DEVELOPMENT OF IN-HOUSE ELISA AND ITS COMPARATIVE EVALUATION WITH RFFIT FOR ESTIMATION OF ANTI-RABIES VACCINAL ANTIBODIES IN DOGS
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Date
2017-10
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KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCIES UNIVERSITY, BIDAR
Abstract
The study was undertaken to develop in-house ELISA and it’s comparision with
RFFIT. The recombinant pETRVL-G plasmid having G gene of Dr. Largi’s strain of
rabies virus was cloned into pFastBacTM1 vector in DH5α E.coli. The recombinant
pFastBacTM1 was confirmed by colony PCR and RE digestion (EcoR I & Sal I) and
transposed into DH10 Bac. Recombinant bacmid was selected based on blue-white
colonies and confirmed by PCR. Further, the DNA extracted and transformed into Sf-21
cells. The resultant recombinant baculovirus were used to obtain P2 and P3 stock virus
and P3 stock used for expression. The expressed recombinant rabies virus G protein was
analysed by SDS-PAGE, western blot and doublet bands of 62 and 59 kDa were
observed. The crude lysate of baculovirus infected Sf-21 cells was used in ELISA and
optimised to arrive at 500ng/100 µl of antigen, 1:100 dilution of serum and 1:15,000
dilution of anti-dog HRPO conjugate. The serum samples from vaccinated dogs (n=247)
were tested by RFFIT and in-house ELISA. By RFFIT, 212 serum samples were found to
have protective neutralising antibody titre accounting for 85.82 per cent protection. By
in-house ELISA , 199 serum samples were found reactive, showing per cent, positivity
(PP) more than 36.29 as cut off accounting for 80.56 per cent protection. The diagnostic
sensitivity and specificity of in-house ELISA was 90.56 and 80 per cent, respectively
with the kappa value of 0.614 revealing good agreement. Usage of purified recombinant
rabies virus G protein may be suitable for in-house ELISA in sero-monitoring of antirabies virus neutralising antibodies.