Detection of peste des petits ruminants virus antigen using immunofiltration and antigen-competition ELISA methods.
Peste des petits ruminants (PPR) is one of the most economically important diseases affecting sheep and goats in India. An immunofiltration-based test has been developed using either mono-specific serum/monoclonal antibodies (mAb) prepared against a recombinant truncated nucleocapsid protein of rinderpest virus (RPV) cross-reactive with PPR virus. This method consists of coating ocular swab eluate from suspected animals onto a nitrocellulose membrane housed in a plastic module, which is allowed to react with suitable dilutions of a mAb or a mono-specific polyclonal antibody. The antigen-antibody complex formed on the membrane is then detected by protein A-colloidal gold conjugate, which forms a pink colour. In the immunofiltration test, concordant results were obtained using either PPRV mAb or mono-specific serum. Another test, an antigen-competition ELISA which relies on the competition between plate-coated recombinant truncated 'N' protein of RPV and the PPRV 'N' protein present in ocular swab eluates (sample) for binding to the mono-specific antibody against N protein of RPV (in liquid phase) was developed. The cut-off value for this test was established using reverse transcription polymerase chain reaction (RT-PCR) positive and negative oculo-nasal swab samples. Linear correlation between percent inhibition (PI) values in antigen-competition ELISA and virus infectivity titres was 0.992. Comparison of the immunofiltration test with the antigen-competition ELISA yielded a sensitivity of 80% and specificity of 100%. These two tests can serve as a screening (immunofiltration) and confirmatory (antigen-competition ELISA) test, respectively, in the diagnosis of PPR in sheep or goats.
Raj
G Dhinakar
GD
Department of Animal Biotechnology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai, India. dhinakarraj@yahoo.com
Rajanathan
T M C
TM
Kumar
C Senthil
CS
Ramathilagam
G
G
Hiremath
Geetha
G
Shaila
M S
MS
D000818:Animals; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral / Q000097:blood; D000956:Antigens, Viral / Q000032:analysis* / Q000276:immunology; D002850:Chromatography, Gel / Q000379:methods / Q000662:veterinary; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary*; D015511:Goat Diseases / Q000175:diagnosis*; D006041:Goats; D009296:Nasal Cavity / Q000821:virology; D029021:Peste-des-Petits-Ruminants / Q000175:diagnosis / Q000662:veterinary*; D018107:Peste-des-petits-ruminants virus / Q000276:immunology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D012680:Sensitivity and Specificity; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis*
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral