Introduction. The main approach to the rabies prevention is the vaccination of domestic and wild carnivores. For the routine evaluation the anti-rabies vaccination effectiveness, World Organization for Animal Health (OIE) recommends various enzyme-linked immunosorbent assays (ELISA).The aim of the study was to design and validate a competitive ELISA (cELISA) test system for the detection of antibodies to the rabies virus (RABV).Materials and methods. The development of the cELISA was carried out following the OIE recommendations.Results. The repeatability of the cELISA results within one laboratory was satisfactory (coefficient of variation 7.95–13.61%). The coefficient of determination (CD) between the results of the virus neutralization reaction (FAVN) and cELISA was 0.988, p < 0.001. The lower threshold for antibody detection was less than 0.02 IU/ml. The cELISA did not demonstrate cross-reactivity against antibodies to canine distemper virus, parainfluenza virus, parvovirus, coronavirus, and canine adenovirus (types I and II). During the study of 137 dog blood sera, diagnostic specificity (DSp) and diagnostic sensitivity (DSe) for the cELISA were 83.1% and 94.9%, respectively, and CD between the cELISA and FAVN results was 0.968, p < 0.001.Discussion. Indirect ELISA test systems for determining the level of antibodies to RABV are not sensitive enough compared to reference tests, unlike cELISA. The developed test system is not inferior for its DSp and DSe to the commercial cELISA BioPro ELISA Rabies Ab (DSp 66.7%, DSe 94.4%).Conclusion. The developed cELISA test system can be used to detect antibodies to RABV in the blood serum of dogs for evaluating the effectiveness of mass vaccination programs.
This article presents the results of a study of the effectiveness of anti-rabies and anti-chlamydia antibodies conjugated with fluorescein, freeze-dried. Immunobiological products lyophilization is one of the most reliable ways to preserve their physicochemical and biological properties, since this is a soft drying method, in which the dried product is frozen, then it is placed in a vacuum chamber, where the solvent is sublimated. Lyophilization allows you to get drugs without losing their shape, structural integrity and more importantly, without losing their biological activity. The study aimed to standardize methods for the purification of FITC conjugates, as well as reagents used in the production and drying of FITC conjugates. We used conjugates of anti-rabies and anti-chlamydia antibodies labeled with fluoresceinisothiocyanate (FITZ) obtained by hyperimmunization of sheep and rams using an immunostimulant. We tested the ability of various concentrations of dextran T-70 to retain the specific activity of fluorescent conjugates of anti-rabies and anti-chlamydia antibodies when added dextran T-70 to them before the lyophilization. To determine the optimal amount of T-70 dextran filler, prepared 1%, 2%, 3%, 4%, and 5% solutions in 10mm phosphate-salt solution. We found that the effectiveness of dextran T-70 as a filler substance in the lyophilization of FITC conjugates depends on its concentration. The optimal stabilizing vehicle for lyophilization of both fluorescent conjugates is 2-3% concentration of dextran T-70 in 10 mM phosphate-buffered saline. Analysis of the results of the quality of FITZ-conjugates allows us to conclude about the high quality of biological products prepared during freeze-drying of the material in the developed temperature regime using dextran T-70 as a filler substance. As a result of the studies, the technology for the preparation of lyophilized FITC conjugates of anti-rabies and anti-chlamydia antibodies for diagnostic test systems was optimized. The specific activity and stability of the physicochemical characteristics of the preparations after lyophilization and during subsequent storage for two years were observed.
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