Where the curve oroosod th* 1 1 m representative ef 50 peroent hemolysis (reading *f 35)# ib would also imt*rs*ot th* coordinate representing th* ameunt ef complement necessary to produce this degree of hemolysis. Th* curve shewn in Graph ^ was obtained from a titration of complement and was carried out as described in th* section on complement titration. The same type of graph oan be prepared for the readings obtained for any titration, or reaction moaaurement, as long as the amount of hemolysis gives readings which are within the range of 20 to 80 poraont and are distributed to both sides of th* 50 percent hemolysis value. When a largo number of determinations was to be made, the plotting of the respective values became a task of considerable magnitude. As will be noted in Graph 3* only two of th* readings are aotually used for the determination of the 50 percent end point. These are th* above and just below th* reading of 35 (50 percent hemolysis). ones just To eli­ minate the necessity of plotting the electrephotomoter readings on graph paper, connecting th* points and determining th* milliliters of comple­ ment fer each titration, a table was prepared which gave th* proportion­ ate distance, between any two complement values, where th* curve would oress th* line representative of 5® percent hemolysis. This table was prepared by using a large sheet of graph paper and making two columns of values from 0 to 70 as shewn in Figure 3_. By extending a straight edge from th* value 1 of th* left column to all of th* values greater than 35 in th* right column, then from 2 to all of th* values over 35# so on through 3l±, th* propertionate distance between these columns (considering th* total distance to be 10 unit spaces) where th* curve intersected th* line ef 50 percent hemolysis (55) determined and recorded. For 37 70 - 60 - llectrophotoaeter Beading 50 - 40 - 30 - 20 10 - - { (50 percent end point) » • i i f I i i i i i i i i i j I t t I | i i i i i .001 .0015 .0020 .0025 .0030 .0035 Milliliter* of Undiluted Complement * Graph 3 The Relationship of the Blectrophotometer Readings to the Amount of Complement 70 -i 70 60 60 1 50 50 3 -i -4 40 35 40 — 4- -4- 35 \ 30 4 30 -1 i 4 20 4 20 3 10 10 figure 3 Chart Used for Determination of Proportionality Values example, if the reading immediately below the 50 percent value was 25 and the one abeve was U5# line cenneoting the two weuld intersect the 50 percent line half-way between the columns, as shown in Figure give a proportionate value ef 5« and By arranging these values as shown in Table III, the proportienate distance where a line, between any two of the readings for different amounts ef oonp lament would cross the 50 per­ cent hemolysis line, could b e determined* Oils eliminated the necessity ef platting the readings and could be used with any amounts of oenplement employed. The use ef the table far the determination of the proportionate distance where the curve cresses the $0 peroent hemolysis value net only eliminated the necessity ef platting the electrephotometer readings, but a also reduced the number ef readings which had to be made. Only two read­ ings were neoessary to dotormino the amount of reaction occurring in a given titration, those being the ones on either side and closest te the 30 peroent hemolysis value. The use of this procedure eliminated the neoessity far the use of a slope value in any of the determinations and avoided the inherent errers which weuld have resulted from the use ef such a value . h. Calculat ion of data:--The use of a large number ef different anti­ gens and antiserums made it necessary to adopt a method for the calcula­ tion of results which would facilitate the easy identification of the various reagents. As will be shewn later, symbols for the individual reagents were used, but for the calculation of the amounts ef complement fixed, either in terms ef milliliters ef undiluted complement er units, additional symbols were needed. The notations suggested by Thompson et^ TABLE 07 FR0PQBTI0HATJ VALUI8 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 1 fio 2|10 3 110 4 10 5 10 6 10 7 10 8 10 9| 1 0 ! llT 9 1 2 '9 9 13 14! 9 9 16 9 17 9 18 9 19 9 20 9 9 21 9 22 23 9 24 9 25 9 26 9 27 9 28 9 29 8 30 8 31 8 32 7 33 6 34 5 1 0 10 10 9 9 9 9 9 9 9 9 9 '9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 8 9 8. 9 8 9 8 9 8 9 8 9 8 9 8 9 8 "9 8 9 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 7 8 7 8 8 7 7 7 7 8 8 7 6 8 7 6 7 6 5 7 6 5 6 5 4 5 4 4 4 2 2 8 8 8 8 8 8 8 7 7 7 7 7 7 7 7 7 7 7 7 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 7 8 7 8 7 8 7 8 7 8 7 7 8 7 7 7 7 7 7 7 7 7 7 7 6 7 6 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 7 8 7 8 8 7 8 8 8 8 7 8 8 8 8 7 8 8 7 7 8 7 7 7 8 7 7 7 6 7 7 7 6 8 8 7 7 7 6 7 7 7 6 6 7 7 6 6 6 7 7 6 6 6 7 7 6 6 6 7 6 6 6 5 6 5 5 6 6 6 6 5 5 5 5 5 5 4 6 6 5 5 4 4 5 4 4 4 4 4 4 4 3 3 4 3 3 3 2 3 3 2 2 2 1 8 8 1 8 1 1 1 7 7 7 7 7j7 7 7 7 7 7 7 7 7 7 7 ? 7 7 7 7 7 6 7 7 7 6 7 7 6 6 7 7 6 6 7 6 6 6 7 6 6 6 7 7 7 7 7 7 7 7 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 5 5 5 5 5 5 5 5 5 4 4 4 4 4 4 3 3 3 3 5 5 5 5 5 5 5 5 '5 5 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 3 3 3 3 3 3 3 3 3_ 3 6 6 6 6 6 6 6 6 6 5 5 5 5 5 5 5 4 4 4 4 4 4 3 3 3 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 4 4 4 4 4 4 4 4 4 4 4""4 4 4 4 4 4 4 4 4 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 .6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 5 5 5 5 4 4 4 4 3 3 5 5 5 5 '4 4 4 3 3 ;3 5 5 5 5 5 4 4 4 4 3 3 5 5 5 5 5 4 4 .4 4 4 3 3 5 5 5 5 5 5 5 4 4 4 4 3 3 3 3 ,5 5 5 5 5 5 4 4 4 4 4 3 3 3 3 5 5 5 5 5 5 5 4 4 4 4 3 3 3 3 5 5 5 5 5 5 5 4 4 4 4 4 4 3 3 3 3 2 2 2 2 2 2 2 2 2 .2 2 5 5 5 5 5 5_. 5 5 4 4 4 4 4 4 4 3 3 3 3 6 6 6 2 2 2 2 2 2 2 1 1 1 2 i2 i :i 6 6 6 6 6 5 5 5 5 5 5 5 5 4 4 4 4 4 4 3 3 .3 3 3 6 5 6 5 6 '5 "5 6 ’ 5 5 5 5 5 5 5 5 5 5j 5 5 ”5 "5 5 5 5 5 5 5 5 5 5 4 5 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 .4 4 4 4 4 4 3 3 3 3 3 3 3 3 3 3 3 3 3 2 6 6 5 5 5 5 5 5 5 5 5 5 2 2 2 2 2 2 2 2 6 66 67 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 4 5 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4_ 4 - 4 4 4 4 4 4 3 4 3 3 4 3 3 3 3 3 3 3 3 3 3 3 3 3 3 68 69 70 5 5 5 5 5 5 5 5 5 5 5 5 5 5 4 ^ 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 -4 -*4 4 4 3 3 3 3 3 3 3 3 3 3 3 '3*'S 3 3 3 3 3 2 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 "l T T 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 ,1 X. 1 1 1 1 0 0 0 0 0 0 2 £ c si. (I9U9 ) were used for this purpsss. Ths origin ef thsss symbols was out1 insd by him and will not bs rspsstsd. A list of thoss ussd in the calculation of ths results given hers are as follows: (1) K — (2) K# = the amount ef complement necessary to produce 50 percent hemolysis. K when serum, antibody, and antigen are absent suoh as in the reaction called ”complement titration." This value is the arbitrary unit, making K# = 1. (3) = (ii) Ka r (5) K when serum alone is present, and K when antigen alone is present. K when both serum and antigen are present. All ef these notations refer to the values when expressed in terms of units of c emp lemont. The same notation was used when the values were in terms ef milliliters of undiluted complement, except that the letter K was primed. Therefore, when expressed in terms of milliliters of undi­ luted complement, the symbols were K*s , K ’a , and a» If specific serums or antigens were involved, the symbols representative of them were inserted in place more than of the "s" or "a". This made itpossible to use one serum or antigen in a group of tests andmaintain its identity. The use of these symbols in the indirect test calculations neces­ sitated the introduction ef an additional notation representing the reaction where both chicken and guinea pig serums were present. This was designated as: (6) K_ _ _ (units) or K* „ ^ (milliliters of undiluted complement; which, when subtracted from the values obtained for (5)# g av# "the titer ef the chicken serum. A dearer_understanding of tho mothod of calculation will ba pessiblo if tho values for a typical titration are used, and a dotailod calculation carriod out. Tho titer of tho complement was dotorminod first. Tho value is always 1; therefore, only tho K'# , which is in torms of siillilitors of undiluted complomont, can bo used since, as will be shown, tho other K T values must bo dividod by it to determine the K, or unit, values. In this oaso, tho K'0 was 0 .0032. Tho values for tho K*a and K* were calculated next by use of tho proportionality table (Table III) and wore tho tests on tho anti comple­ mentary activity of those reagents. Tho symbols for the actual reagents wore substituted for tho waw and ns", respectively. The calculation for vs the influenza antigen (FA) was * KFA = " FA The subtraction of 1 ^ o accounts for tho one unit necessary to produce the 50 percent hemolysis which was measured. calculation was: When tho actual values wore inserted, the actual Kr,. “ .0QLi£> -l r I.!* -1 - 0 J* units. The antioemple.603^ montary values fs r tho guinea pig anti-influonza serum (FGPS) and chicken anti-influenza serum (FCS) wore calculated in tho same manner. KrjPS = KPPS -1 = 1.3 -1 * 0.3 units = «QQ50 -1 = 1.5 -1 = 0.5 units .0032 Those values represented tho anticemplementary activity of tho indivi­ dual reagents, and had to bo taken into consideration in tho calculation of the amount of fixation occurring in any reaction in which they wore involved. The next calculations to consider wore tho control reactions involv ing all possible combinations of reagents normally considered to have no effect ox tho complement. When a specific reaction was being measured, and there was a number of reagents present, control teste had to be made using all combinations of all reagents. This was done to eliminate the possibility that a second reaction was taking place which weuld have seme effeot en the complement. The first of these combinations was the influenza antigen and chicken anti-influenza serum. The antibodies from the ohioken, although capable of combining with the antigen, should net produce acomplexwhich made as acheck en weuld affect the complement, that assumption. andthis test was The anticomplementary values for these two reagents had to be taken into consideration and the calculation made as follows: KFA ♦ FCS s ( H ,.**». PCS -1 ( = ( ( = ( ) _ (k f a * Kpcs) - (O.I4 ♦ 0.5) ) .0050 •00^2 1 .6 - 1 -1 ) - ) ) 0.9 = 0.6 - 0.9 K FA V FCS r - 0.3 units The second control test involved the guinea pig anti-influenza serum (FGPS) and the chicken anti-influenza serum (FCS). This was an important control since, if the material inoculated into the guinea pig to produce the influenza antiserum had contained chicken material of a nonspecific type, the antibodies formed might react with a similar substanoe in the chicken serian. The calculation for this test was; UU Kp3PS * rcs = (K ro?K-* reS -1) : r ( $ & s - = ( 1.8 - C W > S ♦ Krcs> -1 ) - (0-5 * °*5) -1 ) - 0.8 = 0.8 - 0.8 k fgps + PCS = 0.0 The direct test involving the influenza antigen (FA) and guinea pig anti-influenza serum (FGPS) was then calculated in the same manner as the two previous reactions. When a large number of tests was being made by the indirect technique, the direct test had to be standardized and was actually an indicator system, since all indirect tests contained a complete direct test system plus an additional antiserum (chicken) which was the material being tested. The calculations for the direct reaction were as follows: KFA ♦ FGPS = ' ( K *FA ♦ FOPS ( jfT ( .0032 « ( 18.1 ) _ ,) ,v v \ ) " ' FA ♦ * FOPS'* _ ♦ °»3) -1 ) - 0.7 - 17.1 - 0.7 KFA ♦ FGPS = 16.u The indirect test was calculated next. Both the guinee pig and chicken anti-influenza serums were involved in this test and as will be noted, the anticemplementary values for each are taken into consideration, -1 > - (Kpi^Kreps^Kpqs) KFA t TOPS * PCS = * ( j.0? ? ® . -1) - (o.u ♦ 0.3 ♦ 0 .5 ) =( 6.9 - 1 ( .0032 = 5.9 - 1.2 K FA *■ FGPS 41 FCS = U-7 unit* ) ) - 1.2 % Tho difforonoo in the number of units of complement fixed in tho two tosts indicated tho oxtont of binding of tho antigon by tho chickon antiserum. When tho units fixod in tho indiroct tost worn subtracted from tho number obtainod for tho diroot tost, this binding by tho chickon antis or um was dotorminod in torms of units of complainant • This valuo was tho indiroct titor of tho chickon sorum and designated by tho symbol Ic 8 * In torms of tho natations it was equivalent to: Ics * KFA ♦ FGPS “ KFA ♦ FGPS «■ FCS which , whon calculatod with tho actual valuos, was: Xos = l 6 *k - U *7 Ic> s 11.7 units IV. a. TITRATION OF REAGENTS Complainant titrat i o n :— The amount of fixation m s tasts in terms of milliliters of complainant. maasurad in tha It was desirable, however, for oasa of intarpra tat ion, to ccnvart thasa amounts to units. The na - fare, tho datarminatian of tha amount of camp 1 ament nacassary to produca 50 par cant homely sis, or one unit, was nacassary. Tha complement, in tha form of f rat an guinea pig strum, was removed from storage, thawed under flawing tap water, and diluted 1:100 with ionic buffer. A suspension of shaap red blood calls was standardized electrophetometrioally and mixed with an equal volume of hemolysin. thus made sensitive to tha action of complement. The calls ware By mixing varying amounts of complainant wi-th these sensitized calls, the exact amount nacas­ sary to produca 50 percent hemolysis, which would be one unit, could be determined. A series of reactions in which varying amounts of complement ware employed, constituted a complement titration and was prepared as shown in Table IV. When the proportionality table (Table III) was us ad to determine the proportionate distance between the amounts giving the readings of 5U and hB in tho titration shown, a value of 1 was obtained. Therefore, since in this case the interval between ths volumes of comple­ ment was 0.05 , the proportionate amount was 0.1 of this or 0.005, making 0.255 milliliters of the 1:100 dilution of complement necessary for the 50 peroent hemelysis. This was rounded off to 0.26 or 0.0026 milliliters of undiluted complement. TABU IT COMPLEMENT TITRATION Tube Number Reagent 1 2 3 4 5 Complement 1:100 (ml.) 0.15. 0.20 0.25 0.30 0.35 Ionic Buffer (ml.) 0.85 0,80 0.75 0.70 0.65 Sensitised Red Cells (ml.) 1.0 1.0 1.0 1.0 1.0 Electrophotometer Reading 9 20 34 48 57 b. Hemolysin tit ration:--The technique described by Kant (I9U 6 ) -was ua#d to determine tho optimal cone out rat ion of hemolysin. This has boon described by Wadsworth (19^7) *s the concentration beyond which further increase fails to enhance appreciably the hemolytic activity ef comple­ ment. Therefore, if varying concentrations of hemolysin were added to a series of tubes, e'ach containing 1 unit of complement, all tubes contain­ ing 1 or more units of hemolysin would show 50 percent hemolysis, since there would be sufficient hemolysin present to allow all of the comple­ ment activity to be expressed. However, in those tubes containing less than 1 unit of hemolysin, the complement activity would be lessened and the amount ef hemolysis would fall below 50 percent. In other words, as the amount of hemolysin decreases below a certain magnitude, the amount of complement necessary to produce 50 percent hemolysis increases. The hemolysin dilutions were prepared over a range of from 1:100 to l:i;000. Ten r.ill' 1 Iters cf each of the dilutions was mixed with an equal volume of standardized red cells and allowed to stand at room tempe rature for at least 10 minutes (1/ayer et a l ., 19U6b). type shown in Table V were then prepared. Twelve titrations of the These were merely complement titrations with varying amounts of hemolysin by which the effect of the hemolysin concentration could be determined. lhe titrations were con­ ducted in the same manner as described for the complement titration, but the red blood cells were, in each case, sensitized with a different con­ centration of hemolysin. The effect of the change in the hemolysin con­ centration on the complement unit was best seen when the amount of complement necessary for 50 percent hemolysis was plotted against the concentrations of hemolysin, as shown in Graph U* The curve shows that TABLE HEMOLYSIN T TITRATION Complement Complement 1:100 (ml.) .30 ".20>' ' .25 .35 Electrophotometer Reading ---34 48 — 32 45 — — 50 31 — — 51 32 — 49 30 — 29 47 — — 32 48 _ — 51 31 _ 28 48 — 24 44 — — 42 31 27 - Hemolysin diluti on 1:100 1:200 1:300 1:400 1:500 1:600 1:700 1:800 1:900 1:1000 1:2000 1:4000 Unit .40 — — — — — — — — 49 0.0026 0.0026 0.0026 0.0026 0.0026 0.0026 0.0026 0.0026 0.0026 0.0028 0.0032 0.0037 .0040-1 .0035- fr ^ .0030-1 *CJ « .0025C3 .0020- TS'~$ & $ § to C- 00 o* o Q Hemolysin Dilution Graph 4 Graphical Determination of Hemolysin Titer 1 o there was no change in tha amount of coup lament nacassary to prod uc a 50 percent hamolysis at tha hamolysin dilutions of 1:900 or less; however, above this dilution, larger amounts of complement were necessary. The titer, therefore, was considered to be 1 :9 0 0 , or the concentration whi da, when mixed with an equal volume of standardized sheep red blood cells, contained one unit of hemolysin. To insure the presence of sufficient hemolysin, a dilution of 1:500 was used in all tests. This dilution contained approximately 2 units as recommended by vVadsworth (1939)* The titer of the hemolysin was checked every two to three weeks and varied within the range of 1:800 to 1 :1000. The dilution used in the tests was maintained at 1:500, however, since the 2-unit value was approximate and used only as a safeguard against a deficiency of this reagent. v. PREPARATION OF TEST REAGENTS A. Antigen The antigens used in tha studies reported here were Human Influenza virus (PR8 strain) Type A, normal chicken tissue extract, and extracts of lymphoid liver tumors and pectoral muscle tumors from chickens show­ ing visceral lymphomatosis. The Influenza virus was used for the stan­ dardization of the test technique. The normal tissue extract served as a control on the normal tissue reactions of the tumor materials, and the tumor extracts were the test antigens used in attempts to determine the presence of the tumor agent. a. Influenza virus:— The virus* had been cultivated in the allantoic fluid of chick embryos, harvested when the embryos were 12 to 1U days of age, and formalin added in an amount sufficient to make a 0.2 percent solution. This inactivated the virus and served as a preservative. When treated in this manner, the virus suspension remained apparently unaltered for an indefinite period, if stored at 2-U° 0. It was given the name "Flu Antigen" and designated by the symbol FA. b. Normal tissue antigen:--An extract of normal chicken tissue was prepared for use as a control on the normal tissue reaction which might occur when the tumor materials were used in the tests. Hie principal tumor antigens, as will be described later, were prepared from tumorous livers. The control antigen, therefore, needed to contain as nearly as possible, the same tissue components, with the objective of making the * Obtained from Lederle Laboratories Division of the American Cyanamid Con^any, Pearl River, New York. only difference between the two materials the presence of tha agent in the tumor antigen. The liver, spleen and thymus were selected as the tissues whioh would furnish the materials necessary for the production of a satisfac­ tory control normal tissue antigen. The liver supplied normal liver cell components, while the spleen and thymus, due t o their relatively high lymphoid cell content, supplied components of this type of cell. The lymphoid cells of the spleen and thymus were not identical to those found in tha lymphoid tumors, but war* considered to be the best available. Tha tissues were secured from frozen materials that had been stored in tha dry ice chest at -?G° C. They had been removed from chickens which were the progeny of a single mating, designated as mating number D018. These birds had been raised in complete isolation, having been hatched and reared in an isolated pen within a quarantined building. They were approximately 2100 days of age wher. killed, and had shown no evidence of lymphoid tumors or other disease conditions at any time. ^or the actual preparation of the normal tissue antigen, samples of liver, spleen and thymus tissue were removed from storage and thawed under flowing tap water. Jhe tubes were then opened and the tissues placed in the bowl of a Tfaring blendor in the proportions of approxi­ mately 20 grams of liver, 2 grams of thymus, an d 5 grams of spleen. Two hundred aad fifty milliliters of sterile phosphate buffer was added, and the mixture blended for five minutes. The resultant homogenate was them transferred to sterile lustero id centrifuge tubes and spun in the multi­ speed attachment of a refrigerated International Number 2 centrifqge for 10 minutes at 3U,000 g. Three distinct layers were formed in the tubes, tho bottom layer consisting of particulate material, a middle layer of very small particles and materials. soluble materials, and a t op layer of fatty The middle layer was removed by carefully siphoning it from between the other two layers. The top and bottom portions were then dis­ carded and the material whi oh had been s iph oned off transferred to addi­ tional sterile lusteroid tubes. The second group of tubes, containing the original m'ddle layer, was centrifuged for 10 minutes at L600 g and the central portion siphoned off the same as had been done with the first centrifugate. It was then diluted with phosphate buffer until a volume of 250 milliliters was obtained, and 10 milliliter amounts. and transferred to serum vials in 1, 5» The vials were sealed with a flame, and frozen by placing them directly into a mechanical freezer at -35° C. This material, the normal tissue antigen, was designated as NTA, N repre­ senting normal, T-tissue and A-antigan . c. Tumor ant ige n :- -The chickens used as a source of tumorous material had been, with two exceptions, inoculated with filtrates of the RPL strain 12 tumor when they were one day of age. All of the methods of prepara­ tion were designed to furnish a suspension of the tumor agent which could be used to stimulate antibody production and serve as an antigen in the complement fixation reactions . d. Tumor antigen-Ll:--Five chickens served as a source of the tumorous livers and serums used for the preparation of this antigen. When, on physical examination, it was evident that the livers were enlarged, each bird was bled by cardiac puncture, the serum recovered, and dry ice chest. aseptically. stored in a The bird was then killed by electrocution and opened A portion of the liver was removed, minced with scissors and transferred "to pyrax tost tubas. Theso wore ssaled with a flame and stored in tha dry ica char" . At tha tima af preparation of this antigen, which was designated as TA-L1 (tumor antigen - liver l), the tumorous material, tetaling 25 grams, was removed from tha free tar, thawed under running tap water, placed in a Waring blandor with 100 milliliters af phasphate buffer and blended far five minutes. While still in tha blandor, 81 milliliters of serum fram tha same birds was added. Tha mixture was blended for another minute, transferred to lusteroid centrifuge tubas, and spun in the multispeed attachment af a refrigerated International Number 2 centrifuge at 3k,000 g far 10 minutes. The supernatant fluid was siphoned off, placed in glass serum tubes which were sealed, and frozen slowly in an alcohel-dry ioe bath. The temperature of the bath was first adjusted to 0* C, the tubes placed in it and the temperature lowered ene degree a minute until it reached -30*, at which time the tubes were transferred to a dry ice ohest. a. Turner antigen-L2:--In this preparation, an attempt was made to con­ centrate the turner agent and at the same time, decrease as much as pos­ sible the tissue component content. Fifty-two grams ef tumorous liver material was placed in a Waring blandor with approximately nine volumes ef cold phosphate buffer (U5° milliliters), and blended for five minutes. It was then transferred to large glass centrifuge tubes and spun far 20 minutes at 5*000 g in tho angle head ef a refrigerated International Number 2 centrifuge. The supernatant fluid, which was quite turbid, was siphoned off into a flask immersed in ice water, and the sediment, con­ sisting ef the larger particulate materials, was discarded. A portion ef the supernatant fluid was than transferred to six lustaraid tubas having a oapaoity ©f ^ppraximntaly 15 millilitars. Thasa six tubas, which wars tha capacity of tha multi spaed attaohmant head, war© spun for 120 minutas at 30,000 g. Tha suparnatant fluid was pourad aff, tha tubas allowed to drain, and than refilled with an additional 15 millilitars af tha original material. This was rapaatad until a total of four volumss of approximataly 15 millilitars aach, had baen oontrifugad in aach tuba. Tha pollats incraasod in siza with each centrifugation, and after the fourth run, aaoh contained tha sedimented materials from approximately 60 millilitars of tha original material. Each pallet was resuspended in 10 millilitars of phosphate buffer, returned to tha multispaed attaohmant, and spun for five minutes at 9.000 g. Tha supernatant fluid was then siphoned off, and tho sediment of each tube re-extracted with a second 10 millilitars of phosphate buffer. The supernatant fluid from the second extraction was added to that obtained from tha first extraction and tha mixture transferred to serum tubes. Thasa wars frozen with the aid of an alcohol-dry ice bath, as previously described, and stared in tha dry ice chest. f. Turner antigon-L3i— A third turner antigen, 150 grams of tumorous liver tissue. TA-L3. « s prepared from Two thousand millilitars ef phos­ phate buffer wss added to the tumor material, and tha mixture blended for five minutes in tha Waring blandor. The blender capacity was limited to approximately 700 milliliters, so the tumor material was divided into three parts and each portion blended with approximately one-third ef the buffer solution. The three batches wera then mixed in a large flask and 9 6 grams ef Celite Number 5^3 added. This was mixed for five minutes with ft stftrilft stirring rod, and filtered through number 1 Whatman filter p»j-*r and thorn possod through tho continuous flow bowl of ft steam-driven Shftrploft centrifuge at 70,000 g, with tho rftto of flow adjusted to approximately 2,000 milliliters por hour. Aftor all tho matorial had passed through tho centrifuge bowl, the system was washed with 500 milli­ liters of cold buffer solutioa. Tho centrifuge was then stopped, 20 milliliters of sterile glass beads added, tha ialet aad outlet openings plugged, and the bowl shaken vigorously t o free the sediment which had accumulated on the sides of tho bowl. The resulting suspension was transferred to large centrifuge tubes and an additional 100 milliliters of buffer placed in the bowl to remove any sediment still adhering to the walls or the glass beads. centrifuge tubes, which were This was added to the material in the spun in the angle head of a refrigerated centrifuge for 20 minutes at 9.000 g. The supernatant fluid was siphoned off and the sediment resuspended in 50 milliliters of phosphate buffer. The tubes were then recentrifuged and the supernatant fluid mixed with that recovered from the previous spin. After thorough mixing, the material was placed in a batch bowl of the Sharpies centrifuge and spun for 10 minutes at 10,000 g. The supernatant fluid frnn this centrifuga­ tion constituted the tumor antigen-L3 which was placed in serum tvbes, sealed, frozen and stored as described for the L2 antigen. g. Infected chicken serum:--The serums of birds having visceral tumors, which had been induced by the inoculation of RPL strain 12 filtrates, were used as antigens. Fifty-four serum tubes containing this type of serum were removed frem storage, thawed under flowing tap water, pooled, and centrifuged at 5,000 g for 20 minutes. The centrifugation removed precipitates wh ic h formed in same of the samples wham they wara tha wad. Tha supernatant fluid was siphoned af f and placed in sarum tubas which wara sealed, frosen ia an alcohol-dry ica bath and starad in tha dry iaa ohast. This infaotad chickan sarum, which tatalad 920 millilitars aftar tha oentrifugatioa, was designated as ICS and was usad as an antigen in various tasts and far tha inooulatian of animals for tha praduction af antisarums. h. Normal chiokan sarum?— The serums of randomly sala ctad chickens can not ba considarad as free af tha agent af visceral lymphematosis . Normal appearing birds can apparently transmit the infection thrergh tha egg to thair offspring, indicating tha presence of tha agent in tha hen produc­ ing thasa eggs (Cattral, 19149) • Since tha occurrence of turners in infaotad birds reaches a peak at around 200 to 250 days of ago (Waters, 19l47)» * greup of birds which had lived considerably longer witheut shewing infection, would ba a more likely source of agent-free material* A group af birds from one af the susceptible lines af chickens (Lina 15) at tha Regional Poultry Research Laboratory had bean reared in oempleto isolation, and at tha age of 2100 days, had shown no evidence af infec­ tion. Thasa wara tha DQ18 group which had furnished the tis sue s far tha norma 1 tissue antigan. Serum had bean secured from these birds at tho timo they wore sacrificed and was usad as a source of normal ohickon serum. This served as a central an tha infaotad chi oken serum, when used as an antigan and in the inoculations far tha production of antisarums. In ardor to differentiate this normal chickan serum from tha serums taken from birds, whi ch were later usad for inoculations, tha symbol NCSA was usad. A group of 11 tumor antigons was prepared apeoifically far the inooulatlflfa ef chi okems in ax attempt te preduce a. material that mi^it stim­ ulate the preductien ef antibedies which weuld be detectable by the indirect camp lamest f ixaties teohaiqua. Thasa antigens wara preparad frem strain 12 filtrata-inducad tumors with tha excaptica ef one which employed strain 1? pectoral muscle tumors and a second prepared from naturally occurring liver tumors. The pectoral tumor had been induced by tha transplantation of viable tuner colls rather than a filtrate of tha agont (Burmester at_ al., 191*6). i. Tumor antigon-PTl*:--The peoteral muscle tumor antigan, designated TA-PTi* was prepared by mincing 15 grams ef pectoral turner as described by Olson (191*0) and suspending the mince in 135 millilitars of 0.85 per­ cent saline. of fino gauze. This was stirred vigorously and filtered through two layers Tha filtrate, totaling 112 millilitars, was transferred to a largo oontrifuge tubo and 2.25 millilitars of a 10 percent solution of formaldohvde added. This was mixed thoroughly and allowed to stand in tha refrigerator for seven days at I** C. On the seventh day the ma­ terial was placed in serum vials in 10 milliliter volumes, sealed, and stored at -35* C. j. Tumor antigen-LU;--The TA-Li* was prepared in the same manna r as the TA-PTl*. A UO-gram sample of liver tumor was minced and suspended in 3^0 milliliters of saline which was filtered through gauze. The filtrate was divided into three portions ef 100 milliliters each and 10 peroent formaldehyde added in amounts sufficient to make concentrations of 0.05 percent, 0.20 percent, and 0.80 percent. These were allowed to stand in the refrigerator for one week, and then transferred to serum tubes and stored. Th* three material* wara identified according to tha ccneentra- tiaa af tha formaldehyde present, as: TA-IJ4A - 0.05 percent formaldehyde TA-U4B - 0.20 peroent fermaldehyde t a -l U c - 0*80 peroent fermaldehyde lc. Turner antigen-L5;— The TA-L5 w»s prepared in th* same manner as described for TA-L3 except that th* precedure was stepped washing with 300 milliliters *f phesphat* buffer. after the Thesediment remain­ ing after the washing was suspended in appreximately 200 milliliters ef th* buffer and divided into three equal parts. Formaldehyde was then added as described for th* TA-UU, and th* resulting antigens designated as TA-L5A, TA-I/5B, and TA-L5C. 1. Tumor antig en-L7;--The next antigen, TA-L7 was prepared from llO grams ef liver turner which was blended in a Waring blender for three minutes with 500 milliliters ef phesphat* buffer, and the* mixed with an a additional 2100 milliliters ef the buffer. This was filtered through three layers of gauze and the filtrate passed through the centinuous flow bowl ef a Sharpies centrifuge at ^>0,000 g with a flow rate ef 2,000 mil­ liliters per hour. The sediment was discarded and the supernatant fluid passed thr ouf^i the centrifi^e again, this time at 60,000 g with th* same flow rate. Th* supernatant fluid from this con trifug at i in was discarded and th# sediment suspended in 100 milliliters of phesphat* buffer. A clarifying spin of 20 minutes at 2,000 g was carried eut in th* refri­ gerated centrif qg e. The supernatant fluid was then decanted off and divided into twe parts. The first portion, designated as TA-L7A was placed in serum tub*6, sealed and stored. Fermaldehyde solution (10 peroent) was addad to tha sac aid part 1b ait amount sufficient ta make a 0.05 par cent cane ant rati on and was designated as TA-L^B. m. Timor awt igon-L91--This antigan was prepared frem a partian af tha same peel af tumor used far TA-L7* A 100-gram sample was mixed with UOO millilitars af 0.85 percent saline and blended far five minutes in a Waring blender. This was transferred ta large centrifbge tubes, and spun in the refrigerated centrifuge at Li.,000 g far 20 minutes. The supernatant fluid was then decanted aff and allawed te stand in th a rafrigeratar overnight at U® C. It was then re centrifuged at 14,000 g far 20 minutes and the supernatant fluid, which constituted the TA-D3, decanted eff and stared in serum vials in the mechanical freezer at -35* n. Turner antigen-Lyl:— The last of these antigens was prepared frem liver turners wh ich had been recavered frem birds having naturally occur­ ring visceral lymphemates is . Filtrates, prepared fram these tumors, had been used far the inoculation af chirks, producing tuner in a high per­ centage ef cases indicating the presence ef the agent (Burnester, B. R., oral communication). One hundred grams ef the tuner was blended with I4OO milliliters ef O .85 percent saline far five minutes, and the same procedure fallowed as was described for TA-L9* B. Antibody Production Tha aat isaruns usad lit tha tasts war* practiced in rabbits, guinea pigs, ohiokaas and ducks by tha inaculatian ef tha various antigens. Tha rauta, volume and number af iaaculatians varied far tha diffaraat anti­ gens ; however, the precedure usad far the blaadiag af aach kiad af animal remained cocstant. Alsa, tho freezing and storage pracadure was tha sans , regardless af tha ineculum or animal iavalvad. a. Blaadiag Precedur a Bach animal was bled priar t a inaculatian to previde a central material by which the presence af aon-specific reacting materials could ba detected. One ar mare bleedings wara made fallawing tha iaaculatians and tha serums usad as a source af antibodies. tha inoculations wara classified as normal The serums abtaiaad before serums. By combining tha symbols N far normal, R, OP, D, and C far rabbit, guinea pig, duck, and chicken, respectively, and S far serun, each of tha serums could ba easily identified; far example, NRS represented normal rabbit serisn. All bloedimgs were made aseptically by cardiac puncture. Far tha rabbits, a J>0 milliliter syringe fitted with a 1 -inch 18 gauge naadla was usad and approximataly 30 millilitars af bleed removed at esch bleeding. With tha guinea pigs, a 10 milliliter syringe and a 1-^-inch 20 gauge naadla wara usad, while with tha chickens and ducks, a 20 milliliter syringe fitted with a 1^-inch 20 gauge needle was employed. Approximately 8 ta 10 millilitars af blood was taken fram tha guinea pigs and 15 ta 20 millilitars fram tha chickens and ducks. At tha final bleedings, bleed as passible was seoured fram eeah animal. as mush The bleed, after having been tra««ftrreri fr«m the syringe to the test tube, was allowed te clet at rear tempe rature, and them thoreughly chilled by plaoing the tubes im a refrigerater at h° C fer twe heurs. They were them cemtrifuged at 1,000 g fer 15 minutes te cempress the slots and the serums transferred te serum tubes which were immediately sealed with a flame, amd as seen as sufficiemtly ceel, placed in am ilcehel-drv ice bath at appreximately -ApO* C. k. mechanical freerer at -35* Ter sterage. They were that placed im The precedure eutlined here its used fer beth the pre- amd pest-imeculat ien bleedimgs . b . Iweculat jam Precedure Ineculatien techniques varied fer the different animals. lhe met had ised fer each animal will be described and the volume and number ef .necul at ions given later according te the individual antiaerums. 1 . Rabbits;--The inoculations were made intraveneuslv into the marginal ar vein. The ears were washed thoreughly with seap and warm water and he area ever the marginal vein shaved, rinsed with water and swabbed with ettsm which hadj&een dipped into 95 percent alcohol. A one milliliter uberculim syringe amd a 1-inch 2h gatge needle vr jre used fer the imecuatiens. After all the material had been injected, the needle was care- ully removed, slight pressure applied te the area, the ear rinsed with eld water, dried with a soft cloth, amd the rabbit returned te its cage. 2. Guinea pigs;--The intraperiteneal route was used fer all ef the neculations in guinea pigs. The animal was placed en its back, wi dii ts legs extended, and the abdominal wall cleansed with 95 percent alceel. The inoculations were made through the posterior portion ef the bdeminal wall, with the needle directed anteriorly. A one milliliter 63 iberculin syringe amd a 1-inch 2l| gatg e maadla wara usad far thasa leculatiens. Na special care was aaaded in remeving tha maadla ar hand- Lmg tha animal befara returning it ta Its cage. 3 . Chiokans amd ducks:--The same precedixre was used far tha inaculatiam n bath tha chickams amd duaks. The bird was placed an its side an a ible and the area ever tha brachial vaim picked clean af feathers. 'ter cleansing: the area with 95 percent aloehel, inaculatiam was made rte the vein using a ana milliliter tuberculin syringe and a 1-inch 2U tuge needle. Pressure was exerted an the area fellewimg the inecula- Len te central the hemerrhage. c. Inecula t iar s and Bleedings Tha number and velume af injectians will be described aocarding ta te antigen invalved and animal being inecula tad. 1 . Influenza antigen:— Three rabbits were given twe ineoulatiens af le milliliter each at an interval af seven days. Feurteen days later, third ineculatien af 0.5 milliliters was given and bleedings started Lve days fellewing this third ineculatien. Three bleedings wens made : seven-day intervals and the animals bled ta death at the third bleeding. Fifteen guinea pigs were given an initial ineculatien af 0.5 milliLters and f ell awed at five-day intervals by twa ineculatiens ef 0.25 Llliliters each. Ten days after the last ineculatien, bleedings were :arted and marie every seven days with the surviving animals being bled 1 death at tha faurth bleeding. Twa af the animals died fellewing the *cend bleeding and feu r fellewing the third bleeding, as a result af ccessive damage ta the heart and hemerrhage inta tha pericardial s a e . Six chickens were given twe ineculatiens ef erne milliliter each at a interval af seven days. Bleedings were started 10 days leter amd emtimued every seven days umti1 three bleedings had been made. One ef he birds died fellewing the ascend bleeding and shewed excessive cardiac amage. Twe ef the birds were bled te death at the third bleeding. By rrer, three ef the birds were bled te death at the first bleeding. Te facilitate the identif icati en ef these utissruas, the fellewing ymbels were assigned: FRS - rabbit anti-inf1 uenza serum FOPS - guinea pig anti-inf1uenza serum PCS - chicken anti-inf1uenza serum The number ef ineculatiens and bleedings and the ameunts ef serum btained are shewn in Table Via. 2. Nermal tissue ant ig e n :--Twent y feur guinea pigs were given feur aeculatiens ef 0.25 milliliters each at weekly/ intervals and 10 days • H e w i n g the last ineculatien, they were bled. igs died fellewing the first bleeding. Three ef the guinea The 21 survivers were bled te •ath seven days later. Feur chickens and twe ducks were ineculated three times at weekly ntervals with 1.0 milliliter ef the antigen. Ten days later, bleedings ere started and repeated every seven days, with all the birds being led te death at the third bleeding. Symbels were assigned te these materials, as described previeusly, he enly new item being the nermal tissue antigen, which was designated T. These antiserums ware thus designated as: TABLE Via Lniaal a o • IHOCULATIONS WITH INFLUENZA VIRUS No. tabbit 35 2 1 2 1 2 1 1 2 2 2 2 2 2 2 37 50 luinea Pig Bhicken 15 plga K1321G K1316L K1319E E1460C4 K1408G2 K7i7v2 Inoculation_____ al. Total al. 1.0 0.5 1.0 0.5 1.0 0.5 0.5 0.25 1.0 1.0 1.0 1.0 1.0 1.0 Bleeding Bafora Inoc. Af tar Inoc. No. ml? No. al? 2.5 1 15 3 173 2.5 1 15 3 140 2.5 1 15 3 175 1 1 1 1 56 15 15 15 4 2 3 3 1 1 1 247 65 85 95 50 50 50 . «*• 1.0 2.0 2.0 2.0 2.0 — 2.0 _ 2.0 - - • al. of serua recovered ** total al. for aach guinea pig TABLE VIb INOCULATIONS WITH NORMAL TISSUE ANTIGEN ▲nia&l No. Guinea 24 pigs Pig Chicken K13}0G2 El31012 XI310B2 K1413H2 Duck 5 6 No. 3 3 3 3 3 3 Inoculation al. Total al. 0.25 1.0 1.0 1.0 1.0 1.0 1.0 1.0 3.0 3.0 3.0 3.0 3.0 3.0 • al. of aerun racorered ** total al. for aach guinaa pig Bleeding Before Inoc. After Inoc. No. al? No. al? 1 300 126 2 2 2 2 2 1 1 40 42 37 40 10 15 3 3 3 3 3 3 70 85 70 75 163 175 NTGPS - Guinea pip; anti nermal tissue strum NTCS - Chicken antinermal tissue serum NTDS - Duck antinermal tissue serum tabulatien ef these inecula ti tms and bleedings is shewn in Table V I b . 3. Turner antigens?--As was described previeusly, eight turner antigens arm prepared frem liver tuner materials and ene frem pecteral tuner Ls sue. The TA-L2 was used fer the ineculatien ef guinea pigs, and the 1-L1 fer t h e ineculatien ef guinea pigs, ducks and chickens. All ef le ether tuner antigens were used fer the ineculatien ef chickens, with le exceptien ef the TA-L3 which was empleyed enlv as a test antigen. 1 Table VTI the antigens and animals invelved, the number and velume ef teculatiens, the number ef bleedings, and v e l u m ef serum recevered ifere and after ineculatien are tabulated. The antiserums frem these animals had t e be identified, especially te chicken antiserums, and the fellewing symbels were enpleved. T was ted te designate turner antigen as the ineculated material, which was ►H e w e d by the symbel fer the respective animal, such as OP far guinea Lg, D fer duck and C fer chicken, and that was fellewed by S, indicating itiseruir. The symbel fer the respective antigen as LI, L2, PTl_i, et ►tera, was then added. Fer example, the guinea pig antitumer-Ll serum is designated TGPS-L1. The interval between ineculatiens and between the last ineculatien id the first bleeding varied. The guinea pigs were all ineculated at iven-dty intervals and the first bleeding made 10 days after the lest leoulatien. The bleedings were then made at seven-day intervals with LI ef the guinea pigs being bled te death at the secend bleeding. This TABLE VII IH0CULATI0N8 WITH TUMOR ANTIGHJS Inocul a t i o n _________ Bleeding_________ Inoculum Animal Ho* No* ml* Total ml.Before Inoc* After Inoc* _________________ Ho* al* Ho* *4*. TA-L1 Guinea Pig 24 pi 9 '* 0.25 1^0' ~~1 178 2 365 Chicken K1412J2 3 1*0 3.0 3 55 3 105 KI4 OIR3 3 1.0 3*0 3 60 3 110 K1301V 3 1.0 3.0 3 60 3 105 K1310X 3 1.0 3.0 3 60 3 115 Duck 1 3 1.0 3.0 1 10 3 150 2 3 1.0 3.0 1 15 3 160 3 3 1.0 3.0 1 13 3 165 4 3 1.0 3.0 1 15 1 28 !W2_fluiai»'H| 24 piflB 4 0.25 1.0 1 3.47 2 300* ~TA-TT4 Chicken X1466C 9 2.0 18.0 1 25 2 36 X1513I 9 2.0 18.0 1 20 2 40 __ _ K2S»95 9 2.0 18.0 1 20 2 40 “TJUlAA“Chicken * XI5030 3 2.0 6.0 1 25 1 20 " K2P100 9 2.0 18.0 1 25 2 40 _____________________ K5P42 3 2.0 6.0 1 20 1 20 TA-L4B Chicken K2P93 9 2.0 18.0 1 20 2 40 __________ K5P46 9 2.0 18.0 1 20 2 40 TA-L4C Chicken K2P89 9 2.0 18.6 1 20 * 2 40 “ K2P97 9 2.0 18.0 1 15 2 40 __ X5P49 9 2.0 18.0 1 20 2 40 TA-L5A Chicken K2P99 11 2.0 22.0 1 22 2 50 K2P96 11 2.0 22.0 1 25 2 50 E1455Q, 11 2.0 22.0 1 25 2 50 TA-LSB Chicken 1^52 11 2.0 22.0 1 25 2 ' 50 K1473T 11 ‘ 2.0 22.0 1 25 2 50 K1473Z 11 2.0 22.0 1 25 2 50 TA-L5C Chicken K2P90 11 2.0 22.0 1 25 2 50 K1414% 11 2.0 22.0 1 25 2 50 K2P72 11 2.0 22.0 1 25 2 _ 5° “TEA-L7A Chicken K2&99 2 2.0 ' 1 4.0 8.0 1 50 K1455Qg 2 2.0 1 4.0 8.0 1 50 K2P98 2 2.0 1 4.0 8.0 1 50 TA-L7S Chicken X2P42 2 2.0 1 4.0 8.0 1 17 E1473T 2 2.0 1 4.0 8.0 1 24 ri414D2 2 2.0 1 4.0 8.0 1 19 TABLS Inoculum Animal TA-L9 Chicken TA-Lyl Chicken Ho. Ho. K1473L2 11 1 K147303 11 1 K1516L2 11 1 K620Q 10 1 K669L2 10 1 11 K25&K 1 K262B 11 1 K669X2 11 . 1 E1466M 11 1 K1511H2 11 1 K350S 10 1 K620B 10 1 K669C3 10 1 VII (Cont.) IhOCUlatiPP____________ Bleeding _ _ ml. Total ml.Before Inoc. After Inoc. Ho. ml. Ho. ml. 0.5 60 55 2 75 2 2.0 0.5 60 55 7.5 2 2 2.0 0.5 60 30 2 1 7.5 2.0 0.5 3 50 25 7.0 2.0 1 0.5 60 3 1 25 7*0 2.0 ■ 0.5 60 2 70 2 2.0 7.5 0.5 60 60 2 2 2.0 7.5 0.5 60 45 1 7.5 1 2.0 0.5 60 1 55 2 7.5 2.0 0.5 60 2 1 35 7.5 2.0 0.5 60 25 3 7.0 1 2.0 0.5 60 3 25 7.0 1 2.0 0.5 50 3 25 7.0 1 2.0 69 interval ef seven days between bleedings was usad reutinely far all af tha ineculated animals, tha arlv variatieaa being i* the numbar af bleed­ ings made. Tha chickens and disks receiving tha TA-L1 ware ineculated at weakly intervals and tha bleedings started seven days after tha last ineculatien. Tha chickens receiving; tha TA-PTl*, TA-LUA, B, and C, and TA-L5A, B, and C, hawavar, ware ineculated twice weakly and the first bleedings made 10 days after tha last ineculatien. Twa af tha birds receiving the TA-LLA received anly three ineculatiens instead af nine, as was the case wilh the ether birds in this greup. The chickens receiving TA-L9, TA-Ly 1 were ineculated twioe weekly with 0.5 milliliters fer a tetal ef 11 ineculatiens, and than given tha final ineculatien ef 2.0 milliliters after a^ interval ef 10 days. The TA-L7A and B received twe inecula­ tiens of 2.0 milliliters an d a third ineculatien ef JLi.O milliliters at intervals ef seven days. Infected chieken serum:--The serums ef infected birds weuld net centain tha agent in as cencentrated amaunts as the centrifuged materials. Therefere , a larger numbar af ineculatiens was needed ta supulv a oemparable ameunt af agent far antibady stimulat ien. Six guinea pigs and twa ducks ware ineculated 15 sad li± times, respectively, at weekly intervals. The first twe ineculatiens centain ed feur milliliters each; the volume was then reduced te ana milliliter fa- the remaining inecu­ latiens. feven days after the last ineculatiens, they were bled. The ducks were bled te death at the first bleeding, while the guinea pigs were bled te death at the secend bleeding. nated; These anti serums were desig­ 70 ICSGPS - Guinee pig t n t i i t m l chicken serum ««ru» ICSDS - Duok u t i M n m l chicken serum strux The tebuletiens of the ineculetiens end bleedings with the infected end norm el chicken serums ere shewn in Teble VIII. TABLE Till t Inoculum infected Chicken Serum (IC8) Animal No. Guinea Pig 6 pigs 74 Duck 75 Normal Chicken Serum (NCS) Guinea Pig 6 pigs Duck 71 73 • ml. of serum recovered ** total ml. for each guinea pig Inoculation ___ Bleeding_______ No. ml. Total ml. Before Inoc. After Ino No. ml? No. ml 13 2 12 2 12 4.0 1.0 4.0 1.0 4.0 1.0 2 13 2 12 2 12 4.0 1.0 4.0 1.0 4.0 1.0 2 '•*10 INOCULATIONS WITH CHICKEN SERUMS 21.0** 1 48 2 105 20.0 1 30 1 80 20.0 1 30 1 80 21.0** 1 45 2 100 20.0 1 30 1 80 20.0 1 30 1 80 VI. STANDAPDIZATICN OP TECHNIQUE A reaction involving a known virus and specific aatiserum was needed far tha standardization af tha technique with regard ta tha ardsr af addition af tha reagents amd tha temperature amd duratiaa af tha iacubatian pariads. Tha influenza virus was chasan ta sarve as t he antigen , and anti serums were preduced ia rabbits and chickens. Tha rabbit anti- influenza serum was titrated first and than tha antigen standardized at a c eneent re tian* wh era it was tha limiting factar with ragard te tha amount af c amp lament fixed. This was n a a a n a r - ta insure that all af the activity af the chicken anti-influenza serum ceuld be measured whan it was all awed te react with the antigen in an indirect test. The direct reaction invelving the influenza antigen end rabbit antiinf luenza serum was standardized and an indirect test en tha chicken ant i-inf 1tianza serum attempted. If influenza sntibedies were present in the chicken anti -influenza serum, they would carr.bine with the antigen and reduce tha amaunt af complement fixed .' . ’hen the rabbit anti -influenza « * r u - was added. However, a larger amaunt ef complement was fixed when tha chicken anti-inf1 uenza serum was present than eccurred in the direct reactien. Nermal chicken serum, frem the same birds furnishing the antd- influenza serum, was than tested and similar results ebtained, with even greater ameunts af complement fixed than was obtained with the chicken anti-influenza serum. Normal serums frem horses, oews and -ducks were tested but failed te affect the amaunt ef complement fixed by the direct reaction af influenza virus and rabbit anti-influenza serum. Central tests in which rabbit anti-influenza serum was tested against chicken 73 ant i-influenza serum and narmal chlclcan serum showed there was a reaction occurring in both instances which produced a complex capable of fixing approximately 7*5 units ef complement. This fixation was considered due te an antigenic material in the chicken serums and specific antibodies in the rabbit anti-influenza serum because, as has been mentioned, anti­ bodies produced in the chicken are not capable of producing a complementfixing complex with specific antigen. 7/hen the indirect titers of the chicken anti-influenza serum and normal chicken serum were calculated, a negative rather than positive value was obtained. The magnitude of this negative titer increased with increased dilution of the chicken anti-influenza serum but decreased with the higher dilution of the normal chicken serum. was attributed to the following facts. 7 hi r difference Three reactions occurred in the tests where the chicken anti-influenza serum was used. The first was the reaction of the chicken anti-influenza serum with the influenza anti­ gen, which would reduce the amount of antigen available to reset with specific antibodies in the rabbit anti-influenza serum and decreased the final amount of fixation. The second reaction was between the rabbit ant i-influenza serum and th<* antigen which caused fixation in proportion te the emount ef antigen available. The third reaction was the ore between the unknown component in the chicken anti-inrluenza serum and an antibody in the rabbit anti -infl ucnia serum. This third reaction was apparently capable of fixing complement and would add to the final amount of fixation measured. '.Then normal chicken serun was pr-sent, enlv two of the reactions occurred; the one between the rabbit anti-influenza serum and the influenza antigen, and the one between th- unidentified an tib ed ies i* present the in th e rab b it ch ick en an ti-i» flu eaza serum s • Th e t h i r d reactio n , was u s e d , acted in th e o p p o s i t e way fix a tio n by its actio n on th e in flu en za effect present B oth d i l u t i o n was o f th e A d eterm in atio n c o n stitu en t o f the were sm bryos, v iru s. ch ick en »cc*nf any th e resp o n sib le stitu e n t for the in th e of icrun. b is a lla n to ic day of in same a n t i g e n the is and t h e present a norm al of o th e r th an th e se in c u b atio n . in the com ponent a p p a r e n tly an tig en flu id as th e chicken em bryos at When c o n c e n t r a t e d by w ould n e c e s s a r i l y co n tain ed an tib o d ies w h ich th e m aterial re a c te d w ith th e serum s. th at th is was an tig en s in to a h etero p h ile-tv p e are cap ab le rab b its (l9U q ) t h a t (1923). a n ti­ Th e a n t i b o d i e s th is flu id a llan to ic amount o f lessen ed had b e e n grow n i n using early em bryo a s known t o aqueous early as be present of et a l., em bryonic occur r e a c tio n was in fo u rth in th e 19ii5)» stag es the suspensions, th e con­ stim u la tin g a n tib o d y (Z in sser, o n e o r m ore a n t i g e n s w h ic h K richevsky >hile a n t i g e n an tig en of the is e n shown b y S o h e o h tm a n was serum ev id en t. w ith, in flu en za com plem ent. th e ch ick en sub s t a n c e , p ro d u ctio n p r o d u c tio n when i n o c u l a t e d As was a t t e m p t e d . the the decreased th e reactio n reacted fix ed cem peaeat a n t i - i n f 1 uenza an tig en . Snd a p p a r e n t l y Th e h e t e r o p h i l s shicken c o n ta in it p artic le s, ch ick en The p o s s i b i l i t y sid ered . in fo u rteen th v iru s th at sp ecific reactio n s ohickan of th e serum s, from t h e s e n trifu g a tio n , a p o rtio n jo t, n atu re The i n f l u e n z a ap p ro x im ately t h e ir in th is serum w hich and h a r v e s t e d th e in flu en za produced by a s u b s ta n c e in flu en z a th ese r e a c t i o n s b e c a m e more of the ra b b it ant i-in flu en za of o n l y when in c re ase d , o t h e r tw o le ru m an 1 t h e organs has of the ad u lt d etected d ay of It ch icken h etero - in cu b atio n , and b l o o d of c h i c k e n s , w h ich w ould a c c o u n t f o r an tiseru m , to produced tr' th e re a c t w ith few a n i m a l s et a l ,, th eir norm al cap ab le 19U5)• organs or of in do n o t If the th e use because tiv e tite rs in d irect the u n its to for tite r serum in th e o b tain ed . in the The f o r the have th is seru n to of such h a J b e » n showr. t o eccur a t th e serum s, the w ith the p o sitiv e ant i - i nf luenza of v alu es ^.p u n its of th at the in flu en za a n tig en - organs. ch ick en a n t i - if a reactio n th e rab b it was n o t e d , guinea p ig an tig en and react g iv e serum . ran g in g 11 in d i­ w h ic h w ould v alu es com plem ent. c o m b in in g and in te r­ nega­ The aerum , when t e s t e d up to the of should not serum and r a b b i t fix r a b b i t a n t is erum w ere and th u s use w ith elim in ate c ase w ith a reactio n gave n e g a tiv e (Z in sser in t h e i r • ^ :n st determ ine as h ad been th e should an tig en the in e ith e r p ro d u ctio n re actio n ant i- in f lu e n z a serum , re a o tio r., tim e d ire c t th e on c h i c k e n a n t i - i n f l u e n z a H ow ever, seru- in the chicken chicken chicken same chicken te sts t, d irect for one of a h eterep h ile pigs had not form ed a n tib o d ie s in d irec t u n its. correct th a t serum was t e s t e d com bined, ant i- in f lu e n z a -2 .7 is when i n o c u l a t e d was due to pigs p ig s No e v i d e n c e guinea com ponent rab b it g uinea guinea norm al serum . th a t th e the of tests an tiseru r. fo r th e s e r u m a nd w ith th e a s s u m p tio n w ere in d irec t o c c u r r e d when t h e y w e re catin g produce a n tib o d ie s p ig a n ti- in f lu e n z a an ti-in flu en za The r a b b i t an tig en or b lo o d p ro d u ci^ serum s. was a l l o w e d h etero p h ile d ifficu lty , in flu en za imrrun* c h i c k e n em bryo m a t e r i a l , A n im als w hich h a v e t h e reactio n , S uinea of r e a c t i o n e n c o u n t e r e d w he n an tib o d ies co m p lem en t-fix in g th is in o cu latio n of h etero p h ile su b stan ce an tib o d y ty p e th ese h etero p h ile a n tig e n . ferin g the w ith from -0 .2 an ti-in flu en za u n i t s w ere a n t i - i n f luenza This fix atio n w ould sp e c ific an tib o d ies f i x i n g c om.plement . Th e 76 ’i x a t i o n m e a s u r e d w o u l d t h u s ;he f i x a t i o n brouf^it irrt i - i n f l u e n z a nent fix ed uneunt ef bodies frorr th e from t h e and t h e resu ltan t correct in d irec t sequent tests. Fallowing the tests on the individual turner antiserums, tha supply af turner antigen-L2 was practical ly exhausted, and the turner antigen-L3 was breught into use. These twa antigens (TA-L2 and TA-L3) were titrated against the turner antiserums (TAGPS-L1 and TAGPS-L2) ta determine thair relative potency. The amount ef fixation occurring in the tests employ­ ing the IA-L3 ats 20 and 23 units when tested against the LI and L2 antitumer serur.s, respectively, which was slightly less than when the TA-L2 was used, which fixed 26 and 26 units. However, the 21 units was suffi­ cient te allow the use ef the TA-L3 ir. the direct tumor tes'c. The normal tissue antigen (NTA) and guinea pig antir.enr.al tissue serum (K70PS) served as central materials with which the reaction ef ne ran 1 tissue components ef the t m e r materials ceulc be measured. The specific nermal tissue reactien was tested first b y preparing twe-feld serial dilutions ef antigen ranging frem 1:2 te 1:32 and dilutions ef the guinea pig airtinennal tissue serum frem 1:2 te l;l6 . Three sets ef determinatiens were made in which each dilutien ef the ant igea was tested against each dilutien ef the antiserum. The results in the duplicate tests were censtant and the values fer the varieus reactions averaged. A 1:2 dilutien ef the antigen and 1 sL|. dilutien ef the antiserum fixed 12.5 units and was used as a base line fer the determinatien ef the relative ameunts ef nermal tissue reagents in the turner materials. The normal tissue antibecty centent ef the guinea pig antitumer serums was then determined. The ameunt of fixatien by the nermal tissue antigen- guinee pig antitumer serum reaction, when cejr.])ared te the ameunt ebtained with the nermal tissue reagent reactien, indieated the relative cencentratien ef nermal tissue antibedies in the guinea pig antitumer serums, with respeet te their concentration in the guinea pig antinermal tissue serum. The number ef units fixed by the twe guinea pig antitumer serums was practically identical, being 12.6 and 12.9 units fer the LI and L2 antiserums, respectively. When cempared te the 12.5 units ebtained with the guinea pig antinermal tissue serum, tissue it was evident that the nermal antibedy centent in all three ef the antiserums was appreximately the same. The next stop was the testing ef the three antiserums (NT1PS, TA'iPS-Ll and TAG-PS-L2) against turner antigen (TA-L3). The cencentratien ef nermal tissue antibedies in these antiserums had ba*n shewn te be the same. When allowed te react with turner antigen, the antitumer serums should fix mere caipleneat if anti.tumer cell component antibedies er antituner agent antibedies w«r* present. Tha l a m l tissue antibedy oaataat af tha aatis arums had baan osmparsd w h e n all af tham wars at a dilutian af 1:1).; the refare, tha s u m dilutien was U6ed aad tha anti sarums tastad against varying canoantratians af tha tumar antigen-L3* As shawn in Tabla XVII, the f ixatien by tha anti tumar sarums was mueh graatar than with tha antinarmal tissue sarum, baing mara than twica as graat whan tha antigan was at the 1:8 dilutien. ta the reactien ef antibedies This diffaranea was attributad in the guinea pig antitumer s e r u m which reacted with a specific m t e r i a l in the turner antigen which was net pre­ sent in tha nermal tissue antigen. These reactians ware due either ta tumar call constituents end specific antibedies, ar tumar agent and its specific antibedy. reactians. Ne methed was knewn t differentiate between these There was tha pessibility, haw aver, that if chicken anti- sarums eeuld be ebtained which centained anlv agent antibedies, they ceuld be used ta differentiate tha partien af the reactien due ta tha agent, but this wauld be dependant upan tha demenstratian ef antibedies in chicken sarum, which was ana af tha main ebjectives af tha tests. Tha guinea pig antiserums had been used at a dilutien af 1:1) far tha eampari sen af their nermal tissue antibedy centent. Other dilutiens, - namely 1:2 and 1:3, we're alse prepared and all three dilutiens ef tha guinea pig antitumer and antinermal tissue serums tasted against a 1:8 dilutien af the tumar antigen-L3» The 1:1) dilutiens af the antiserums gave titers which eempared faverably with cemparable reactians eenducted previeusly. In tha reactians with tha 1:8 dilutiens af the antiserums, there was the expected decrease in fixatian with decreased antiserum cenaant rat ien. Hew ever, when tha 1:2 dilutien af antis arum was involved. V TABLB XVII TITRATION 07 TUMOR ANTIGKN-L3 AGAINST GUINEA PIG ANTITUMOR-L1, ANTITUM0R~L2,AND ANTINORMAL TISSUS SERUMS Antieerun NTGP8 (1:4) TOPS-LI (1:4) TQPS-L2 (1:4) Teat No, 1:4 TA -L3 1:8 Units Fixed 10.8 1:16 (1 ) 15.5 (2) 15.2 11.3 8.0 (3) 15.1 1Q.9 8.4 Are. 15.3 11.0 8.2 (1 ) (2 ) >26.0 24.1 13.1 >26.0 23.9 13.6 (3) >26.0 23.4 13.5 Are. >26.0 23.8 13.4 (1 ) (2) >26.0 24.2 13.0 >26.0 24.1 12.9 (3) >26.0 24.6 13.3 Are. >26.0 24.3 13.1 8.1 very little increase in the ameunt ef fixation eccurred aver wha t had been ebtained with tha 1 :U dilutian. Thasa results shewed that whan tha antigen was at tha 1:8 dilutian and the antiserum dilutian was at 1:U, bath reagents ware at appreximat ely apt in urn oencentratien and tha anti­ gen was a limitl ng facter. Tha ref era, if chicken antitumer serums ware tasted in the indirect test, using these eancentretiens in the direet reactien, any affect they might have an tha antigen weuld be detectable. The standard direct turner test thus inve lved TA-L3 at a 1 :8 dilutien and TA3PS-L1 ar TA0PS-L2 at a I jJLj. dilutian. When nermal tissue materials were used in direct tests (far the indirect titratien af chicken anti­ serum), the antigen (NTA)" was diluted 1:2 and tha anti serum (NTGPS) diluted 1:L}. «. Indirect cemplement fixati an tests an chicken antiserums far the detaotlan af turns r agent a ntlbad ies;--The next step was the testing af chicken antiserums indirectly, using tha standardized tumar direct reaetian. Tha chieken antiserums preduoed by tha ineculatien af tha LI tumar antigen ware prepared in dilutiens af 1:2 and 1:2* and tasted with direct tumar reactians empleying tumar antigen-Ll and guinea pig antitumer serums LI and L2. Nermal sarums, which had been ebtained frem the res­ pective birds prler ta their ineculatien, were alsa tasted. The results af thasa tests are given in Table XVIII and indicate that three af tha faur chieken antitumer serums apparently pessessed antibedies in law canc entratian, the highest titar being 2.1+ units. haw aver , were witheut affect. Tha nermal serums, The small values af 0.1, 0.2 and 0*3 units feund in many af tha central tests ware f aund ta be due ta irregularities imrelving the antic ample me ntary activities af tha individual reagents. TABU m i l INDIRECT TITXR Of CHIC D S ASTITUMOR AITIGIH-Ll SERUMS WHIN TESTED WITH TA-L3 (ANTIGEN) AID TGP8-L1 AID 12 (ANTISERUMS) II THE DIRECT R1ACTIOI Direct Reaction TA - L3 ♦ TOPS -LI TA - L3 + TOPS -12 K1412J2 (1) 0.2 Indirect Titer of Chieken Serum TCS -11* ICS* Are. (3) (3) (2) (2) (1) 1.5 1.3 0.3 0.2 0.2 1.2 I1401R3 0.1 0.3 0.1 0.2 0.1 0.4 0.3 0.3 K1301W 0,0 0.1 0.1 0.1 2.2 2.4 2.5 2.4 I1310X 0.1 0.2 0.0 0.1 1.8 1.7 1.7 1.7 K1412J2 0.0 0.1 0.1 0.1 1.3 1.5 1.6 1.5 K1401R3 0.1 0.2 0.2 0.2 0.3 0.4 0.3 0.3 I1301W 0.2 0.3 0.3 0.3 1.9 1.9 2.0 1.9 KI310X 0.1 0.1 0.0 0.1 1.7 1.9 1.8 1.8 Chicken No, •Chicken serum need at dilution 1:2 Are i;3 The direet reactien was standard, and since the titers ef the chicken antiserums were the enly values ef impertamce in these tests, they were the emly enes used in the evaluatian ef the indirect test results. It will be ebserved in Table XVIII that the type ef guinea pig antiserum empleyed had ne effeet en the ehickam anti serum titer. This was expeeted since the aetual react ien being measured invelved the chicken astiserum and the antigen, while the guinea pig antiserum was, ity , a part ef the indicater system. in real-, The nature ef the reactiam by the chieken antiserxnns, hew ever, was net knewn. The turner antigen centained agent, nermal tissue eell eempenents and turner cell cempenents. Any ef these might have stimulated antibedy fermati en in the chicken and been respensible fer the indirect titers ebserved. If nermal tissue anti­ bedies were present and respensible fer the titers ebtained, the substitutien ef nermal tissue antigen (NTA) fer the turner antigen (TA-L3) the direct test sheu Id give pesitive titers fer the chicken antitumer serum ef appreximately the same magnitude as was ebtained with the turner antigen. This was dene, and as shewn in Table XIX, titers almest iden­ tical te these feund with the tumar antigen were abtained. The re fere, the antibedies in the chicken antitumer serums apparently had been predue ed by n e m a l tissue cempenemts ef the turner antigen, and antiserums frem chiokens which had beat ineculated with nermal tissue antigen sheu Id give similar reactians. This was feund te be the ease when chicken anti­ nermal tissue serums were tested using the same direct reactians as empleyed in the testing ef the chicken anti turner serums. these tests are shewn in Taible X X . The results ef Slightly higher chicken antinermal tissue serum titers were ebtained when nermal tissue antigen was cam bin ed TABU XIX INEIB1CT TITXB Of CHIC U N AXTITUMOR ANTIOXH-L1 8XRUMS WHXR TXSTXD WITH ISA (ANTICM) AND TOP9-L1 AND L2 (ANTISXRUMS) IN TH1 DIRECT RXACTIOV Direct Reaction NTA * TOPS -LI NTA + TOPS -12 K1412J2 (1) 0.0 Indirect Titer of Chicken Sens ICS* TCS -LI* (2) (3) Are. (1) (2) (3) 0.1 1.4 0.1 1.5 0.1 1.4 KI4 0 IR3 0.1 0.2 0.1 0.1 0.1 0.3 0.0 0.2 I1301W 0.1 0.0 0.0 0.0 1.7 1.9 1.8 1.8 K1310X 0.3 0.2 0.3 0.3 1.7 1.6 1.6 1.6 n412J2 0.1 0.2 0.2 0.2 1.3 1.4 1.4 1.4 H401&3 0.0 0.1 0.0 0.0 0.1 0.2 0.2 0.2 X1301W 0.3 0.1 0.2 0.2 2.4 2.1 2.0 2.2 X1310X 0.1 0.2 0.1 0.1 1.9 1.8 1.8 1.8 Chicken No, Are, 1.4 •Chicken eeruae used at dilution 1:2 107 TABU XX INDIRECT TITER OF CHICKEN ANTINOBMAL TISSUE SERUMS WHEN TESTED WITH NOBMAL TISSUE AND TUMOR ANTIGENS AND GUINEA PIG ANTINOBMAL TISSUE AND ANTITUMOR SEBDMS IN THE DIRECT REACTION Direct Reaction Chicken No. Indirect Titer of NTC8* (1) ' (2) (3f ~ Are. 1.8 2.8 1.2 1.6 2.7 0.4 1.3 Unite K1310G2 1131012 K1310D2 K1413% 1.4 2.4 0.5 1.3 1.7 2.8 0.3 1.5 X1310G2 I1310D2 K1413% 2.4 3.0 0.7 2.3 2.4 3.2 1.0 1.9 2.2 2.9 1.0 2.0 2.3 3.1 0.9 2.1 TA - L3 4 NTGPS K131002 I1310X2 K1310D2 11413% 1.6 1.9 0.0 1.7 1.3 2.1 0.3 1.7 1.3 1.8 0.0 1.8 1.4 1.9 0.1 1.7 NTA ♦ NTGPS K1310G2 11310% I1310D, 11413% 2.6 3.1 0.2 2.2 2.8 3.6 0.4 2.0 2.6 3.2 0.4 2.6 2.7 3.3 0.3 2.3 TA -13 + TOPS -LI NTA * TOPS -LI ki310X3 •NTCS used at dilution 1:2 0.5 with wither guinea pig antineranl tisaue er antitumer an tig ea-LI serums in the direet reactien, hut the differences were net sufficient te incH. cate any specificity ef the chioken antineimal tissue serums. The supply ef guinea pig antitumer serums was quite limited and sinee ne difference had been neted between them in any ef th e reactians, ealy ene ef them - the guinea pig antitumer antigen-Ll serum - was used in these tests. It was evident that the antibedies demenstrated te be present in the chioken antituner antigen-Ll serums were specific fer tissue mate­ rials but net the turner agent. The narked reactien ef the guinea pig antitumer serums with turner antigen, as cespared te their reactien with nermal tissue antigen in direet reactions, had indicated the presence ef specific materials ethm* than n e m a l tissue, but the chicken antiserums apparently reacted with enly the nermal tissue pertien. Therefore, it was necessary te ebtain chicken antiserur which, when tested against turner antigen, weuld cause mere fixation than when tested against n e m a l tissue antigen. By using the direct reactions TA-L$ ♦ TIPS-LI, and NTA o TGPS-LI fer the testing ef chicken anti turner serums, the presence ef mere fixatien by the fbnrer reaetien weuld indicate the presence ef agent antibedies in the chicken serum. The subsequent tests, the refers, smpleyed these twe direct reactiens, the farmer being referred te as the tuner direct reaetien and the latter as the nermal tissue direct reactien. Ducks had been inoculated with turner and nermal tissue antigens (TA-L1 and NTA) and w iih nermal and infeoted chicken serums (NCS and ICS). Duck antiserums aet similar te the chic In n antiserums in that they preduee ne fixatien when used in direct te:ts. Duck antinermal antitumer serums were tested by the indirect technique. tissue and The NTDS and NCSDS acted as centrals, u d tha TDS-Ll and ICSDS sarvad as patamtisl saureas af tumar agaat antibedy. badias had b a m As o a a ba saan im Tabla XXI , anti - praduced in all the duoks imaoulatad with the narmal tissua ar tumar ratarial except duck number 2, which was in tha g reup that had received tha tumar antigen-Ll. The titers af tha duck anti- tuaar and antinermal tissua sarums ware a ppreximat ely tha same w h a n tasted with either tha, tumar ar narmal ing again that tissua direct retcti ens, indicat­ enly narmal tissue antibedies ware present. The duck antinermal and ant i-infeoted chicken serum serums failed ta shew any raaati as indicating the absence af antibedy f a m a t i a n . As was mentiened previeuslv, antibedies specific far the agent. infected chicken serum might can tain This serum, having been secvred frem birds shewing visoaral turners, had been used as a patential saurca af agent in previaus experiments. Hawever, it was net knawn if birds, whan shewing advanced tumar invalvament, preduoed specific tumar agent arrtibadias. Whan the serums af infected birds wore pealed far the praductian af tha ICS antigen, samples af thasa having the largest valumes had been saved and refresen. Thasa sarums ware remeved frem stersge and used as chioken antitumer serums in indirect tests. As will ba saan in Tabla X XI I, n m e ef "these serums exhibited pasitive titars when tested with tha tumar direct reactien, which indicated the absence ef either tissue ar agent antibedies. The lack af any reactien with tha turner direct reactien made it unnecessary te cenriuct tests an them with narmal tissue direct reactien. Chickens ware ineculated with tumar antigens prepared in variaus ways in an attempt ta stimulate agent antibedy preducti en. The variaus TABU XXX IHDIRECT TITX& OF DUCK ANTISEHDMS VHBI TXSTXD WITH BORKAL TISSUE ABB TUMOB ANTIGEBS ABB GUINEA PIG ABTITUMOR-L1 SEBUM IB THE DIRECT REACTIOB Duck Ant1serum BIDS (1:2) Test Duck __Bo.__ Bo. 5 6 TD8 .LI (1:2) 1 2 3 4 BCSD8 (1:2) 74 75 1CSDS (1:2) 71 73 BDS (1:2) Pool (1) (2) Are. (1) (2) Are. (1) (B) (1) (2) Are. (1) (2) Are. (1) (2) Are. (1) (2) Are, (1) (2) Are* (1) (2) Are. (1) (2) At s . (1) (2) Are. Direct Reaction TA -1*3 + TOPS -LI* BTA ♦ TGPS^Ll* Units 4.5 4.6 4.1 4.7 4.3 4.6 2.8 3.3 2.6 2.9 2.7 3.1 3.7 4.5 4.1 4.4 3.8 4.4 0.2 0.1 0.0 0.6 0.4 0.1 1.5 2.1 2.0 1.8 2.0 1.6 3.9 4.1 4.7 4.3 4.1 4.4 0.1 0.0 0.4 0.1 0.2 0.1 0.1 0.3 0.0 0.1 0.0 0.2 0.1 0.2 0.0 0.0 0.0 0.1 0.1 0.5 0.4 0.0 0.4 0.0 0.0 0.0 0.2 0.1 0.1 0.0 * Direct reactions used to test duck antiserums 112 TABLE XXII INDIRECT TITERS OF INDIVIDUAL INFECTED CHICKEN SERUMS WHEN TESTED WITH TUMOR ANTIGEN-L3 AND GUINEA PIG ANTITUMORL1 SERUM IN THE DIRECT REACTION Chicken Serum (ICS) dilution 1:1 1:2 1:4 K2P25 0.0 0.1 0.0 K1517H 0.1 0.1 0.0 K1401E 0.0 0.0 0.0 K151SH 0.2 0.1 0.3 K1463P 0.2 0.0 0.3 K15110 0.0 0.0 0.2 K1508Z 0.2 0.1 0.2 K1405R 0.0 0.0 0.0 K1511N 0.0 0.1 0.1 KL507Z 0.8 0.1 0.1 E1519D2 0.3 0.2 0.3 K1504M2 0.0 0.1 0.0 KL414Ag 0.1 0.0 0.0 K1521C~ 0.0 0.1 0.0 Chicken No. chi cken direct antitajmor serums w e r e react: err. Th e t i t e r s .a 111 , variec Teble vious tests, the t i t e r s we r e reaction w a s employed. It wa s , bodies specific therefo re , tumor agent antibodies. decreasing the e t h e r than test or for t h e available Tho removal tho normal pr e s e n c e o f tho but had depend agen t. The net reaction on the -s. s u s p e n s i o n materials of normad an tig on cou Id not bo from g u i n e a c o u l d not be used pig of v a l u e complement fixation lymphoma­ development of techniques of t he no w agent and these stimulated fron r e a c ti on . the t u m o r .-uinea m e t h o d f o r t h e e l i m i n a t i o n of re action. in the tissue rr.atorial for t h e a d so rp ti an anti serums r e mo ve d f o l l o w i n g t h o since all of tho 3y r e m o v i n g tho pig a n t i tumor seru m, reaction would present in of t h e a n t i g e n s i n t e r f e r e d w i t h the d o ter mi nat ion from. the guinea b y a n o m a 1 tis su e »• rmal t i s s u e de tectable c eat rifuga t ie n t e c h n i q u e s a possible which tis­ produced a n t i ­ avian v i s c e r a l tissue a n t i b o d i e s c o n s i d e r e d as in ir p r e ­ the unr. il produced of a of an agent of a t u m o r a g e n t - a g e n t a.ntibedy of c o m p l e m e n t bodies will of t h e n o r m a l tissue ca se chick eas h a d of e i t h e r t h e a g e n t o f nor ma l t i s s u e a n t i b o d i e s were that de ve lop men t for t h e d e t e c t i o n »nti8 *rum was same w h e n es s h e w a t un er d i r e c t r e a c t i o n w a s do not a l l o w s u f f i c i e n t s epara tioi tissue materials pig the and t u m o r t i s s u e m a t e r i a l s The specifi c a n t i b o d i e s purification had beer, tho lie m e t h o d w a s k a e w n wh ic h w o u l d b e tissue detection concluded material these e m p l o y e d . f o r the tosis normal As approximately used as w h e n t h - for t i s s u e turner and r. armtl ti csue ebta ine d f or t hese a n t i s e r u m s , 0 te U . l units. f rem sue di r e c t t e s t e d with, the occur, even no f ix at io n though the re an tiger. . similar te the normal t i s s u e of t h * n o n r a l ti ssue a nti o f t he ti ssue c o m p o n e n t s adsorption, and t h o portion r e m a i n in g TABLE XXIII INDIRECT TITER OF VARIOUS CHICKEN ANTITUMOR SERUMS WHEN TESTED WITH NORMAL TISSUE AND TUMOR DIRECT REACTIONS Chicken Antiserum TA -PT4 TA -L4A TA -L4B TA -L4C TA -L5A TA -L5B TA -L5C TA -L7A TA —L7B TA -L9 TA -lyl Chicken No. K1466C K1513I K2P95 X15030 K2P100 K5P42 K2P93 K5P46 K2P89 K2P97 “ KSP49 K2P99 K2P96 X1455Q K5P52 K1473T K1473Z K2P90 ~ K1414E2 K2P72 K2P99 Kl45»i2 K2P98 K2P42 K1473T K1414D2 K1473L2 K147302 K1516L2 K620Q, K669Lo K258K K262B K669X2 K1466M K1511H2 K350S K620B K669C3 TA -L3 Direct Reaction TOPS -L2 NTA 4 Units 2.1 3.7 1.7 0.2 1.9 0.3 1.4 0.1 2.6 0.7 3.4 1.7 2.1 1.4 2.6 0.4 3.6 0.7 2.1 0.2 3.6 1.6 2.4 0.7 1.9 0.2 1.6 0.3 2.7 3.8 0.7 1.5 0.2 0.1 2.7 3.6 1.2 3.8 0.2 4 _ TOPS -L2 2.4 4.1 2.0 0.0 2.0 0.4 1.9 0.3 3.0 1.2 2.9 1.9 1.7 1.1 2.2 0.0 3.1 1.1 1.7 0.3 2.9 2.0 2.3 0.5 2.2 0.1 1.9 0.1 3.0 3.4 0.6 1.9 0.0 0.2 3.0 3.4 0.9 3.5 0.0 _____ 115 w ould c•Tibiae w i t h normal tissu* vided tissue antibodies, s y ste m which would f i x co m p lem en t. a source culty. o f normal The r e d b l o o d matoly 500 d a y s chicken cells of ago wore u s e d . entirety, tissu e m aterials leaving that tho had b e e n a d s o r b e d packed c h ick e n re d tho by shaking and a llo w e d t o repeated a total noted in tho f i r s t five until none was first ic. te used tho titers especially guinea pig tests increase content. adsorptions w ith considerable testing for ^0 normal tissue bo removed normal The g u i n e a tissue pig • nt itum or antigen pig i t was normal t i s s u e J>7m and evident, antisoruw believed to was less o f 1L These were tumor a n t i g e n ­ The as s hown removed, reaction of t h e s e r urn ° e r might its be d u e in which would agent antibody i f t h i s w *r« t n n contain in had b e e n adsorption, determ ine C. 16 m i l l i l i t e r s antibody reaction, chicken in anti serums w e re compared antibody serum. remaining, a f t e r to Volumes serum w o re r e c o v e r e d . adsorption, testing suspended at pig 10 i r e rea sin g l y s e r u m and d iffi­ could bo for the m inutes seventh ad so rp tio n . only method a v a i l a b l e of ch ick en c o l l s were b u t became adsorbed guinea u s e f u l n e s s when The cells colls. pro­ of approxi - A gglutination of the c e lls p r o p o r t i o n to t u m o r a g e n t - a g e n t its The incubate antigen-Ll obtained before from t h e g u i n e a cells. pig antinorm al antitum or direct that red b leed on t h e r e d 10 t i m e s . observed a f t e r th e p ig a n titu m e r serum, a greater blood or s i x Vthmn t h e t i t o r b o f t h o T a b l e XXIV, the of for th o guinea in chickens this an d a n t i tumor a n t i g o n - L l serum s w are mixed w i t h This was gu i n e a elim inate an t i s o r u n u n c h a n g e d e x c e p t volumes of fo r tho normal normal cells mixing w i t h th o a n t i scrum, c o u ld tissue m illilitors red b leed w h ic h would The c h i c k e n a n ti normal serums Chickem from a p p aren tfy w a s h e d a n d meo s u r e d a n d a ’t e r in t h e i r tissue forming a c*n;pl«te wa s agent a n tib o d ie s . TABLE XXIV TITER OF GUINEA PIG ANTINORMAL TISSUE AND ANTITUMO&-L1 SERUMS BEFORE AND AFTER ADSORPTION WITH CHICKEN RED BLOOD CELLS Antiserum Treatment Antigen Dilution 1:2 Before Adsorption TA -L3 1:8 11.7 10.4 6.2 26.1 24.1 15.4 NTA 1:2 15.2 12.6 8.6 15.2 12.4 8.8 TA -L3 1:8 7.8 5.9 4,6 14.5 11.2 8.3 NTA 1:2 8.2 6.6 5.4 7.1 5.3 4.0 After Adsorption i TGPS -LI 1:2 1:4 1:8 NTGPS 1:4 1:8 117 The c h i o k e n a m t l turner te ste d w ith thm u m a d a e r b a d m a t e r i a l s adsarbod g u in ea pig antiturar reaetiem s. As th* chicken s e r u m s eeulc b e wham t h e shawm i n umadserbad Th* r e d u c t i o n indicated mennal Th* v:ith XXV, in amti s e r u m had b e a m chiok*n red turner *f the direct th a chicken ? n a t e r i a l s e n d ma t tian m aeessity ar the ebtaining af antisarum s fraa # r ne used cell?, dir*et ebteiae d reacti*m s was reduced r e m e v a l was n e t c h i c k e n i n t i s s r u m s with b a t h reectiem s cenclusively antitum er serums turner a g e n t . This af reaetians. am tibadias but th e ir direct the t i t a r the t i t e r s im t h e d i r e c t tissue wham em pleyiag tha chamga i n im t h * fixatian * f ma r ma l bleed absame* a f turner a g e n t a n t i b e d i e s the little * f t h a air.eunt a f hi gha a t t i t e r s t h * t u r n e r a m d mar mal t i s s u e m a t e d w h a n o am pa r e d t * sim ilar tite r s tis s u e and th* a n t i b a d i a s tissue Table had g iv e n tha were again t e s t e d , aarum ia t h a t t h e c oncem trat ian by ad s* rp ti* m cam plata. scrums whiah ti.* dam en s t r a t e d t h a t were s p e c i f ia f a r marma 1 f u r t h e r emphasized th* in the ch ick en a n titu m e r serums, mare p u r i f i e d r e a g e n t s f *r u s * a f na r s: al t ir s u e e e n t * mi n a t i a n . in th* end preduc- TABLE XXV TITERS OF VARIOUS CHICKEN ANTITUMOR SERUMS WHEN ADSORBED GUINEA PIG ANTI TUMOR SERUM USED IN DIRECT REACTIONS Chicken Chicken Antiserum No, TA -PT4 K1466C _ Direct Reaction TA -L3 + TOPS -Li* _ K1513I Unite 1.7 1.8 3.1 3.0 TA -L4A K2P100 1.4 1.1 TA -L4B K2P93 0.7 1.0 TA -L4C K2P89 1.8 2.0 2.1 2.6 1.6 1.4 1.3 1.6 2.4 2.6 K1473Z 3.3 2.9 TA -L5C K1414S2 1.2 0.8 TA —L7A K2P99 2.1 2.4 K2P98 1.1 1.4 TA -L7B K1473T 1.8 1.7 TA —L9 K1473L2 1.2 1.8 K1516L2 3.0 2.4 K620 0 3.0 3.4 0.9 1.2 K1466M 1.7 2.1 K1511H2 2.8 2.4 K5P49 TA -L5A K2P99 K2P96 TA -L5B TA -Lyl K5P52 K258K __________________ K620B_____________ * NTA + TGPS -LI* 2.6_______________ 3.2________ Direct reaction used for testing chicken antitumor serums SUMMARY VIII. Av ia n visceral lymphomatosis, m a j o r e o a n o m i c pr o b l a n the control procedures for large (F u r t h cable sca le its The application. Wi t h the to be de ve lo p such em p l o y e d . techniques were d e s c r i b e d by Rice be of t h e obtained hemolytic for calculated fer a an t i - i n f luenza reagents were slope and group as (1QU2) used prac ti ca l in techniques d e ve lop men t test w a s seleoted ma d e to for appli­ of a f o r use routin e l s b o r a t o r y a d a p t e d to the t e s t i n g is not wi l l A tests a c c o r d i n g to t h e e t a l . ( 1 9 U 7 )* c o m p l e m e n t tit rat io ns . and a value, slope The s l o p e antige n could net was a l s o or rabbit ^cu rth g r o u p w h e n both of these or a n t i s e r u m was 0 . 2 0 g i v e n b y Ma yer were in w h i c h are c a l c u l a t e d by the influenze W h e n eit he r t h e a n t i g e n as t h e optima l t e c h n i q u e must c o n s i s t e n t v a l u e f o r the of t i t r a t i m s w h e n specific of f i x i n g c o m p l e m e n t fix ati cn co nd uc t dia g­ of c h i c k e n combine w i t h capable ( 1 9 U 6 ) and Mayer value s , cons ide rab ly a b o v e the Rice tut are not developments complement s e r u m was p r es en t, present. is olation of an i n f e c t i o u s a g e n t in te rms o f u n i t s w h i c h slope. id en tic al resistance a n d isolation in the chicken indirect the c o m p l e m e n t is m e a s u r e d u se test, Attempts is a a test. resulting complex The chickens, N o ^effective m e t h o d f o r infection f i x a t i cn c a n n o t be Antibodi es p r o d u c e d of the p o s s i b i l i t y of t h e The c o m p l e m e n t complement fixation l(jliaPb) . of subsequent characterization, a n t i g e n but t he (Rice, Genetic incidence of v a r i o u s d i s e a s e s , serum. is k n o w n . 1933) disease i n d us tr y. the considered. in an at te m p t nosis influence and B r ee di s, test was the p o u l t r y of t h i s .disease may to to a ma l i g n a n t ob t a i n e d . et a_l. present, (19U6b) W h e n both the a n t i g e n and was anti s e r u m w e r e present , the 0 .2 1 , b u t v a r i e d f r o m 0.15 to 0 .2 7 . The i n c o n s i s t e n c y of t h e s l o p e culation ef the technique mination un it s in w h i c h of the of fi x a t i o n , the s l o p e titer of t h e c o m p l e m e n t duce a v e r a g e of t h e d e t e r m i n a t i o n s values, n e c e s s i t a t e d the calculation of c o m p l e m e n t measured w i t h an against ceuld be p l o t t e d d i r e c t l y against t h e s e v a l u e s was va l u e s co mp lement, be manner. propo rt io ra t e and d i v i d i n g n u m b e r of un it s f i x e d tests. guinea deter­ normal of' a ’i s t a n c - These antigens we r e of hemolysis. any used f o r a n t i s e r u m chickens and d u c k s . a l s o used a s antigens in Norrra 1 and tests and f o r of c o m p l e ­ o f t he se two v o l u m e s percent hemolysis read in gs . of fell, B y determining h e m o l y s i s w h e n a g iv en unit v a l u e , the calculated. and t u m o r a n t i g e n s w e r e pigs, log s c a l e of p r o p o r t i o n a l ity between could b e The any reaction occurred taole fer 50 pro­ of complement The a m o u n t it by tho c c m p l e m e n t by the r e a c t i o n tissue, needed t o The a c t u a l plottirg the a m o u n t o f c c m p l e m e n t n e c e s s a r y for 5 - percent In fl ue nz a, The q ui te e a s i l y b y the v o l u m e d e t e r m i n e d fr o m the e ]e c t r e p h o t ameter p r es en t done percent 5 ^ p e r c e n t h e m o l y s i s when where the amount necessary re ac ti on w a s cal­ d e v e l o p m e n t of a would be readings. by the p r e p a r a t i o n the th e t o the h e m o l y s i s , ir. a s i m i l a r eliminated f r o m which c o u l d be give d et er mi ne d for t h e ele ct rophotoma te r a n d s i n c e t h e were proportional could b e used not n e c e s s a r y . This c o u l d readings me n t n e c e s s a r y to was d e t e r m i n i n g t h e a m o u n t that e x a c t l y 5^ p e r c e n t h e m o l y s i s . hemolysis wrg were of c o m p l e m e n t c o n s i s t e d of t e s t i n g v a r y i n g a m o u n t s and plotting the volume w hi ch used pro du ct io n infected inoculation. ir ir chicken the v a r i o u s rabbits, serums w e r e I n f l u e n z a antig-r. procedure. Itebbit and a direct was of phile anti s e r u m . anti serum. Th ese if found chicken inf l u e n z a v i r u s duce the h e t e r o p h i l s was canpleted by of i n c u b a t i o n and The first s e r u m as an the- d e t e r m i n a t i o n tests antigen on o r i g i n a t e d f r o m the the materials could b e and seru ms as a n t i g e n s , chicke n act as f o u n d t o be c o n c l u d e d that t h e not times sufficient to of h e t e r e in tumor due t o co nc er t ratior stimulate s p e c i f i c a n t i g e n in the specif ic antigens, normal ra bb i t anti go* embryos in used to and pro­ temperatures in je c t e d and since chicks* s e r u m serum. 1 ive r t umor c h i c k e n se ru m . similar reactions uuir e-;» pig an t i serums, were tested and a g a i n using normal sli-ht reactions tissue materials. It w a s , of the agent in infected c h i c k e n antibody presence the t o be a d d e d . us irr detected tissue antigen. and in the *'ouni not as the a n t i g e n f o r t he i n f e c t e d tis su e was op ti ma l were made not as s ter. da rdizat i jn o f t he ‘ e c h n i q u o of t h e produced w i t h normal fere, the r e a c t i o n a g a i n s t g u i n e a p i g a* t i-inf ecte d c h i c k e n substituted noted but were reaction, presen t in w h i c h the r e a g e n t s w e r e ob ta in ed w i t h n o r m a l in f e c t e d to were luinea .pigs were S l i g h t r e a c t i o n s w er* n o t e d but were could be cue direct spe ci fi c a n t i b o d i e s tumor materials r e a c t i o n b e t w e e n these extract was a n d had and tests i n f l u e n z a anti s e r u m in the an ti b o d i e s to be propagation. order original h a d b**en p r o d u c e d b y h e t e r o p h i l s antibodies, th<* in t h e chicken occurred serum with antibodies used s t a n d a r d i z e tho t e s t the a m o u n t o f f i x a t i o n w a s i n c r e a s e d influenza i n f l u e n z a a n t i g e n ma te r i a l for the No technique, This was a n t i g e n in the When over w h a t h a d b e e n the c a s e us ed to serum was standardized. indirect decreased, should h a v e ch i c k e n ar.ti-influenza was t e s t e d b y the instead the test and a n t i s e r u m s w o r e formation in the g u i n e a of t u m o r g u i n e a pig we r e the res*r ur. or to pig anti s e r u m . Th e s t a n d a r d i z a t i o n of a d i r a c t ant it u m o r s* ru m s c o u l d b e sence antigen af g u i n e a p i g a nt in or m a l tent was of the g u i n e a pig an ti n o rm a l sp ec if ic re action was considered antibodty, direct due t o or t u m o r Chicken normal present titers, and had antisorvjr.s ag ain Thus, i dent ical with th os e the a n t i b o d i e s p r o d u c e d as a result tion had not antibedies b u t w ith the either antibodies the agent specific and g u i n e a quite tested. of normal The lew, pig of of a r auction and for could its then:. a n t it um er s e r u m . showed t h a t a n t i b o d i e s of t h e in a n ti ge n. tho i n d i re ct Noriral dire ct titers c h i c k e n anti s er um had t i ss ue s t i m u l a t i o n . .Then gu i ne a other th an inoculation h a d b e e n ex p- cte d. — This s p e c i f i c the nor ma l ch ic ke ns , The inocul a ti rr. pigs we r e litt le o r no of normal used. apparently b eea tiss ue inoculated with had normal ch ic ke n and o b t a i n e d were A normal tissue ty p e t i ss ue reaction found w h e n the t u m o r a n t i g e n had be e n b e e n ex p e c t e d . a n ti ge n, and c ons iderabl r gr e a t e r tumor antigen in the t u m o r con­ Turner a n t i g e n indicated the presence inactivated a portion for the a n ti bo dy t e s t e d with a s t a n d a r d i z e d t u m o r although the alm ost and tumor a n t i g e n substituted tas te d rea gen ts o t h e r t h a n that w h i c h systems ser um s w e r e antigen was c h ic ke n tissue tis su e m a t e r i a l s . cell c o n p o n e n t s reaction employing This turner on e o f two antitumor c h i c k e n s e r um were the s e ru m was pig a n t i t u m e r s e r u m than w i t h the tissue serum. the the p r e ­ its t i t e r w i t h the t i t e r the normal antis erums, the g u i n e a between be a c c o u n t e d for b y t he se chicke n co n s i d e r a t i o r pig anti tu mo r and b y c o m p a r i n g tissue serum, t h e n used f o r t e s t i n g guinea Gu i n e a into pig a n t i t u m e r s e r u m was d e t e r m i n e d . r e a c t i o n was n o t e d with The test ed had t o tak e of n o r m a l t i s s u e r e a c t i o n s , against normal tissue r e a c t i o n w i t h w h i c h the rea c­ turner b e e n produced, tissue reaction tiss ue into its homologous host was c o n s i d e r e d much production than would tha presence lass l i k e l y to of t um or stimulata antibody coll components agent w h i c h w o u l d b e c o n s i d e r e d f o r e i g n m a t e r i a l . finding, antiserums f r o m chickens which t i ss ue a n t i g e n wore tained for the normal tissue titrated. c hi ck e n direct of the concluded l ymp ho mat o sis the stimulated antibody Ducks wore i n f e c t e d and stimulated antibody antiserums, the c o m p l e t e of two w a y s . of t u m o r tissue serum absence and in o r the dire ct in fe ct ed The dud: t o exhibit been test. they might ti ss u e a n t i g e n , and normal and tissue antigens the chicken by a similar ar.t i -inf ec te d and --nti- an^ reac ti on, in hi bi t tho could h a v e i nd ica tin g reacted formed d u r i n g the reaction an ti bo d i e s second therefore, e n t i g e n h ad n o t i n h i b i t the r-a ctior The se co n d This It w a s , p r e v i o u s l y u s e d & s an ant ige n, was If ant i b e 'ti.es had this way,, also or formation. serum, react with tumor the sa m e m a g n i ­ t umo r a n t i g e n was a c c o m p a n i e d This s e r u m would tumor ob­ ch icken. ir. direct m e t h o d . g u i n e a pig a n t i b o d i e s . pr-sent w h i c h in the normal sa m e ti t e r s w e r e but as ha 4 b e - n f o u n d w i t h of a n t i b o d y dev elo pm en t, i n o c u l a t e d wi th of a p p r o x i m a t e l y The tumor an ti g e n . on this serums w i t h either tha t u m o r a n a normal serums f a i l e d infected ch ick en the ag en t serums. formation, normal no rr a 1 c h i c k e n te st e d b y ch i c k e n As a ch oc k anti tumor s e r u m s . p r o d u c t i o n in the & r e a c t i o n wi th reaction with The chick«t inocu ' at “d w i t h normal tiss ue a n d w ere tude as the t i t e r s that be e n A p p r o x i m a t e l y the an ti n a r m a l reaction, had o r tumor in either advance! stages of t h e a g e n t w i t h could a r i s e in the guinea if ament w e r e pi g anti serum, r e a c t i o n of t h e a n t i g e n a .-id a n t i b o d y possibility was chick er. s - r u m h a d b e e n us ed as an eliminate:! when an tig en in the direct test s; t h er ef o r e, serum, if an in direct titer it w o u l d h a v e b e e n were due to not t h e case, however, since was tested in t he i n di re ct react ion . procedures stimulate of tumor antigens in an a t t e m p t specific chicken antitumor r e a c t i v i t y whe n The tite rs p a ni ed b y a •f tire g u i n e a r em ov -d the si mil ar pig a po rt i o n detection it type, the This det ec t' d w h e n it centrifugation of t h e a g e n t w h i c h w e u l d inoculated into exhibited chickens. varying The degrees of 3.8 units, but as had b e e n found, in all a r n ctirr: with normal eli mi na ti - n normal in the of but did chickens s pe ci fi c for s c o o t ,- it. isorp ti on r-d blood ceils not f a c i l i t a t e chicken antitumer that th' were an ti g e n w a s antigen. chicker. tissue antibodies v.&s co n c l u d e d noire w h i c h tie t u m o r nor s.a 1 ti ss u e anti tumor s e r u m with o r the chicken reactions employing tumor antigen. the r. eveiopmer.t 1 y n p h ora t © s i s , upon a suspension r e a c t i o n a ga in st a n t i b o d i e s but dependent to of a g e n t a n t i b o d i e s t h e s e fi nd ing s, tissue dir ec t could b e using v a r i o u s p r e p a r e d w i t h t her from M.l of this p r ep ar ed format! a" w h e n tested with rang'd previ ous te sts serums was inf ected o f s p e n t a n ti b o d i e s . no a n t i b o d i e s to pr oc u d e i-ntibot/ for t h e the p r e s e n c e was A group •btained se ru ms . vr«n h ad pro du ce d normal the au *r.t ofv i s c * r a 1 of a c o m p l e m e n t f i xat ion te st hue n o r m 1 tissue r„ateri« Is. is CONCLUSIONS 1. the The hemolytic presence s ho ws w i d e v a r i a t i o n and charges in of v a r i o u s t e s t r e a g e n t s , m a k i n g it u n s u i t a b l e for u s e in the calculation 2. liters fixation, of u n d i l u t e d 3« can of the a m o u n t s Complement eliminate to the t h e use be us ed determined slope the can g r a p hi ca ll y then contains production of h e t e r o p h i l o p h i l e a n t i m e n of the fi x e d . in t e r m s be c o n v e r t e d of m i l l i ­ to u n i t s and calculations. test a n t i r e n for formation of complement complement, of /'/hen the not sl ope v a l u e chicken chicken tissue of c o m p l e m e n t a n t i b o d i e s wi nc h serum. Cuinea pips material, fix in g rabbits antiserum, roo ct with the Ho n o t form due hetero- this anti bo-ly . U. agent Infected antibody reaction when 5. formation Tumor antigen 6. tumor pigs, s t i m u l a t e s the to the stimulate the and t - give f ai ls of a specific pip antis erum. formation f r o m normal presence production op antibodies chicken tissue of t u m o r cel l in g u i n e a antibodies, components and not stimulates the agent. T he formation due not t u m o r guinea distinguishable but a r e a p p a r e n t l y the t u m o r in gu in e a t e s t e d with pigs w h i c h are normal chicken serum does inoculation of a n t i b o d i e s ti ss u e ag en t antigen, antibodies. of c h i c k e n s which and rea ct there with tumor antirens s i m i l a r l y w i t h t u m o r a n t i g e n or is no evidence of the production of 7. tion of the Th e u t i l i z a t i o n a^ent ©f avian davelapment af tachmiquas tion af the a.^ent. of the viscaral far the coir.plement fixati art lymphair* tosi s wi l l increased test in the depend upan purificati on and detec­ tha concent ra 127 BIBLIOGRAPHY Bi ester, H. S., and L. ' . S c h w s r t e D i e e a s e s of P o u l t r y , Ed. 3, Iowa St*-*-*: C o l l e y ® 1952, pp. U 5 5 - 5 0 7 . Bit tn er , J. 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