Veterynarna biotehnologija – Veterinary biotechnology, 2016, 29, 196-204 [in Ukrainian].

NYCHYK S.A., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., POLUPAN I.M., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., MAZUR N.V., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., HOMENKO JA.V., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.

Institute of Veterinary Medicine of the NAAS

SPYRYDONOV V.G., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.

Ukrainian laboratory of quality and safety of agricultural products

SENSITIVITY AND SPECIFICITY OF ANTI-RABIES IMMUNOGLOBULINS OBTAINES FROM QUAIL EGGS YOLK ANTIBODY (Ig Y)

Introduction. Despite significant advances in medicine and biotechnology, rabies is a problem in many countries. One of the rabies issues is application of FAT (fluorescent antibody test) in laboratory diagnosis, which is considered to be the gold standard test. Prospective method of obtaining specific immunoglobulin for rabies diagnosis is isolating Ig Y from the bird’s eggs. The method of producing Ig Y antibody has some advantages over the production of antibodies from mammals because there is no need to bleed the animals, it is easy to purify a large amount of antibodies, and it is feasible to produce specific antibody to a small amount of antigen that is poorly immunogenic in mammalian hosts.

The goal of the work. The study of sensitivity and specificity of fluorescent rabies immunoglobulin (Ig Y) isolated from quail eggs.

Materials and methods. We used 4 six months old quails (m= 300 g) for immunization. 2 antigen samples: 52 G Wistar strain (vaccine IndiRab, India) and strain CVS-11 (obtained in the laboratory, the virus titer was 6.23±0.11 TCID₅₀ / 0.05 ml) strains for immunization. For the first immunization we used 0.5 ml of each sample and added equal volume of complete Freund's adjuvant (Thermo). Antigen preparations were injected intramuscularly. We repeated immunizations on the 10th and 20th day. Antigens were prepared similarly as for the first immunization, but adding an incomplete Freund’s adjuvant. For the determination of rabies virus specific antibodies titer we collected quail eggs (from 45 days after the first immunization), isolated yolk antibodies, which were tested in indirect ELISA. Virus neutralization activity was tested in mouse neutralization test (MNT). Sensitivity and specificity samples were performed in FAT. Simultaneously, we determined a working dilution of conjugates (1:2, 1:4, 1:8, 1:16).

Results of research and discussion. After the separation, purification and concentration two samples of Ig Y were obtained: sample #1 from G 52 Wistar strain in 30 mg/ml concentration, sample #2 from CVS-11 strain in 19.5 mg/ml concentration. We observed a positive correlation in ELISA between used antigen and antibody levels indicating specificity of antibodies to rabies virus. At the same time established the increase of antibody titer when used CVS-11 strain. We obtained similar results studying virus neutralization activity of these samples in MNT. Thus, sample 1 had specific potency at the level 26.25 IU/ml, and sample 2 – 81.25 2 IU/ml compared to standard of rabies immunoglobulin. Study in FAT of samples FITC-labeled Ig Y showed specificity of Ig Y antigen to rabies virus strain CVS-11 (working dilution of 1:2).

Conclusions and prospects for further research. Obtained two samples of Ig Y were obtained: sample #1 from G 52 Wistar strain in 30 mg/ml concentration, sample #2 from CVS-11 strain in 19.5 mg/ml concentration. In MNT and ELISA we observed a positive correlation between used antigen and antibody levels indicating the specificity of antibodies to rabies virus. Study of FITC-labeled Ig Y samples in FAT showed specificity of Ig Y antigen to rabies virus strain CVS-11 (working dilution of 1:2).

Keywords: rabies, strain, rabies globulin, Ig Y, ELISA, FITC.

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