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M «v! “1"" p- u-w “- r lllllilllllllllllIllllllllllllllllllllllllIlllllllllllllIIHI 31293 007843 LIBRARY Nicklaus State University This is to certify that the thesis entitled DEVELOPMENT OF AN ELISA FOR THE EVALUATION OF SERUM IMMUNE RESPONSES IN PIGS EXPOSED TO ACTINOBACILLUS PLEUROPNEUMONIAE presented by Tesfaye Belay has been accepted towards fulfillment of the requirements for Master's degree in Microbiology 977W/37m Major professor Datem 0-7639 MS U i: an Affirmative Action/Equal Opportunity Institution PLACE N RETURN BOX to remove thle checkout from your record. TO AVOID FINES return on or before due due. DATE DUE DATE DUE DATE DUE ——]l__JCj ’ ' fl " " ‘“—--H-*‘**n-.L-w-w-- .3, “' "'lIHIIIl DEVELOPMENT OF AN ELISA FOR.THE EVALUATION OF SERUM IMMUNE RESPONSES IN PIGS EXPOSED TO ACTINOBACILLUS PLEUROPNEUMONIAE by Tesfaye Belay A THESIS submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1989 aoolgfib ABSTRACT DEVELOPMENT OF AN ELISA FOR THE EVALUATION OF SERUM IMMUNE RESPONSES IN PIGS EXPOSED TO ACTINOBACILLUS PLEUROPNEUMONIAE by Tesfaye Belay The purpose of this thesis research was to analyze the immune responses of pigs to Actinobacillus (Haemcphilus) pleuropneumoniae (App), the causative agent of contagious porcine pleuropneumonia. To detect and quantitate antibodies to App, a: sensitive and reproducible enzyme-linked immunosorbent assay (ELISA) procedure was developed using App outer membranes as the antigens. The ELISA then was used to measure serum antibodies to App serotypes 1 and 5 in vaccinated, infected, and unexposed pigs. It was found that 1) the magnitude of the immune responses of animals experimentally infected with either App serotype was dose dependent, and that these animals raised significant ELISA antibody titers against both the challenge serotype and heterologous serotype; 2) animals vaccinated with either an App serotype 5 outer membrane vaccine or a commercial bacterin also raised high antibody responses, which were protective against subsequent challenge with either App serotype 5 or 1; and 3) piglets born of an immune sow had significant serum antibody titers to App at birth, which declined continuously to background levels by 5 to 7 weeks after birth. DEDICATED TO MY PARENTS ii ACKNOWLEDGEMENTS I wish to express my appreciation.to my major advisor, Dr. Martha H Mulks, for her guidance in my research and the writing of the thesis. Thanks are due to Dr. Brad Thacker for his assistance in animal studies and statistical analysis of my data. I also extend my thanks to my other committee members, Drs. Robert Brubaker, Walter Esselman and Ronald Patterson for their advice in my study. I wish to express my appreciation for the assistantship awarded to me from the College of Osteopathic Medicine and World Health Organization that made it possible for me to continue my studies at Michigan State University. iii Table of contents List of tables .............. . ......................... vi List of figures ...... .. ......... ........ .vii Introduction .......... . ............. 1 Literature review H .......... . ........................ 2 Chapter I Development of an ELISA procedure ........ . ............. 13 Materials and methods .................................. 14 Results ................................................. 18 Discussion ...... . ...... . .................. ........27 Chapter _I_I Evaluation of immune responses of App vaccinated pigs...32 Materials and methods 33 Experimental design ..................................... 33 Results ..... . .............. . ........ . .......... 36 Discussion ......... . ............. ...51 Chapter III Evaluation of immune responses of App serotypes infected pigs................. ...... .. ..... . ...... ......58 Materials and methods.. ............................. ....58 Experimental design.. ................................... 58 Results ............ . .......................... 60 Discussion.. ......... ... ............................... .72 Chapter fl Evaluation of passive immunity in piglets of a serotype 5 infected sow77 iv Materials and methods . ....... ............ 77 Experimental design. .................................... 77 Results......... ......................... . ..... .... ..... 78 Discussion................................ .............. 82 Conclusion ............................................. 86 Bibliography..... ....................................... 91 LIST OF TABLES Chapter I Page Table 1. Comparison of ELISA and complement fixation (CF) App 6-week serotiters of pigs from SPF and non-SPF herdSOOOOO......OOOOOOOOOOO ...... O ...... 24 Chapter I_ Table 2. Comparison of ELISA and complement fixation (CF) App serotiters in pigs six-weeks after vaccination...... 38 3. Comparison of serotiters of pigs from 3 vaccination trials vaccinated with a commercial bacterin.........40 4. ELISA serotiters and protection against experimental challenge in pigs immunized with purified outer membranes of App serotype 5 in Freunds' incomplete adjuvant..................................49 5. ELISA serotiters and protection after experimental challenge in pigs vaccinated with a commercial whole cell bacterin App serotypes 1, 5 and 7 ......... 50 Chapter III Table 6. Clinical and hematological outcomes of experimental infection of SPF pigs with various dosages of App serotype 5 ...... .. ....... 63 7. Complement fixation serotiters six weeks after experimental infection of SPF pigs with various dosages Of serotype SOOOOOOOOOOOO......OOOOOOOO0.0.0.64 8. ELISA and CF serotiters after 6 weeks of experimental infection of pigs with various dosages of App serotype 1.............. ..... . ................... 69 Chapter IX Table 9. Comparison of ELISA and complement fixation serum titers of piglets farrowed from a surviving sow.” ..... u. ........ . .................. 80 vi 10. Comparison of ELISA titers against App serotype 5 or serotype 1 and complement fixation titers in exposed pigSe .........OOOOOOOOOOOOOOOOOO....... 00000 90 vii List of figures Page Chapter I a Figure 1 Mean optical densities i 1 standard deviation versus serum dilutions of positive and negative control sera against serotype 1 outer membranes ...... 25 2 Mean optical densities : standard deviation versus serum dilutions of positive and negative control sera assayed against serotype 5 outer membranes.................................... ........ 26 Chapter II Figure 3 Serum immune responses of App serotype 5 outer membrane subunit vaccinated SPF pigs against serotypes 1 and 5 outer membranes. n.u.n. ....... .44 4 Serum immune responses of commercial vaccine immunized SPF pigs against serotypes 1 and 5 outer membrane antigensu”.n.u.n.u.n.u.n45 5 Serum immune responses of commercial vaccine immunized non-SPF pigs against serotypes 1 and 5 outer membrane antigens.u.n.n. ........ ”.u46 Chapter III Figure 6 Serum immune responses of various doses of serotype 5 infected pigs to serotype 5 outer membrane antigenSe ............OOOOOOOOOOOOIOO ..... .0 ......... 65 7 Serum immune responses of various doses of serotype 5 infected pigs to serotype 1 outer membrane antigens.............. .......... . ..... 66 8 Serum immune response of an App serotype 1 infected Lug against serotype 1 and serotype 5 outer membrane antigens. Samples assayed in triplicates and repeated 3 times on different days ............ ....70 viii 9 Serum immune responses of an App serotype 1 infected pig against serotype 1 and serotype 5 outer membrane antigens. Samples assayed in triplicates and repeated 3 times on different days..... ....................... 71 Chapter IX Figure 10 Passive immunity of piglets against serotypes 1 and 5 outer membrane antigens. ..... 81 ix INTRODUCTION Porcine contagious pleuropneumonia is a respiratory disease of swine caused by Actinobacillus pleuropneumoniae (App) (1,2). This disease is economically important in pig raising countries around the world '(1,2,3,4). The major economic losses are due to high mortality rate, deteriorated growth rate of infected.pigs and increased veterinary fees (4,5). Although several studies have been conducted to develop better methods for the prevention and control of the disease, there are no completely effective vaccines. Various serological tests have been developed for App serotyping (e4; co-agglutination) and/or detection of App antibodies (complement fixation). The cell surface components of App that contribute to induction of protective immunity in pigs have not been completely defined although circulating antibodies to capsule, lipopolysaccharides (LPS), outer membrane proteins (OMPs) and hemolysins have been detected. The purpose of this investigation was to develop a reproducible and sensitive enzyme-linked immunosorbent assay (ELISA) procedure and to utilize this ELISA to analyze the serum immune responses of infected pigs, vaccinated pigs, and passively immune piglets. LITERATURE REVIEW Actinobacillus pleuropneumoniae (App), previously known as Haemophilus pleuropneumoniae (pr) is a gram negative, nonmotile, nonspore forming, frequently encapsulated pleomorphic rod, with predominantly coccobacillary forms. It grows on enriched culture media, requiring V factor (nicotinamide adenine dinucleotide, NAD) but not X factor (hemin). On sheep blood agar, colonies usually produce a zone of B-hemolysis (6). Recently,the genus name Haemophilus was replaced by Actinobacillus based on data demonstrating that isolates share greater DNA homology, outer membrane protein profiles on gel electrophoresis and morphological and biochemical relatedness with members of the genus Actinobacillus than members of the genus Haemophilus (7AM. Porcine pleuropneumonia can be either acute fibrino- hemorrhagic or chronic localized necrotizing pneumonia with pleuritis in natural or experimental pig infections (1,2). Although all ages may be affected, pigs about 3 to 5 months of age are the most vulnerable (3,9). The spread of the disease into a new herd is by importation of acutely infected pigs or asymptomatic App carriers or chronically infected pigs (1-3,9). The disease is transmitted via respiratory routes by direct contact from pig to pig'(1,2,8).1High fever, severe respiratory distress, coughing and anorexia are some of the clinical symptoms of porcine pleuropneumonia.(2,8). 3 Mortality varies greatly (0.4 to 100%) depending on the immune status of the herd. Morbidity is also high (10 to 100%) as measured by a decrease in growth rate and decreased body weight gain of pigs from birth to market or slaughter stage (3-5). Recovery from clinical disease has been enhanced by treatment with antibiotics (3,9) although antibiotic resistant strains have been isolated (10-11). Serotyping and antigen characterization 9; App: At present 12 serotypes have been described based upon capsular antigenic serotype-specificity (12-20). Nontypeable strains have also been reported.(5p20L.Early studies on serotyping described serotype-specific antigens to be capsule, LPS or both (14-18 ). More recently, Inzana et al.(21), have demonstrated that the capsular polymer is the serotype- specific antigen for serotype 5. From cross reactions between serotypes, it has been concluded that serotypes of App possess type specific and common species specific antigenic determinants on their cell surface antigens (12,20). Heterogeneity within a single serotype has also been described (12).‘Tests that have been used to determine the serotype include tube agglutination (13) slide agglutination (13,16 13), coagglutination (19,22), «counterimmuno- electrophoresis, immunodiffusion (14,21,23), direct immunofluorescence and indirect fluorescence (13,23,24) and complement fixation (14,25). The heterogeneity within a 4 species and cross-reactions between serotypes are a problem for both serodiagnosis and vaccination and thus require further development of specific and sensitive tests. The pattern of geographic distributions of App serotypes varies around the world (3,13,26). Serotypes 1, 5 and 7 are common in the United States.(13). Serotypes 2,3,4 and 9 are found in Canada while serotype 2 is common in Western European countries (3,12,16,24,26). There are also App isolate reports from Japan, Australia and Taiwan (26). A Western blot procedure has been used in our laboratory to detect porcine antibody to App outer membrane proteins (OMPs), LPS and capsular polysaccharide antigens. We have identified major OMP antigens with estimated molecular weights of 16-18 kDa, 28-29 kDa, 39-43 kDa, 40-42 kDa, 66 kDa and 97 kDa by comparison with standards of known molecular weights (BioRad) separated by SDS-PAGE and stained with Coomassie blue-silver (27). Similar findings were shown by Rapp et a1.(28,29). High molecular weight capsular polysaccharides, rough type LPS (12-14 kDa) in all serotypes and laddered smooth type LPS in serotype 4 and 7, have also been identified. There are differences in the 0MP and LPS profiles between serotypes but, in general, little variation is seen among isolates of the same serotype (30). ggphpggpgsIgi The pathogenesis of porcine pleuropneumonia has not been fully elucidated, particularly the conditions allowing for the proliferation of the organism 5 in the lower respiratory tract and development.of’clinical diseases. Many stress situations such as transportation, extreme weather conditions and possibly bacterial and viral respiratory infections may be significant in allowing App to escape the natural defenses of the upper respiratory tract of a pig and invade the lung (3,8). Rapid multiplication of encapsulated bacteria and release of toxic products, including endotoxin, cause alveolar and vascular damage followed by death of alveolar macrophages and neutrophils facilitating the lesion development (31,32). In the acute clinical form of porcine pleuropnuemonia, the lesions are histologically characterized by hemorrhagic necrosis, exudation of fibrin, infiltration of an unidentified population of degenerated mononuclear cells and thrombosis of pulmonary vessels (2,32-34). In chronic infection, severe clinical disease is not a common phenomenon but pigs show reduced appetite, decreased growth and chronic cough (1,2). Pathological studies have indicated that there is fibrinous pleuritis and necrosis with variable size and distribution in lungs of chronically App infected pigs (1,2). Virulence factors: Capsule, LPS and.hemolysins and/or cytotoxins are the known virulence factors involved in App infection (1,31-45). Hemolysins: Three types of extracellular hemolysins have been described in App serotypes that are responsible for the necrosis feature of chronic and acute porcine contagious 6 pleuropnuemonia (34-40,43-45). It has been shown that App serotype 2 broth cultures contain an extracelullar hemolysin known as a heat-stable carbohydrate with hemolytic activity, an anti-phagocytic, and a cytotoxic substance to swine macrophage and neutrophil cells (36-40). In contrast, other studies have suggested that bacteria free-culture supernatants of App serotypes contain heat-labiLe extracellular protein hemolysins and has been shown that they are cytotoxic to neutrophils and contribute to extensive necrosis seen in pulmonary lesions of diseased pigs (34,35, 43-46). Recently, a 105 kDa Ca2+induced protein extracellular hemolysin and a 27 kDa cohemolysin protein have been identified in several App serotypes (43-45). Furthermore, the 12 App serotypes have been categorized into groups depending on requirement of Ca2+ for hemolysin biosynthesis and/or activity (45). Capsule: It has been shown that capsule is required for colonization of App in the host. Its role is most likely to protect bacterial cells from bactericidal action of complement and antibody thus allowing proliferation during initial phases of infection (21,41,42). Experimental animals intratracheally inoculated with capsular materials did not develop pulmonary lesions. Therefore, it is suggested that capsule is not responsible for lesion development in the lungs of App infected pigs (33, 41,42). Endotoxin/LPS: There have been eeveral studies to 7 determine:the role of endotoxin in the pathogenesis of App infection ( 31-35, 41). Rough LPS extracted from App serotype 5, for example, induced lesions of typical acute App infections in pigs; smooth LPS also induced lesions, although to a lesser degree; suggesting involvement of LPS in pathogenesis (41). App appears to produce endotoxin like other gram-negative bacteria and its endotoxin is partially responsible for the massive inflammatory edema, congestion of alveolar capillaries and blood vessels and intravascular fibrinous thrombosis in the lungs of infected pigs (l,3,21,26,33,4l). In most studies of pathogenesis so far carried out, there is no evidence of the involvement of adhesins in attachment to respiratory mucosal cells in App infection. Recently, however, pili-like structures in App, Itmultocida and.Ihhaemolypica have been suggested by Carlos Pijoan ( International Conference on the Haemophilus, Actinobacillus, Pasteurella group of organisms, Guelph, Canada 1989:18). Involvement of outer membrane proteins of App in pathogenesis has not been reported but it was recently shown that outer membrane proteins of 76 and 105 kda were expressed in iron-restricted App growth conditions (47). The function of these proteins is speculated as receptors of iron-carrying compounds in App serotypes (47). Comparative studies in pathogenesis (42,48,49 ) have suggested that different serotypes of App exhibit differences 8 in degree of virulence when injected into the lower airway of pigs. This could be due to differences in capsular and lipopolysaccharide chemical composition. The difference in virulence among App of serotypes 1, 5 and 7 has been shown to be low, but serotype 3 is less virulent than.1.(48).‘The lack of virulence of some serotypes and/or strains is probably due to insufficient capsular material to avoid destruction or removal in the respiratory tract by host defense mechanism (26,41-42,48-49). The heat-labile protein hemolysins (105 and 27 kDas), the heat-stable carbohydrate hemolysin or cytotoxins, and LPS may have a major role in damaging tissue cells directly or as a result of damage to macrophages and polymorphonuclear cells. Damaged or killed phagocytic cells may release their toxic contents to the environment, causing severe damage to tissues that can lead to intensive lesion development. These, therefore, appear to be necessary in the development of lesions in App infection. The capsule and possibly the outer membrane proteins are probably necessary for the establishment of infection in pigs. Immunity and control: What constitutes a protective immune response to App infection has not been well defined, although protective immunity can occur after naturally or experimentally induced App infections as well as in vaccinated animals (50-52). Studies have shown that animals which have been infected with App either naturally or by 9 experimental intranasal or intratracheal inoculations with live organisms, are in general immune to further infections with organisms of the same serotype and are at least partially resistant to challenge with organisms of heterologous serotypes (30,53). In contrast, immunization with formalin-killed organisms usually induces only partial protection against challenge with the homologous serotype, and no significant protection against heterologous serotypes is observed (53). The results of several experiments strongly suggest that the immune response of animals to parenteral vaccination is different from the response seen after infection (30,53,54L. The important part of the defense mechanism against App infection could be a local barrier preventing the agent from penetrating the respiratory mucosa. During infection antibodies against deep-seated common antigens of most serotypes may be produced that increase resistance to reinfection. By parenteral vaccination little local antibody response may be elicited or it may stimulate anticapsular antibody production to protect against homologous but not heterologous infection. Evaluation of secretory immune responses in pigs is also underway in our laboratory to understand immunity in resistance to App infection. The transfer of immunoglobulins across the placenta of a pig to its fetus is not a common process (56). However, it has been shown that antibodies to App are present in sera of 10 piglets (57, 58). The passive transfer of immunoglobulins in piglets occurs by absorption of colostral immunoglobulins at birth (57). Transfer of humoral antibodies from sows to piglets may protect them against infection of App during the first weeks of age because it was shown that mortality and morbidity rates in colostrum-deprived piglets were almost 100% after four days from birth, but these rates were low among suckled piglets from immune sows (57y. The protective efficacy of anticapsular antibodies has been investigated in pigs and mice by Rosendal et.a1.(59). After immunization of pigs with two different capsule extracts as vaccines, partial protection and reduced mortality were observed during a subsequent challenge with a homologous serotype of App. It was also demonstrated that passively administered specific monoclonal anticapsular antibodies prevented pigs from death due to induced App infection (60). Complete protection against infection, however, has not been provided by antibody to capsule (21,59,60). Fenwick, et a1.(61) investigated the potential protective effects of immunity against common lipopolysaccharide core antigens of gram negative bacteria by inducing App infections in pigs which had received a vaccine of an Rc mutant of E; gpII 35. This Rc mutant IIEQII contains a mutation in UDP-galactose epimerase which makes it unable to incorporate exogenous galactose into LPS resulting 11 in exposure of the LPS core (61-63). There were increased titers to I; gpII J5 after vaccination and protection against App re-infection in pigs suggesting the exposure of cross- reacting immunodeterminants of App. In another study, immunogenicity of oligosaccharides of App serotype 5 was improved in experimental animals by conjugation of its LPS to tetanus toxoids (62). Neutralizing antibodies to hemolysin have been demonstrated in the serum of both pigs and rabbits immunized with concentrated culture supernatant (hemolysin) as well as in the sera of pigs that have survived natural infection with App serotype 1 (35, 40). Protection studies by using hemolysins as vaccine components have been developed in our laboratory. Our data in these vaccination trials showed that bacterin vaccines containing hemolysin were more effective than similar vaccines without hemolysin ( Mulks, unpublished data). Several studies have shown that variations in degree of protection are obtained with different vaccines depending on the quality of vaccine (age of culture for vaccine), route of immunization, type of adjuvant incorporated, dose of vaccine and other factors (50-52,55,59,62-64).IELis suggested that vaccination with bacterins prevents the acute form of porcine pleuropneumonia and improves the rate of weight gain, and decreases the magnitude of pleuritis at slaughter of chronically infected pigs (50-52). Other means of controlling 12 outbreaks of porcine pleuropneumonia include ventilation of facilities in which animals are housed, minimization of temperature changes, avoidance of overcrowding of pigs in barns, avoidance of introduction of sera-positive pigs to a new herd, elimination of infected animals from the herd and antibiotic therapy (3,4,65). Despite the many reports and studies conducted on App, porcine pleuropneumonia continues to cause economic losses for the pig industry; A more complete understanding of the pathogenesis of App infection, especially the role of specific virulence factors: a more effective, cross-reactive vaccine: and a sensitive and specific serodiagnostic test that permits rapid evaluation of immune status and identification of asymptomatic animals are all needed in order to prevent and control the disease. CHAPTERI DEVELOPMENT OF AN ELISA PROCEDURE ELISA" a widely used type of serological assay, is beginning to be used for the detection of antibodies in the serum of swine infected or vaccinated with App (21,25,61,66). Several different antigens, including capsular polysaccharide, LPS, and whole cells, have been tried. Our procedure used purified outer membranes of App. To develop a sensitive and reproducible ELISA assay, we tested and standardized several variables to optimize ELISA reactions and minimize background and nonspecific reactions and define titer within the context of our particular ELISA assay. Variables tested included a) selection of solid phase by comparison of different plates b) comparison of coating buffers, coating procedure and optimum antigen concentration for coating. c) antiserum dilutions d) choice of conjugated enzyme-substrate system e) temperature and incubation times. 13 MATERIAIS AND METHODS Bacterial Strains: App strain No.178, serotype 5, received from V. Rapp, Iowa State University, and strain No.27088, serotype 1, from the American Type Culture Collection, were used throughout the serological investigation. Chemicals and media: Brain Heart Infusion was bought from Difco Laboratories, Detroit, Mi. Bovine serum albumin (BSA), Tween 20, NAD, DTT, lysosyme, sucrose, Trizma base, and Sodium phosphate salts were obtained from Sigma Chemical Company, ST. Louis, MO. Oxalic acid, sodium carbonate, sodium bicarbonate, and sodium chloride were purchased from J. T. Baker Inc. Pillisburg, N.J. Serum preparation : Blood from experimental pigs for serum preparation was obtained by jugular vein puncture using the vacutainer system. Blood was allowed to clot at room temperature for one hour. The serum was harvested by centrifugation and stored at -20°C until used. Complement fixation Test: Complement Fixation antibody titers were determined at the Iowa Veternary Diagnostic Laboratory, formalin-killed whole cells of App serotypes of 1, 5 and 7 as antigen was used.(67). The maximum complement fixation titer measured is 1:128. Titers less than 1:8 are considered insignificant. Statistical Analysis: The significance of differences in serum antibody titers between experimental groups were analyzed by Students't-test or Least Significant Difference 14 15 (LSD), a multiple comparison of variance with a multicomputer program, StatistixR(NH Analytical Software, St. Paul, Minnesota). For statistical analysis, reciprocal ELISA titers were transformed as follows: <60 = 0, 60 = 1, 120 = 2, 240 = 3, 480 =3 4, 960 = 5, 1920 8 6, 3840 = 7, 7680 =3 8. The actual geometric mean ELISA titers shown in Tables were calculated from the arithmetic mean of transformed titers by the following relationship: Arithmetic mean titer is multiplied by logarithm of 2, then the inverse logarithm of the product is multiplied.by:HL The same formula was used for the standard deviation calculation. Preparation of outer membranes by sucrose gradient. We used a modified Osborn and Munson method to separate inner and outer membranes of App Serotypes (68). Cultures of both strains, within two subcultures of isolation from pigs, were grown in Brain Heart Infusion Broth (BHI) supplemented with NAD (long/per ml) overnight at 37°C lhl a water bath shaker. Cells were harvested by centrifugation at 13400 X g for 15 minutes at 4°C and resuspended in 40 m1 of phosphate buffer (.033M pH 7.00), and centrifuged at 13400 x g for 15 minutes at 4°C. The cell pellets were rapidly resuspended in a 10 ml cold solution of (.75M sucrose - 0.1M Tris-acetate (pH 7.8)-0.2mM DTT), and freshly prepared cold lysosyme (150 ug/ml of cell suspension) was added. After 5 minutes at room temperature, conversion to spheroplast form was completed by slowly adding 2 volumes of 5mM EDTA ( pH 7.5) - 0.2mM DTT. Cell suspensions were centrifuged at 16000 X g for 15 minutes, and the pellet was resuspended in 2.5ml of .25M sucrose - .OIM Tris-acetate ( pH 7.8) 5mM EDTA ( pH 7.5) - 0.2mM DTT and sonicated on ice until the suspensions were translucent.‘The sonicated cell suspensions were centrifuged for one hour at 140,000 X g in a type 70.1'Ti rotor to pellet the membranes. Sucrose solutions of 55%, 45% and 40% by w/w in 5mM EDTA, pH 7.5, were layered in 12ml ultraclear tubes and 10-15 drops (.about 0.5ml) of membrane suspension was layered on top of the gradient. Centrifugation was carried out at 140,000 X g for 20-24 hours 16 17 in a type SW-41 rotor. The outer membrane bands were collected with a pasteur pipette from the 45-55% sucrose interface. Protein concentration was determined by the Bio Rad protein assay procedure (69). The outer membrane preparations were assayed for quality control by standard immunoblotting against strongly positive pig sera. Preparations were aliquoted and stored at -20°C until used for ELISA plate coating as antigens. RESULTS 1. Optimization of ELISA: Development of the ELISA procedure was based on the methods described by Engvall and Perlman (70). a) Solid phase: Microtiter plates from several different sources were tested as solid phase for binding App outer membrane antigens. All of the plates showed reproducible results when assayed against ascertained positive and negative pig sera. The polystyrene microtiter Gibco plate (Gibco Laboratories, Grand Island, NAL) was selected for further use because of its excellent binding capacity for antigen and minimum non-specific binding, as well as its availability and low cost. b0 Coating conditions and antigen concentration: Four different buffer solutions were tested as antigen diluents for coating the plates. These were 1) phosphate buffered saline (PBS), (.1M sodium phosphate + .15M NaCl, pH 7.5); 2) carbonate buffer (0.5M sodium carbonate, pH 9.6); 3) Tris- buffered saline (TBS) (.02M Tris-acetate, pH 7.5, plus 0.5M NaCl): and 4) neutral phosphate; ( .1M sodium phosphate, pH 7.0). {Among the 4 buffers tested, the commonly used carbonate buffer was selected for further use. Concentrations-of outer membrane antigens ranging from 0.01 to 2.00 ug protein per well in carbonate buffer were tested by checkerboard titration for ELISA plate coating. Optimal antigen coating level per well was determined to be 1 18 19 ug protein in 100 ul coating buffer per well. Incubation of plates overnight at 37°C was found to be a more consistent method of coating antigens than incubation at 4°C. After coating, plates were washed twice with Tris-buffered saline plus 0.05% Tween 20 (TTBS), then wells were blocked with 3% BSA in TBS for 1 hour at room temperature. Plates were again washed 2 times with TTBS, dried throughly at 37°C for 3 hours, wrapped and stored until used. Coated and blocked plates wrapped with aluminium foil can be stored at -20°C for one month. There was no loss of activity when compared to fresh, one day old and one week old plates. On the other hand, plates coated by incubation at 4° C gave variable optical density (OD) values when assayed after two days or 2 weeks. Outer membrane preparations could be stored for up to six months at -20° C. c) Serum dilutions: Normal saline was used as a serum diluent in our experiments. Positive and negative control sera at 1:25 or 1:60 as an initial dilution, each with a series of eight two-fold dilutions, were examined. With the 1:25 to 1:3200 dilution series, we found inconsistent OD values at the higher dilutions. The use of this serum dilution series also created a problem of rapid substrate color development and high OD values exceeding the maximum limits of the programed ELISA reading at 405. The 1:60 to 17680 two-fold dilution series, however, was found to give consistent results. There was a linear decrease in OD values 20 with the dilution series, and the threshhold value,(oo mononwoo m canon soaonoso mcocawd CD CD CD I l SERUM TITER a C: 03% V IMO I Olommmojpm m ellemmmoitm ._ we I o l i I. l .- l l I l Emmxm nHoaHo mu monaa Haaaao Homooauoo on ooaaonoHoH lI>00230H omso I .0 $00 I H\_/W W NV Ntwo I t/w w/p 6 0.5 I LNOT > b b b > we I o I i u I i J. o 3 m o ... m m 5mM—Am nHoano m" monaa Hasaao nomocauou on I>OOZIZMOF umeo .. Am 0 .. N O/W moo I O H q/w//L llllllllll Av NHmo I a\\% H “/u I. D D DIIIIIIIIW . 0.5 I a a a LNG? > > ... > b we I o n I u I I ._ O L N u e. m m Emmxm mHoano q" monas Haaaao nouocauom on