CONCENTRATION AND ACIQ-ALKALINE EFFECTS ON RIBONUCLEIC ACID INHIBITION OF ANTIth ANTIBODIES Thais for fho Degree of Fit D. MICHIGAN STATE UNIVERSITY Lois Leona Canrad 19.62 This is to certify that the KALINE EFFECTS HIBITION OF ANTI-Rh ANTIBODIES presented by LOIS LEONA CONRAD has been accepted towards fulfillment of the requirements for PH.D. degme in BIOLOGI CAL SCIENCE \ Major professor 4.7 Date May 3’ 1962 \ LIBRARY Michigan ‘5‘“: University «WW7 mu. .. -\.' . ,.r .yy. r I “ _Crv1/V vu‘n‘, In), -..,. t- V.“ t~.1t.—‘"t,f!-.‘I-“.7 . 7:4 .0 ON RIBONUCLII] wLLV ani TT'X L? ANTI» n ANTIBODIES By Lois Leona Conrad I I AN ABSTRACT OF A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY leision of Biological Science 1962 'II T. . S .. . 1: :I. an aw” t - II. WI. :- . I _ G I.-. 3 r“ n. S C x“ .3 17; w: — n1. .x... .a + rd. v w; .: a... .J p. I I. .v .3 a. h v. E .I . 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V uv—y.~r‘r‘1v- fawn ‘-r.l.l\}ih...\ ‘1 ;I‘ I‘ CONCENTRATIC. ‘é-J ’ 5 -¢ d D ANTI—Rh ON RIBONUCLEIC AC1 Lois Leona Conrad ~ A THESIS Submitted to Michigan State University in partial fulfillment of he for the degree of .; ECTS ANTIBODIES requirements ACKNOWLEDGMENTS Most deeply and most directly, I am indebted to Dr. Emanuel Hackel, my advisor, for his many suggestions, F patience and guidance during this thesis work. Also, I wish to express my appreciation to the other members of my committee, Dr. Rollin H. Baker, r. Richard U. Byerrum, Dr. Herman L. King, Dr. Chester A. Lawson and Dr. John M. Mason, for their helpful suggestions and criticism. I am further grateful to Dr. Amos Cahan of Knickerbocker Biolog- icals and Dr. Philip Levine and Robert T. McGee of Ortho Pharmaceutical Corporation who donated the serum used in this experimentation, as well as to all others who assisted me in any way during the writing of this thesis. ii TABLE LIST OF TABLES. . . . . LIST OF GRAPHS. . . . . LIST OF FIGURES . . . . CHAPTER I. INTRODUCTION. . 0 OF CONTENTS II. REVIEW OF THE LITERATURE. History of the Rh Factor. Nature of Rh Antigens . Nature of Rh Antibodies Hemagglutination Inhibition III. Materials and Procedure . . Results . . . Discussion. . IV. SUMMARY . . . . BIBLIOGRAPHY. . . . . . Methods THE EXPERIMENTATION . Used. Studies Page iv vi Table II. III. IV. VI. VII. VIII. IX. AMP AMP AMP UMP UMP UMP CMP CMP CMP versus versus versus versus versus versus versus versus versus LIST OF TABLES Anti-D; Anti-C; Anti-E; Anti-D; Anti-C; Anti—E; Anti-D; Anti—C; Anti-E; 9‘ 9‘ % % 7‘ 3% ‘76 76 7% Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition Page 47 48 49 50 51 52 53 54 55 LIST OF GRAPHS Graph Page Ia. AMP versus Anti-D; % Concentration. 56 Ila. AMP versus Anti-C; % Concentration. 57 [_ IIIa. AMP versus Anti-E; % Concentration. 58 ‘ IVa. UMP versus Anti-D; % Concentration. 59 Va. UMP versus Anti-C; % Concentration. 60 V18. UMP versus Anti-E; % Concentration. 61 VIIa. CMP versus Anti-D; % Concentration. 62 VIIIa. CMP versus Anti-C; % Concentration. 63 IXa. CMP versus Anti-E; % Concentration. 64 lb. AMP versus Anti-D; pH of Inhibitor. 65 IIb. AMP versus Anti-C; pH of Inhibitor. 66 IIIb. AMP versus Anti-E; pH of Inhibitor. 67 IVb. UMP versus Anti-D; pH of Inhibitor. 68 Vb. UMP versus Anti-C; pH of Inhibitor. 69 VIb. UMP versus Anti-E; pH of Inhibitor. 7O VIIb. CMP versus Anti-D; pH of Inhibitor. 71 VIIIb. CMP versus Anti-C; pH of Inhibitor. 72 IXb. CMP versus Anti-E; pH of Inhibitor. 73 Figure 1. LIST OF FIGURES Page The structure of 3'—monophosphates is illus- trated. The 2'-monophosphates differ in that the phosphate group is attached to the 2'- carbon of the sugar rather than the 3'-carbon. 3 Sample illustration of the test set-up and scoring procedure. . . . . . . . . . . . . . . 32 vi D T CHAP INTRODUCTION A number of investigators, Kabat (56) and Morgan 1“ et al. (2, 3, 4, 39, 40), have found the technique of spe~ cific serological inhibition valuable in characterization of A, B, H and Lewis substances from various sources. Through such a technique it was possible for these investigators, during purification procedures of blood group substances, to determine in which body fluids and/or tissues these sub— stances were distributed. In addition, this technique was of value, durirg the assays. in determining when degrada- tion of the blood group substances was occurring. It was possible by this knowledge to avoid procedures that would cause degradation. These investigators demonstrated that isolated blood group substances can prevent their specific antibodies from causing agglutination. Hackel et a1. (46, 48) have discovered chemical substances other than isolated blood group substances which would specifically produce inhibition with anti-Rh and Lutheran sera. By adding one of these chemical substances to a serum with a specific antibody, it was possible for them to demonstrate that the resulting hemagglutination reacti: was decreased. In other words, erythrocytes added to an antibody by which they would be agglutinated, were not as strongly agglutinated in the presence of these substances as would have been the case uithout the addition of the chemical substances. Their investigations included testing sixty differ- ent reagents consisting of sugars, amino acids, short chain polypeptides, purines, pyrimidines, desoxyribonucleic acid (DN ) derivatives and ribonucleic acid (RNA) derivatives. They found one nuclcoside (cytidine) and three nucleotides (adenylic, cvtidylic and uridylic acids), all ribonucleic acid derivatives, effective in the inhibition of anti-Rh and anti-Lutheran sera. As pointed out by Hackel et al. (48, p. AC7), "With respect to the conceptual scheme which places ribonucleic acid in the key position between desoxy- ribonucleic acid as genetic material and protein as phaeno- genetic material, there have been few demonstrations of specificity attributable to ribonucleic acid." It was the purpose of this study to conduct a fur— ther investigation of three of these inhibitors discovered by Rachel et al. (48), nameIV, adenosine-Z'—3'—moncphosphate (AMP), cytidine-Z'-3'—moncphosphate (CMP) and uridine-2'—3'— rzmncnhcsphate (UMP) and their effects at various concentra— b These substances are all derivatives of ribonucleic at:ids which occur, primarily, in the cytoplasm of living ce2lls and are also the main component in some viruses. Riborn1cleic acids are thought to play an active role in protein synthesis of the cell. They consist of a nitrogenous base (either a purine or pyrimidine), a sugar (d-ribose) and a phosphoric acid group. The bases used in this par- ticular study were either adenine, cytosine or uracil. Figure 1 illustrates the chemical structures of the inhib- itors used in this investigation. Mixtures of 2'-3' deriv— atives were employed by Hackel in his early investigations and were used in this study. 5““ Khan o¢°‘\; “ “ I c\ N ~41: '2 I? I' /c\ "'I‘ ‘5/7 }cu &\c,. 1" ti; HT, . £1“ 1 4- 1 Hc‘fi/c\:‘ (1’ on o,c\:‘}cu 3‘ 5. HOCH o | uo—P—ou uocu o l lJb’ \\., I ,h>/ ‘\\ v *‘C H H I 0 4c H H C l\é._é/I , . I\',...,g/I H '5' I" H GYTIDINE-a-PHOSPHATE H I 2' H cl) OH 0 on I HO—P—OH Ho-P-OH I I o o ADENOSlNE-3'-PHOSPHATE .umome -3'-PuosPHATE Fig. l. The structure of 3'-monophosphates is illustrated. The 2'-monophosphates differ in that the phosphate group is attached to the 2'-carbon _of the sugar rather than the 3'—carbon. It was thought that through a further investigation of the effects of these inhibitors by varying their concen- tration and pH, it might be possible to gain a better under- standing of the chemical nature of the antigens involved, as well as the immunological specificity. It is realized. however, that the problem chosen by this author is but one of many facets of the total situation involving antigen- antibody reactions. CHAPTER II REVIFW OF TE? LITFLATCFC of the E H o :11 5... U) ( f O *‘3 c<3 :3. '11 £0 0 .1"? O ’1 H y} rv (D *‘S p: l Darrow (29), in 1938, made a review of the "‘4 ture and an analysis of published data concern' y) '3 T4 to E '3 <<: (’1‘ } 1 () disease of the newborn. He was one of the first to conclude that an appropriate etiological explanation for this disease (D could be attributed to an antigen-antibody reaction. Th kind of reaction involved was determined th following year in 1939. The beginning of an understandini of the an factor was the work of Levine and Stetson {66) in 1939. Thev studied the blood of a woman who had given birth to a fetus which had been dead for six weeks prior to delivery. An ‘unusual antibody was found in this patient's serum which zagglutinated the red b cod cells of 80% of the other irdi- tJiduais tested having the same A-E-Q grgup as that o T Ejatient. it was thought, by them, that the reten;ion of 4 F‘“ +h \ v n ‘r} $ ,7. (- UI sift? JHUQLd..- "Vite Tl: Ill ;\ CZhe dead fetus was responsible a_ ntibodies. They concluded that the fetus had irherited 1 c'. “‘ ' " . 1' -.‘ ‘: .-+~~ .- ' 8J1 antigen from the Iatner wnion caused tn-s maternat im— nvurnization. ! -F$EI~.=.fi£—"=!—_:Z.Et -......"""=.r'-' - 32115.! '- _~_— .‘ 3' However, the true implication of their discovery and interpretations were not clear at the time. As men- tioned by Race and Sanger (76, p. 1165, "had Levine and Stetson given a name to the blood group system which they had dis- covered, it, and not Rh, would have been the title to [their] chapter and of a thousand other publications.f . In 1940, Landsteiner and Wiener (58) injected rab- bits and guinea pigs with blood cells from the rhesus monkey (Macaca mullata). They found after absorption of the rabbit serum to remove antibodies characteristic of the species that there was left an agglutinable factor which aggluti- nated not only the monkey red blood cells but also 85% of the blood of white people tested in New York City. The authors named this new agglutinin, anti-Rh, after the rhesus monkey whose blood cells had produced the original antibody. Thus, those people whose blood cells were agglutinated by this antibody were called Rh positive, whereas those, whose cells were not agglutinated by this antibody, were known as Rh negative individuals. Wiener and Peters (88) studied the blood of three patients who suffered severe transfusion reactions in spite of receiving compatible A-B-O group blood. They demonstrated the presence of antibodies whose action resembled that of :Eantibodies in the original anti-Rh serum. Davidsohn and Toharsky (30) and Murray (73) demon- .S‘trated through absorption experiments, antigenic differences between.the Rh factors in the blood of man and that of the rhesus monkey. Essentially, they found that the animal antisera, after absorption with human Rh positive cells, also contained agglutinins for rhesus erythrocytes. Fisk and Ford (37) also confirmed the earlier fact that there were differences in their demonstrations with cord blood of 312 infants. All of them gave positive reactions with serum derived from guinea pigs, whereas 8.8% gave negative L" W reactions to one human serum, and 13.6% to another. Thus, it was obvious that the anti-Rh serum derived from animals was related to the human anti-Rh variety, but was not identical to it. In fact, for this very reason, anti-Rh serum derived from animals cannot be used today in accurate typing of human bloods for the Rh factor. In 1941, Levine, Katzin and Burnham (64) examined bloods of sixteen women who had given birth to fetuses thought to have died of erythroblastosis fetalis (hemolytic disease of the newborn attributable to the Rh factor). They found that fourteen of these women were Rh negative, six had demonstrable agglutinins and that all of the fathers ,and fetuses tested were Rh positive. Later the same year, Levine, Katzin and Vogel (65) =Esummarized their observations and presented blood studies CIDn 153 women who had given birth to infants with hemrlytic dj.sease. Ninety-three per cent of these patients were Rh neegative and seven per cent were Rh positive. Of 141 women tested, 42 showed the presence of anti-Rh agglutinins. Many, who had no agglutinins, had not been pregnant for over a year; furthermore, all of the 80 fathers and 76 affected infants were Rh positive. From the observations cited above and similar ones by other investigators (8, SO, 60, 78), the role of the Rh factor in blood transfusions and hemolytic disease of the newborn became increasingly apparent. It was evident that the Rh agglutinin was not a naturally occurring antibody but was produced by Rh nega— tive individuals upon introduction of the antigen into their systems. This was brought out by the fact that Rh negative individuals who had received one or more blood transfusions displayed Rh positive antibodies. Also, it became clear that the phenomenon of erythro- blastosis fetalis occurred in a situation where the Rh neg- ative mother was carrying an Rh positive fetus, with the Rh positive factor necessarily inherited from the father. It was thought that the Rh antigens of the red blood cells of the fetus, in some way, crossed the placenta, escaping .1nto the maternal circulation. Since the mother lacked I antibodies would be produced against these ‘these antigens, :Ebreign intruders. These antibodies, then, could pass back t::hrough the placenta into the circulation of the fetus where triey combined with and coated the red blood cells of the .feetus, destroying them by hemolysis. It was noted that one or two incompatible preg— nancies were usually required to immunize the mother which meant that the first and second Rh positive infants were quite frequently unaffected. It is now thought thet this phenomenon concerning the effect of the antibody on the fetus depends upon the titer (quantity) and avidity 5w (strength) of the antibody present in the maternal cir- culation. 1....-1 __ Besides this, it was discovered that many of these Rh negative women exposed to this kind of antigenic stimuli never became immunized. It would seem that it was due to the fact that the maternal circulation was not exposed to the antigen on the ery hrocytes of the fetus; but why and (0* how -his occurs in some cases and not in others is not known as yet. Researchers began to realize that there was appar- ently more than one variety of Rh factor. In 1941, Levine et al. (65) made mention that not all Rh antibodies found in all human serums were identical. They found blood that was Rh positive to anti-Rh serum and also contained Rh agglutinins of a different specificity. Landsteiner and Niener (59) described and verified this and commented on the fact that human serums varied to some extent in their ezzbility to cause agglutination. Still another variety was ciiscovered by Race et a1. (79) in 1943. 10 In 1943 Fisher, cited in Race and Sanger (76), pos- tulated allelic genes which would produce antigens and in appropriate individuals would cause the production of anti- bodies. He further postulated that these antibodies, pro— duced as a result of antigens that originated from allelic genes, would give an antithetical reaction. Thus, he recog- nized the fact that two of the anti-Rh sera described by Levine g£_gl. (65) and also by Landsteiner and Wiener (59) were antithetical and gave them the names, anti-C and anti-c. Since the two remaining sera, the original anti-Rh described by Landsteiner and Wiener in 1940 (58) and that discovered by Race, did not react in an antithetical manner, he further postulated that they, too, had antithetical forms. He called the original Rh antibody, anti—D, and the latter one, anti-E. The postulated antithetical forms he labelled anti-d and anti-e. Anti-e was discovered by Mourant in 1945 (72) as predicted. Diamond, cited in Race and Sanger (76), in 1946, first reported anti-d followed by two other examples reported by Haberman g£_al. (43) in 1948. However, later, consider- able doubt was raised as to the specificity of the anti-d sera. Fisher theorized that there were three genes with C::ontrasting alleles responsible for the antigens C, c, D, (i, E and e found on the red blood cells. He felt that these grenes were closely linked. Wiener disagreed with this, 11 pointing out that instead of three genes, only one gene with multiple alleles could be responsible for the expres— sion of the Rh antigens on the red blood cells. However, as Race and :anger (76, p.3FE' later stated in 1958, "The existence of three sites where Mendelian substitution can go on seems to us unassailable, and to argue whether the three sites are to be placed within or without the boundary of one gene 3 appears particularly unprofitable at the present time when 1..-... no one seems to know what the boundaries of a gene are." In addition to the three main antibodies and the antithetical forms already mentioned, subgroups of these were found, most of them being fairly rare. ”h‘s author shall limit the discussion to those already mentioned since these are the only ones considered in the experimenta- tion of this paper. A very thorough discussion of the other variant forms of anti-Rh can be found in Race and Sanger (76). Nomenclature in early investigations presented no problem, but, with the addition of more and more informa— tion, complications began to arise which necessitated a more standardized nomenclature. The following is an ex— ample of the terminology systems which are now widely used: 12 Gene Combinations Rh Antigens Fisher Wiener Fisher Wiener DCe R1 D Rho DcE 32 c rh' Dce RO E rh" p DCE RZ d Hro i dce r 0 hr 9 H k dCe R e hr ‘ 1 ch R. dCE R y II. Nature of Rh Antigens As has been pointed out previously, Rh antigens are present in erythrocytes of Rh positive and negative individuals. Investigators such as Bernstein and Israel (10), Diamond (32), Potter (74), and Stratton (82) have demonstrated the presence of Rh antigens in fetuses at various stages of development, this being indicative of the fact that Rh antigens appear early in life. Fisk and Ford (37) found that the Rh antigens in infants differ somewhat from those found in adults. Animal anti—Rh serum, derived from guinea pigs, was shown to ag— glutinate the red blood cells of infants up to one month of age whether the infant was Rh positive or negative. This was not the case with adults. 13 More recently in 1961, Levine and co—Norkers' find- ings (67) suggested that there is a "D—like" antigen in rhesus monkey, human Rh-positive and human Rh—negative red blood cells. They indicated that guinea pig reagents produced by injection of the above cells or their extracts defined an agglutinable property which differs distinctly P= from the human D antigenic determinant. The human anti-D will not agglutinate Rh negative cells, whereas in this ) experimentation they were able to recover eluates from Rh positive, Rh negative and rhesus monkey cells that gave D-like specificity. They concluded that the term "rhesus factor,’ as applied to the human D (Rho) antigen, appears to be a misnomer. There have been investigations for the Rh antigen on body fluids and tissues other than erythrocytes. Levine and Katzin (62) were unable to find Rh antigens in saliva, sperm cells and seminal fluid. Wiener and Forer (89) con- firmed this absence and concluded that the antigens were only present in erythrocytes. Boorman and Dodd (7) reported the antigen's presence in the liver, spleen and salivary glands of Rh positive individuals and also in the saliva of 27 of the 51 Rh positive individuals they tested. How- ever, this has never been confirmed. Witebsky and Mohn (92) reported finding the Rh antigen in amniotic fluid in four-fifths of all pregnancies in which the fetus was Rh positive and none in the Rh negative fetuses. 14 Several investigators have undertaken the task of separating the Rh antigen from the red cell stromata through hemolysis. Belkin and Wiener (6) subjected erythrocytes to hemolysis and claimed that they obtained Rh antigen along with A and B substances. They mentioned that the inhibition and absorption titers obtained for the various Rh proper- ties were consiStently lower than the titers obtained for A—B-O substances. These findings suggested to them that the number of Rh "haptens" per cell may be less than the number of A, B or O "haptens." Calvin gt_§l. (l7) separated erythrocyte stromata into a protein fraction which they called stromatin and a lipoprotein known as elinin. From elinin they separated an ether-soluble fraction containing a still greater concentration of Rh antigen and proportion- ately lower concentration of A and B antigens. In 1947 Carter (23) reported an ether-soluble frac— tion separated from group O Rh positive cells. This frac- tion was non—antigenic in experimental animals but was anti- genic when injected simultaneously with a protein carrier. She claimed that this substance specifically inhibited the agglutinins present in anti-Rh serum and it resisted inacti- vation by heat. The substance was thought to be probably the Rh "hapten" in impure form. Price et al. (75) attempted to purify Carter's sub— stance and described the pure "hapten" as an acid, optically inactive, soluble in alkali with a melting point of 156.9° - 15 157.2°. It exhibited activity in dilutions of 1:5000 as measured by the complement fixation test with anti-Rho serum. According to Landsteiner (57), a hapten is a spe- cific protein-free substance which, although active in vitro, induces no, or only slight, antibody response. Thus, a hapten performs as an antigen in that it combines with an F} antibody in vivo and in vitro, but unlike an antigen it, i of itself, will not elicit the production of antibodies. It was with this in mind that several investigators (16, 41, ;_ 42, 49, 53, 68) treated patients; with demonstrable Rh anti- bodies during pregnancy, with an Rh "hapten" using Carter's and/or Price's methods. It was thought that if this was truly Rh hapten, then an Rh negative pregnant woman actively sensitized at the time of treatment would be desensitized, the maternal titer falling in response to treatment with Rh hapten. The results of this treatment were quite variable. In some cases the "Rh hapten" apparently seemed to help, in other cases, there was no response whatsoever. It be- came more and more apparent it could not be concluded that this "Rh hapten therapy" was the determining factor as to whether the mother gave birth to a normal child or the child died of erythroblastosis. Besides this, the previously mentioned experiments of Belkin, Carter and Calvin were not found to be reproduc- ible by other investigators. It also became apparent that 16 what they labelled "Rh hapten" was, in reality, some other substance, non-specific in nature. In 1950, Stratton and Renton (83) followed Carter's original and modified methods for extracting Rh hapten. In all cases, control Rh negative cells were extracted at the same time as the Rh positive cells. They found that Fe the Rh positive cell extracts had a very slight inhibitory ,9 effect on Rh antisera, but also, that extracts from Rh neg- ative cells proved equally inhibitory. It was concluded that the phenomenon was of a non—specific character. Evans and co-workers (36) made a study of the Rh factor in elinin. They could not obtain an active fraction of crude preparations of elinin by chemical or enzymatic means, ncr could they repeat the work of Carter in ex- tracting an active substance from red blood cells by alco— hcl and ether. These later studies helped confirm the belief that an Rh hapten or antigen actually had not been isolated as first claimed by the earlier investigators. Thus, a pur- ified form of Rh antigen has yet to be found. There have been other studies such as that of Lubinski and Portnuff (69) who did an investigation of heat and forma- lin upon the Rh agglutinogen. They discovered that the addition of formalin to red blood cell suspensions reduced their agglutin-ability for anti-Rh serum much more than for A or B sera. It was also found that heating red blood 17 cells to 55° C for five to twenty minutes caused them to lose their reactivity with agglutinating as well as with blocking anti-Rh sera, whereas the anti-A, anti—B anti-M and anti-N sera were not affected. They speculated that these results might be due to the fact that the A, B, M and N agglutinogens are on the surface or the Rh agglutinogens :7 are less numerous and/or there is a difference in chemical E structure. III. Nature of Rh Antibodies Rh antibodies do not occur naturally but are pro- duced as a result of the introduction of Rh antigens into the circulatory system of a susceptible individual, namely, one who is negative for the Rh antigen introduced. Two types of Rh antibodies are recognized in vitro. They are (l) anti-Rh agglutinins which unite with Rh posi- tive erythrocytes suspended in saline and (2) blocking or incomplete antibodies which unite with Rh erythrocytes but do not cause agglutination unless the cells are suspended in a protein-like material such as albumin or plasma. It is thought that, in vivo, both types of antibody cause the same kind of reaction, namely, hemolysis of the erythrocytes following the union with their specific antigens on the erythrocytes. For the most part, these Rh antibodies occur in the serum of Rh negative individuals who have had an 18 incompatible blood transfusion or in Rh negative pregnant women carrying Rh positive fetuses. In addition to this, Witebsky and Heide (90, 91) have demonstrated Rh antibcdies recently delivered women who had the Potter (74) stated that it seemed probable that agglutinating antibodies were the earliest varieties formed in response to stimulation by Rh antigen. Blocking anti- bodies appeared later and were thought to be evidence of a greater degree of immunization and they frequently per- sisted much longer in the blood. However, either type of antibody produced hemolytic disease or could be responsible for transfusion reactions. It has been suggested by Wiener (86) that differ- ences in action between Rh agglutinating and blocking anti- bodies might be due to the number of combining groups (sites on the antibody where a chemical union could be affected with the corresponding antigen) which make up each antibody. The agglutinating antibody was thought to have two combin- ing sites (bivalent) causing agglutination when each site was attached to a red blood cell. On the other hand, block- ing antibodies were thought to have one combining site (univ- alent) which can attach to one erythrocyte but because of the lack of a second site it cannot attach to-a second eryth- rocyte in order to hold two cells together. The demonstration that incomplete antibody in a l9 suitable medium could cause agglutination called into ques- tion Wiener's theory on the univalence of the incomplete antibody. To overcome this, Wiener gt_gl. (87) proposed that plasma or serum media caused the erythrocytes to "stick" together because of a substance called "conglutinin" within the media. In other words. it did not bring about proper a; agglutination. ) A different approach for the detection of incomplete ; antibody was the antiglobulin or Coombs test described by ; Coombs g£_§l. (27) in 1945. It was found that erythrocytes r coated with incomplete Rh antibodies would agglutinate upon exposure to anti-human globulin. These anti—human globulins were produced by immunizing rabbits with globulins of human serum. This is a test widely used today for detection of incomplete antibodies. Diamond and Abelson (33) have shown that agglutinat- ing and blocking antibodies are similar in that they may unite with erythrocytes at room or icebox temperature, but the reaction is more rapid at 37° C. However; blocking antibodies are more thermostable than agglutinating anti- bodies, according to both Diamond (33) and Coombs (28). Coombs and Race (28) state that blocking antibodies will not go through a collodion filter known to be perme- able to proteins with a molecular weight of 30,000. They also found that the electrophoretic migration of Rh posi- tive cells exposed to either kind of antibody was the same. 2O Boyd (11) demonstrated that Rh agglutinins were destroyed by exposure to pressures of 3000-4000 atmospheres for twenty-four hours, whereas blocking antibodies required pressures in excess of this for their activity to be inhib- ited. In 1947, Coombs and Mourant (26) suggested from serological evidence that blocking antibodies were present in the gamma globulin fraction of human serum with the pos- sibility of there being small amounts in the alpha and beta fractions. In the late 1940's, Witebsky and Mohn (93). through dialysis of certain sera containing Rh antibodies, not only found blocking and saline agglutinating antibodies but a supposedly third order of antibody (reactive only in the aJitiglobulin test) in their various globulin fractions and Stipernatants. Hill g£_§l. (51) used the Reid-Jones fractionation lne‘thod (80) utilizing ion-exchange resin materials also PGBSIJlting in a third order of antibody which they termed "Cilfiyptagglutinoids." However, whether these antibodies represent a weaker reacting antibody detected only by means 017 the more sensitive anti—globulin test or whether they are a true "third order antibody" has not been convincingly demonstrated. Cann and co-workers (21), in 1952, employed elec- t1"Dphoresis convection in the fractionation of Rh antibody. 21 A series of top fractions of the sera were removed by suc- cessive runs at progressively lowered pH, ranging from 8.1 to 5.3. Using four sera with Rh antibodies, they indicated that the saline and blocking antibodies were found not only in gamma globulin fractions of human serum but may also be associated with proteins possessing mobilities of alpha and beta globulins. The same year Sturgeon and Brown (84) concluded that the electrophoresis convection technique did not serve to separate the agglutinating antibodies from the blocking antibodies. The electrophoresis convection data tended to show that both saline agglutinating and blocking antibodies sire distributed in two fractions of different mobility, one cxf which is gamma globulin and the other, beta globulin. ifliey felt that from an immunological standpoint the total aaritibody in the serum represented a spectrum with the saline agglutinating antibody at one end, and the blocking antibody Elt the other end. However, they felt that the heterogeneity 5111 electrophoretic properties was unrelated to the hetero- 駀3r1eity in antibody properties. In 1953, Jankovic and Kuijnen (55), using three aJ’Iti-D sera containing both agglutinating and blocking Eir‘TIibody, separated them through the use of paper electro- IDkICDresis. They found agglutinating and blocking antibody (>11:Ly in the gamma globulin fraction. They could not con- I:xiir‘m the observations of Cann et a1. (21) that other 22 globulin fractions also contain Rh antibodies. The authors felt that this discrepancy was due to the fact that a better separation of globulin fractions was possible by means of paper electrophoresis than by the electrophoresis convec- tion method. In 1959, Abelson and Rawson (l) employed yet another method known as exchange chromotography. They found that the incomplete antibodies were removed in a broad band, whereas the saline agglutinins were found in fewer aliquots of the eluting solutions. This, they felt, was in accord- ance with the theory that incomplete agglutinins represent a spectrum of molecules with slight variations while the safline-active antibodies may be more nearly homogeneous. Campbell et a1. (19) claimed that they isolated tlie agglutinating antibodies from the blocking antibodies They stated that the Rh saline ‘bjl ultracentrifugation. agglutinins sedimented at a faster rate than the blocking tarps of antibodies. They came to the conclusion that the‘ IQII saline agglutinins consist of molecules of a greater n10lecular weight than the blocking type. More recent studies lléi‘fe confirmed this plus the fact that saline antibodies “VGBIPe associated with protein of a molecular weight near :l-’ C3C)0,000, whereas incomplete antibodies possessed a molecu— 1‘511’ weight of 160,000 which is that of normal gamma globulin. Chan and Deutsch (25), in 1960, did a study of the <23k1€3mical and biological activities of Rh antibodies which 23 had been reported in the past. Additional information gained from this study was that saline agglutinins were destroyed by treatment with 2-mercaptoethanol, whereas the incomplete antibody was not affected. This caused a loss in direct agglutinating activity but reactivity was demonstrated with the Coombs test. The incomplete antibody, destroyed by papain digestion also resulted in a loss of the usual agglutinin reactions but maintained Coombs reactivity. They speculated that the loss of direct r agglutinin activity could be attributed to the fact that the larger molecules, having been broken down into smaller ones, had fewer sites to offer for complete combination vvith antigen, but enough to affect Coombs reactivity. We find, then, in a review of the properties of fiki antibodies, that the investigators have been able to ecffect a separation of the antibodies through various phys- i.051l and chemical methods. There was agreement that these airituibodies are found in the globulin fraction of serum as (3131308ed to albumin fractions, and, at first, there was disagreement as to which globulin fractions contain the a1"1‘t-Zibodies. However, it is now agreed upon by most investi- é§537t<>rs that antibodies are found primarily in the gamma glObulin fraction of serum. There has been some attempt on the part of several tzC) differentiate the molecular size of the two types of Ell“Ht-ibody which has resulted in apparent agreement that the IIIIIIIl-._ 24 saline agglutinin has a molecular weight of approximately 1,000,000, whereas the incomplete antibody has a molecular weight of about 160,000. From the aforementioned information, it may be ob- served that although there have been many reports concern- ing the physical and immunological properties of both the Rh antigens and antibodies, very little is known about the chemical properties of either. IV. Hemagglutination Inhibition Studies Landsteiner (57) stated that immune antibodies all liave the property of specificity in common, i.e., they re- eact, as a rule, only with the antigens that were used for immunization. Here was a clue to a method for elucidating the ciaemical nature and structure of antigens of unknown com- EHDsition, namely, the hemagglutination inhibition test. 5P11§.s test has been used effectively in the isolation and S’tlldy of some blood group substances. It was employed by Dd<>1?gan et al. in the purification of A, B, H and Lewis sub- St“ances. Also, Kabat (56) and Boyd (12) have summarized irlklibition studies of other investigators and described t h e inhibition technique . The inhibition reaction results from the union of E1)“ artificial antigen or haptenic substance with the anti— 130fly in question. This artificial antigen or haptenic 25 substance is thought to combine with the antibody by vir- tue of its similarity to the original antigen. In the case of hemagglutination the reaction takes place as follows: A chemical or haptenic substance is added to a serum with a known antibody. To this are added red blood cells with the antigen specific for the known antibody. If agglutina— tion of the red blood cells does not take place as expected, it could then be assumed that the chemical substance has inhibited agglutination of the red blood cells. ale is that the chemical substance or hapten must exhibit some of the properties of the original antigen in order to combine with the antibody. Thus, in turn, it leaves .less, or no, antibody free for combination with antigens C)” the red blood cells. I The work of Morgan and his associates (2, 3, 3., 40) 2151s demonstrated that blood group substances A, B, R and ! Ihlxsxes. They foand that acid hydrclvsates of preparation: a . . , - g _ . 4. . ~ . '“—~ “ d and neWis substances from ovarian cyst fluid con- €ii.ned hexosanine (35%), L-fucose (13¢), galactose (17%) 51r3- \ / T‘\ \ x P \ I, \\ \ \ 60 ‘1 \ I, x‘ \ I I- \ _____ J 50 m— 40 ‘— 3 O T- I/ 20 «I- ‘\ /-—— -\ I I. x/ ‘ / I0 I*' \/ 0 I I I 1r 4I 6.6 6.8 7.0 7.2 7.4 8 0 pH OF INHIBITOR °lo INHIBITION ._ 63 GRAPH VIIIJ. CMP VERSUS ANTI -C °lo CONCENTRATION 2—-—---—--———- 6 4—¢¢::¢$rf 6——-----—- . I00~I~ I 90 .0— I- J 80‘1” ’7 I- 70 4»- r .60 «- I- 50 ~- I-"’"~\ /’ \\ h— ’/ \\ ’—-—-‘ 40 Hr”, 6.6 6.8 7.0 7.2 7.4 pH OF INHIBITOR "I Irllfl' °/o INHIBITION 64 GRAPH IXa CMP VERSUS ANTI -E 2——-—-~----—— 6 4—+-4—+-—+-+-—+—+—+— °lo CONCENTRATION 8_._.__...__.. IOO "- l T i "I?- 6.6 6.8 I 7.0 7.2 pH OF INHIBITOR 7.4 L 8.0 65 GRAPH Ib AMP VERSUS ANTI -D PH OF INHIBITOR —-————— 7‘2 \IO’O) _...__..__.. 80 _______ .6 .8 -—§-——+—+—+———§-— 74 ._.__._..__.__ .O °/o INHIBITION 0 + I fi‘ 2 4 6 B °/o CONCENTRATION OF INHIBITOR °/o INHIBITION I00 90 80 7O 60 SO 66 GRAPH IIb AMP VERSUS ANTI -C PH OF INHIBITOR 6.6 - “m 7.2 68%4 *fi % # 74 F = = 7.0 80 _______ E I. F- ” r- ’1’...” / T I’ / ’- / // / / b—-—_———__ /I J 1 T T J». 2 4 6 8 °/o CONCENTRATION OF INHIBITOR °/o INHIBITION 67 GRAPH III 5 AMP VERSUS ANTI -E pH OF INHIBITOR 72 74 c A L V V 7 W 80...- ! i v‘ IOO T O + 2 4 0/0 CONCENTRATION OF J T 6 8 INHIBITOR .Jh. ‘3: T "-‘—"M~— .- 1 'nu -..,_;__-___ .0 _ 68 GRAPH IVb UMP VERSUS AN Tl -D PH OF INHIBITOR 6.6—-—'- ‘- 7.2 6.8 ¢ ‘r F + § 74 : c c 70 .. .. . so _______ I00 "I" 90 +- 80 HI— 70 “I— / 60 «I- f I .- °/o INHIBITION T \ \ ~II— I 40 / ’l’ / 30 "II- o’ / 20 «’/ O 4 + 2 4 6 a °/o CONCENTRATION OF INHIBITOR .I. °/o INHIBITION I00 90 80 7O 6O 69 GRAPH Vb UMP VERS US ANTI -C -——-- — 72 PH OF INHIBITOR A A L L k A .A L f fir f f r _ V v— v 80 ——————— °/o CONCENTRATION OF INHIBITOR 7O GRAPH VIb UMP VERSUS A NTI - E PH OF INHIBITOR 6.6 - - —-- 7.2 68““? # wL # t 74 t c 4 7.0 .. .. 30 _______ IOO *~ 90 "I' Z 9 I: :2 I .7: 0 \ O O 4 T 2 4 6 8 °/o CONCENTRATION OF INHIBITOR .II. IOO 90 80 °/o INHIBITION 71 GRAPH VII b CMP VERSUS ANTI -D 39’?” 00:0: PH OF INHIBITOR —- —-——— 72 _W_____ O _______ l 4 V T 4 6 °/o CONCENTRATION OF INHIBITOR °/o INHIBITION I00 90 80 7O 60 50 4O 30 2O 72 GRAPH VIII b CMP VERSUS ANTI -C PH OF INHIBITOR 6.6—-—-_ 7.2—__— 7IO—.——-c-—--- 8 _______ r; I i d I I. "J L, ’I / /’ -I ‘ / / “~“\ /;'/ ./ T ,/ '/ I/ V/ I/ - ,/ l/ ’/ I I fi‘ 2 4 6 8 °/o CONCENTRATION OF INHIBITOR °/o INHIBITION I00 90 80 70 60 50 4O 30 20 73 GRAPH IXb CMP VERSUS ANTI -E PH OF INHIBITOR 66" - 7.2 6.8 :4 +2 : 4 74 —¢ ¢ : 7.0 ~ -- -- 8.0 _______ I. r /”—. I— // x’ “z +- ’,” F "/' I— ’v/ .. "/ I I. '/ / / /’ P- /// .- ’/ ’/’ V ’/ I- a/ ’/ ’/ P T I 4 2 4 6 8 °/o CONCENTRATION OF INHIBITOR CHAPTER IV SUMMARY The present study is an extension of Hackel and co-workers' (46, 48) investigations Of three ribonucleic acids, namely, adenylic, cytidylic and uridylic acids, all 2'-3' mixtures. The original work involved the inhibitory effects Of these substances on various antisera at a 2% concentration and a pH 6.8. This investigation included adenylic, cytidylic and uridylic acids at concentrations of 2%, 4%, 6% and 8% at the following pH's: 6.6, 6.8, 7.0, 7.2, 7.4 and 8.0. The Rh antisera involved were anti—D, anti-C and anti-E. The inhibitory effects were tested by use Of the hemagglutination inhibition. Increased inhibition Of all three ribonucleic acid derivatives with anti-Rh sera was demonstrated with in- crease in concentration. It was postulated that the more inhibitor molecules in solution, the more antibody—inhib- itor combinations would occur. This would leave less antibody to combine with the specific antigen on the eryth- rocytes. Also, the results indicated that a monomolecular reaction was taking place between antibody and inhibitor molecule because of the steady rise in inhibition as the concentration of the inhibitor substance was increased 7A % ‘ Me: % _IJ _‘—_ __.-‘9 I I M 75 from 2% up to 8%. This investigation further demonstrated that al- kaline pH's Of all three inhibitors resulted in an increase of their inhibitory effectiveness. It was speculated that a certain amount Of hydroxyl ions must be of more importance than hydrogen ions in effecting this inhibition reaction. It was further postulated that the ability Of the hydroxyl ion to remove hydrogen atoms from the inhibitor molecule or antibody molecule could effect a chemical combination. Suggested bonds were hydrogen bonds through amino and/or carboxyl groups, nitrogen-nitrogen bonds, and oxygen bonds through phosphate groups. Also, the specificity of an antibody for an antigen may depend not only upon the determinant groups within each molecule but also upon the spatial arrangement of these groups. Thus, the fact that one inhibitor was more ef- fective than another could be explained further by the spatial arrangements Of the determinant groups within the molecules. For example, the weaker inhibition Of one in- hibitor compared to another could be due to the failure of oppositely charged groups to correspond perfectly in position because of differences in the spatial arrangements of the determinant groups within each individual inhibitor molecule. Taking into consideration both increased concen- tration and alkaline pH, adenylic acid was found to be the i _i,i _‘ .i‘, i- . . ‘5" ‘1‘} . 7.1.. 76 most effective inhibitor with anti-D, anti-C and anti-E sera. Cytidylic and uridylic acid were almost equal in inhibitory effects. All of the inhibitors were most ef- fective with anti-E and anti-D sera, respectively, and were least effective with anti-C. Even though this investigation has demonstrated further increase of antigen mimicry of these ribonucleic acid derivatives in terms of the inhibition theory, there is much to be learned. For example, to be assured of the specificity of these reactions, further investigation is needed with these inhibitors at variOus concentrations and pH's other than anti-Rh sera. It would be interesting to know the effects of concentration and pH's on isomers of the 2'-5' mixtures used in this study. .Also, further in- vestigation of the individual differences between samples Of the same kind of serum is needed--is it a true effect or not? Thus, it would appear that further eXperimentation would be quite helpful in further elucidating the exact role that ribonucleic acid derivatives play in inhibition of anti-Rh sera. 10. BIBLIOGRAPHY Abelson, Neva M., and Rawson, Arnold J. 1959. Studies of blood group antibodies. II. Fractionation of Rh antibodies by anion-cation cellulose exchange chrom— otography. Journal of Immunology, 83:49-57. Aminoff, D., Morgan, W. T., and Watkins, W. M. 1950. The isolation and properties of.the human-blood-group A substance. Biochemistry Journal, 46:426-439. Annison, E. F., and Morgan, W. T 1952. The isola- tion and properties of Lewis (Le ) human blood-group substance. Biochemistry Journal, 50:460-472. ‘. wast—“T 4...- - . 1952. The isolation and prop- erties of the human blood-group H substance. Biochem- istry Journal, 52:247-260. Baar, H. S. 1945. The Race-Wiener test in haemolytic disease of the newborn. Nature, 155:789. Belkin, Ruth B., and Wiener, Alexander S. 1944. Dem- onstration of the properties A, B, M, N and Rh in red- cell stromata. Proceedings of the Society of Experi- mental Biology and Medicine, 56:214. Boorman, K. E., and Dodd, B. E. 1943. The group- specific substances A, B, M, N, and Rh: Their occur— rence in tissues and body fluids. Journal of Pathology and Bacteriology, 55:529. , and Mollison, P. L. 1944. The incidence Of haemolytic disease of the foetus (eryth- roblastosis foetalis) in different families: The value Of serological tests in diagnosis and prognosis. Jour- nal of Obstetrics and Gynaecology, 51:1. , and Morgan, w. T. 1945. 'Enhance- ment Of the action of immune haemagglutinins by human serum. Nature, 156:663. Bornstein, S., and Israel, M. 1942. Agglutinogens in fetal erythrocytes. Proceedings of the Society of Experimental Biology and Medicine, 49:718. 77 11. 12. 13. 14. 15- 16. 17. 18. 19- 20. 21. 22. 78 Boyd, W. C. 1946. The effect of high pressures on hemagglutinating antibodies. Journal of Experimental Medicine, 85:401. . 1947. Fundamentals Of immunology. 505 pp. New York: Interscience Publishers, Inc. , and Reeves, Egli. 1961. Specific inhibi- tion of anti-D antibody by colominic acid. Nature, 191:511 , Boyd, L. G., and Warshaver, E. R. 1945. Spontaneous loss Of specificity of absorbed immune hemagglutinating serum. Journal Of Immunology, 51:191. , McMaster, M. H., and WaszczenkO-Zacharczenko, E. 1959. Specific inhibition of anti-Rh serum by "unnatural" sugars. Nature, 184:989. Brockland, Betty. 1949. The preparation and use Of Rh hapten. American Journal of Medical Technology, 15:179-184. Calvin, Melvin, Evans, Robert, et al. 1946. Rh anti- gen and hapten. Nature of antigen and its isolation from erythrocyte stroma. Proceedings of the Society of Biology and Medicine, 61:416. Cameron, James W., and Diamond, Louis K. 1945. Chem- ical, clinical and immunological studies on the prod- ucts of human plasma fractionation. XXIX. Serum albumin as a diluent for Rh typing reagents. Journal of Clinical Investigation, 24:795-801. Campbell, Don H., Sturgeon, Phillip, and Vinograd, Jerome. 1955. Separation of complete and incomplete Rh antibodies by centrifugation. Science, 122:1091- 1093. Cann, J. R., Brown, R. A., Gajdusek, D. G., et a1. 1951. Fractionation of Rh antiserum by electrophore- sis-convection. Journal Of Immunology, 66:157. , Kirkwood, J. G., et a1. 1952. Studies Of antibody distribution in the serum pro- teins of several Rh antisera. Journal of Immunology, 68:245-251. Cappell, D. F., and McFarlane, M. N. 1946. Spontane- ous loss of specificity Of absorbed anti-Rh sera. Lancet, 1:558. 25. 24. 25. 26. 27. 28. 29. 30. 31. 32. 55. 34. 79 Carter, Bettina B. 1947. Preliminary report on a substance which inhibits anti-Rh serum. American Journal of Clinical Pathology, 17:646-649. 1949. Rh hapten: Its preparation, assay and nature. Journal Of Immunology, 61:79-88. Chan, P. C., and Deutsch, H. F. 1960. lmmunochem- ical studies of human serum Rh agglutinins. Journal of Immunology, 85:57-46. Coombs, R. R. A., and Mourant, A. E. 1947. On cer- tain properties of antisera prepared against human serum and its various protein fractions: Their use in the detection Of sensitization Of human red cells with "incomplete"Rh antibody, and on the nature Of this antibody. Journal Of Pathology and Bacteriology, 59:105-111. , and Race, R. R. 1945. A new test for the detection of weak and "incomplete" Rh agglut- inins. British Journal of Experimental Pathology, 26:255-266. , and Race, R. R. 1945. Rh factor: Further Observations on "incomplete" or "blocking" Rh antibody. Nature, 156:255. Darrow, R. R. 1958. lcterus gravis (erythroblastosis) neonatorum. Archives Of Pathology, 25:578. Davidsohn, 1., and TOharsky, B. 1942. The Rh blood factor: An antigenic analysis. American Journal Of Clinical Pathology: 12:454—441. , and Stern, Kurt. 1948. Interpretation of Rh antibodies. American Journal Of Clinical Pathology, 18:690-6990 Diamond, L. K. 1945. The clinical importance of the Rh blood type. New England Journal Of Medicine, 252: 447. , and Abelson, Neva. 1945. The importance Of Rh inhibitor substance in anti-Rh serums. Journal of Clinical investigation, 24:122-126. , and Denton, Ronald L. 1945. Rh agglutina- tion in various media with particular reference to the value Of albumin. Journal of Laboratory and Clinical Medicine, 50:821-850. 35. 36. 37. 38. 39- 40. 41. 42. 43. 44. 45. 46. 47. 80 Dodd, Matthew C., Bigley, Nancy J., and Geyer, Vir- ginia B. 1960. Specific inhibition of Rh D) anti— body by sialic acids. Science, 152:1598-1399. Evans, Robert S., Moskowitz, Merwin, and Calvin, Mel- vin. 1950. Studies on antigens Of human red cells, 11. Serological properties of Rh factor in elinin. Proceedings of the Society Of Experimental Biology and Medicine, 73:657—642. the Rh agglutinogen Of human blood. American Journal of Clinical Pathology, 12:545. . Fruton, Joseph S., and Simmonds, Sofia. 1959. Gen- eral biochemistry. Second edition, 1077 pp. New York: John Wiley & Sons, Inc. Gibbons, R. A., and Morgan, W. T. 1952. Serological properties of human blood-group A and B substances. Nature, 170:77. . 1954. The isolation and prop- erties of substances of human origin possessing blood group B specificity. Biochemistry Journal, 57: 285- 295. Goldsmith, J. W. 1950. Experiences with Rh hapten. American Journal of Obstetrics and Gynecology, 59:172- Greenwalt, T. J. 1949. Preliminary report of exper- _iences with Rh hapten. Journal of Laboratory and Clinical Medicine, 54:1605-1604. Haberman, W. 1948. The demonstration and character- ization of the anti-d agglutinin and antigen predicted by Fisher and Race. Blood, 5:682. Hackel, Emanuel. 1960. Quantitative aspects of anti- Rh inhibition. Paper presented at American Society of Human Genetics. Unpublished. . 1961. Inhibition studies on anti-Rh anti- bodies. Rome (in press). , and Spolyar, K. S. 1960. Anti- -c and anti- -e inhibition by ribonucleic acid derivatives. VOX Sang, 5: 517- 522. , and Smolker, R. E. 1960. Effect of ribo- nuclease on erythrocyte antigens. Nature, 187:1056- 1037. ‘7 48. 49. 50. 51. 52. 53. 54. 55. 58. 59. 81 , and Fenske, S. A. 1958.1nhibi- tion of anti- Rh and anti- Lutheran sera by ribonucleic acid derivatives. Vox Sang, 5: 402-408. Hamilton, E. G., and Brockland, Mary E. 1950. The results Of treatment with Rh hapten. American Journal Of Obstetrics and Gynecology, 60:815-819. H111, J. M., Haberman, S., and Velez, O. A. 1945. The preparation Of potent anti-Rh typing serum by injection Of Rh positive blood into previously iso- immunized individuals. Journal Of the American Med- ical Association, 128:944. , and Guy, R. 1949. Further evi- dence for antibodies of third order: Fractionation Of agglutinins, blocking antibodies and cryptagglutin- Oids by physico-chemical methods. American Journal Of Clinical Pathology, 19:154-140. Howe, C. J. 1951. The influenza virus receptor and blood group antigens of human erythrocyte stroma. Journal Of Immunology, 66:9. , and Rustigan, R. 1950. Studies with the Rho D antigen. Journal Of Immunology, 64:505-515. Hubinont, P. O. 1948. Action of heating on Rh-pos- itive human red cells. Nature, 161:642. Jankovic, B. D., and Krijnen, H. W. 1955. Serolog- ical activity of globulin fractions Of anti-D sera separated by paper electrophoresis. Nature, 171:982- 985. Kabat, E. A. 1956. Blood group substances. 330 pp. New York: Academic Press. Landsteiner, Karl. 1947. The Specificity of sero- logical reactions, 510 pp. Cambridge: Harvard Uni- versity Press. , and Wiener, A. S. 1940. An agglutinable factor in human blood recognized by immune sera for rhesus blood. Proceedings Of the Society Of Experi- mental Biology and Medicine, 45:225. . 1941. Studies on an agglut- inogen (Rh hI in human blood reacting with anti—rhesus sera and with human isoantibodies. Journal of Experi- mental Medicine, 74:509-520. 60. 61. 62. 63. 64. 65. 66.- b8. 69. YO. 82 Levine, P. 1945. The pathogenesis Of erythroblastosis fetalis; a review. Journal of Pediatrics, 25:656. . 1960. Blood group antigens and antibodies as applied to blood transfusion, 72 pp. New Jersey: Ortho Pharmaceutical Corporation. , and Katzin, E. M. 1940. lsoimmunization in pregnancy and the varieties of isoagglutinins Ob- served. Proceedings Of the Society of Experimental Biology and Medicine, 45:545. , and Burnham, L. 1940. Atypical 9 warm isoagglutinins. Proceedings of the Society Of Experimental Biology and Medicine, 45:546. , . 1941. lsoimmuniza- 9 _— tion in pregnancy, its possible bearing on etiology Of erythroblastosis foetalis. Journal Of the American Medical Association, 116:825. , and Vogel, P. 1941. I ... 9 9 The role Of isoimmunization in the parthogenesis Of erythroblastosis fetalis. American Journal Of 0b- stetrics and Gynecology, 42:925-937. , and Stetson, R. E. 1959. An unusual case Of intra-group agglutination. Journal of the American Medical Association, 115:126—127. , Cellano, Marino, Fenichel, Richard, et al. 1961. A "D-like" antigen in rhesus monkey, human Rh positive and human Rh negative red blood cells. Jour- nal of Immunology, 87:414. Loughry, J., and Carter, B. B. 1948. Report of a case of erythroblastosis fetalis treated with Rh - hapten. American Journal Of Obstetrics and Gynecol- ogy. 55:1051-1052. Lubinski, Hubert H., and Portnuff, Joseph C. 1947. The influence of heat and formalin upon the Rh agglut- inogen. Journal of Laboratory and Clinical Medicine, 32:178-180. Mohn, J. F., and Witebsky, Ernest. 1948. The demon- stration of a third type of Rh antibody with blocking properties. Journal of Laboratory and Clinical Med- 1cine, 33:1360-1368. 1‘3} 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. .fi 83 Moskowitz, M., Dandliker, W. B., Calvin, M., and Evans, R. S. 1950. The separation from human erythrocytes of a water soluble fraction containing the Rh, A, and B factors. Journal Of Immunology, 65:585. Mourant, A. E. 1945. A new rhesus antibody. Nature, 155:542. Murray, J. 1952. Rh antigens of human and monkey blood. Journal of Immunology, 68:515. Potter, E. L. 1948. Rh--lts relation to congenital hemolytic disease and to intragroup transfusion reac— tions, 344 pp. Chicago: The Yearbook Publishers, lnc. Price, Charles C., Reed, David B., et a1. 1948. The isolation Of a substance with Rh hapten activity. Journal Of the American Chemical Society, 70:5527- 3528. Race, R. R., and Sanger, Ruth. 1959. Blood groups in man, 577 pp. Third edition. Springfield, Illinois: Charles C. Thomas Publ. , and Taylor, G. L. 1943. A serum that dis— closes the genotype Of some Rh positive people. Nature, 152:500. , , et a1. 1945. The Rh factor and erythroblastosis. British Medical Journal, 2:289. . 1945. Recognition Of Rh 9 ’ __...— _ genotypes in man. Nature, 152:563- Reid. Allen F., and Jones, Frances. 1949. Production of therapeutic fractions Of human blood serum. Amer- ican Journal of Clinical Pathology, 19:10-15. Benton, P. H., and Hancock, Jeanne A. 1958. .The influence of antigen strength agglutinin and incom- plete antibody on agglutination by anti-D sera. British Journal Of Hematology, 4:89-9b. Stratton, F. 1945. Demonstration Of the Rh factor in the blood of a 48 mm. embryo. Nature, 152:449. , and Renton, P. H. 1950. The Rh hapten. Lancet, 258:526. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 84 Sturgeon, P., and Brown, R. A. 1952. Studies Of a "complete" and an "incomplete" Rh antiserum with ggeégde and -D-/-D- cells. Journal Of Immunology, Wiener, A. S. 1944. A new test (blocking test) for Rh sensitization. Proceedings Of the Society of Ex- perimental Biology and Medicine, 56:175. . 1945. Conglutination test for Rh sensiti- zation. Journal Of Laboratory and Clinical Medicine, 30:662-667. Sonn, E. B., and Belkin, R. B. 1944. Nature ______v of Rh agglutination reactions. Science, 100:98. , and Peters, H. R. 1940. Hemolytic reac- tions following transfusions of blood of the homolo- gous group with three cases, in which the same agglut- inogen was responsible. Annals of Internal Medicine, 13:2506-2522. and Forer, S. 1941.‘ A human serum contain- _' .- ing four distinct isoagglutinins. Proceedings of the Society of Experimental Biology and Medicine, 47:215. Witebsky, E., and Heide, Ann. 1945. Further investi- gations on presence Of Rh antibodies in breast milk. Proceedings of the Society of Experimental Biology and Medicine, 52:280-281. , Anderson, G. W., and Heide, Ann. 1942. Demonstration of Rh antibodies in breast milk. Pro- ceedings Of the Society of Experimental Biology and Medicine, 49:179—185. , and MOhn, J. 1945. Investigations on the occurrence of Rh substances in amniotic fluid. Jour— nal Of Experimental Medicine, 82:145. . 1948. Studies on Rh anti- bodies. Journal Of Laboratory and Clinical Medicine, 33:1353-1369. , , et a1. 1946. A simple method for the concentration of Rh agglutinins. Proceedings of the Society of Experimental Biology and Med1c1ne, 61:1-50 . . . K ("lvllnl. «I'IIII, . RGOM USE ONLY, 9W4 a I TIMER SSE [LIMIT "IIIIIIIIILIIIIIIIIIIIIIIIIIIIIIIIIIs