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CRWAD clinical immunology paper award
Veterinary Immunology and Immunopathology, 21 ( 1989 ) 379-380 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
379
Announcements CRWAD CLINICAL IMMUNOLOGY PAPER AWARD The award of US $100.- for the best clinical immunology paper presented during the 69th Annual Meeting of the Conference of Research Workers in Animal Diseases (CRWAD) in Chicago, given jointly by the American Association of Veterinary Immunologists and Elsevier, has been won by Dr. B. Shankarappa. An abstract of his paper is given below.
ABSTRACT
Monoclonal Antibody-Mediated Competitive Enzyme-Linked Immunosorbent Assay for Potomac Horse Fever BASAVARAJU SHANKARAPPA, SUKANTA K. DUTTA and BONNIE MATTINGLY
Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, MD 20742 (U.S.A.) Potomac horse fever, caused by Ehrlichia risticii, has assumed importance in view of extensive serological prevalence in north America, the threat of morbidity, and the restrictions on horse movements. Due to marked variations in the presentation and severity of clinical signs and the lack of distinction between other febrile and diarrheal diseases, serological diagnosis was the logical sequel. Indirect immunofluorescence test (IFA) (Ristic et al., 1986) suffered from a lack of sensitivity and highly subjective nature of evaluation. Enzyme-linked immunosorbent assay (ELISA) (Dutta et al., 1987; Pretzman et al., 1987), due to high background reactivity, necessitated quantitation of color in making definitive diagnosis of low reactivities. These limitations of the above methods dictated confirmation of the disease in border-line cases by seroconversion. We have utilized the specificity and sensitivity of monoclonal antibody, HybI (Shankarappa and Dutta, 1988), in developing a competitive ELISA (CELISA) with a diagnostic value (Shankarappa et al., 1989 ). The procedure involved incubating the mixture of test sera and monoclonal antibody with the antigen, followed by probing for the bound monoclonal antibody. The inhibition of binding of HybI was apparently dependent on the antibody titer. The ratio between the optical density of negative and positive sera peaked at a dilution of 1 : 100 and 1 : 10 000 of sera and HybI, respectively. Under optimum conditions for differentiating the positive from negative samples, the mean optical density of 30 experimental horse sera was 0.855 in the pre-infection samples as compared to 0.158 at 3 weeks post-infection. Analysis of sequential sera demonstrated the capability to detect horse antibodies at 14 days or earlier following the infection, which is the approximate time-frame the clinical signs of the disease become evident. The presence of inhibitioncould be demonstrated till 900 days post-infection. However, a better estimate of the sensitivity was demonstrated when CELISA could conclusively detect the presence of antibodies in 8 of the 11 acute-phase samples of field sera, which were negative by IFA, and were confirmed to be cases of Potomac horse fever by virtue of seroconversion in the convalescent-phase samples. Five of these samples were barely positive by ELISA. A marked tendency of positive and negative CELISA values to cluster separately was an added advantage in conclusive inferences. Although negative values were spread out, the positive values clustered closely and there was a region of demarcation, facilitating grouping of samples into positive and non-positive. Validation for the usage of CEL-
379
Announcements CRWAD CLINICAL IMMUNOLOGY PAPER AWARD The award of US $100.- for the best clinical immunology paper presented during the 69th Annual Meeting of the Conference of Research Workers in Animal Diseases (CRWAD) in Chicago, given jointly by the American Association of Veterinary Immunologists and Elsevier, has been won by Dr. B. Shankarappa. An abstract of his paper is given below.
ABSTRACT
Monoclonal Antibody-Mediated Competitive Enzyme-Linked Immunosorbent Assay for Potomac Horse Fever BASAVARAJU SHANKARAPPA, SUKANTA K. DUTTA and BONNIE MATTINGLY
Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, MD 20742 (U.S.A.) Potomac horse fever, caused by Ehrlichia risticii, has assumed importance in view of extensive serological prevalence in north America, the threat of morbidity, and the restrictions on horse movements. Due to marked variations in the presentation and severity of clinical signs and the lack of distinction between other febrile and diarrheal diseases, serological diagnosis was the logical sequel. Indirect immunofluorescence test (IFA) (Ristic et al., 1986) suffered from a lack of sensitivity and highly subjective nature of evaluation. Enzyme-linked immunosorbent assay (ELISA) (Dutta et al., 1987; Pretzman et al., 1987), due to high background reactivity, necessitated quantitation of color in making definitive diagnosis of low reactivities. These limitations of the above methods dictated confirmation of the disease in border-line cases by seroconversion. We have utilized the specificity and sensitivity of monoclonal antibody, HybI (Shankarappa and Dutta, 1988), in developing a competitive ELISA (CELISA) with a diagnostic value (Shankarappa et al., 1989 ). The procedure involved incubating the mixture of test sera and monoclonal antibody with the antigen, followed by probing for the bound monoclonal antibody. The inhibition of binding of HybI was apparently dependent on the antibody titer. The ratio between the optical density of negative and positive sera peaked at a dilution of 1 : 100 and 1 : 10 000 of sera and HybI, respectively. Under optimum conditions for differentiating the positive from negative samples, the mean optical density of 30 experimental horse sera was 0.855 in the pre-infection samples as compared to 0.158 at 3 weeks post-infection. Analysis of sequential sera demonstrated the capability to detect horse antibodies at 14 days or earlier following the infection, which is the approximate time-frame the clinical signs of the disease become evident. The presence of inhibitioncould be demonstrated till 900 days post-infection. However, a better estimate of the sensitivity was demonstrated when CELISA could conclusively detect the presence of antibodies in 8 of the 11 acute-phase samples of field sera, which were negative by IFA, and were confirmed to be cases of Potomac horse fever by virtue of seroconversion in the convalescent-phase samples. Five of these samples were barely positive by ELISA. A marked tendency of positive and negative CELISA values to cluster separately was an added advantage in conclusive inferences. Although negative values were spread out, the positive values clustered closely and there was a region of demarcation, facilitating grouping of samples into positive and non-positive. Validation for the usage of CEL-