Avidin-Biotin recombinant nucleoprotein competitive ELISA for the detection of peste des petits ruminants virus antibodies in sheep and goats.

The present study describes the development of a truncated recombinant peste des petits ruminants virus (PPRV) nucleoprotein (rPPRV-NPN) and its polyclonal antibodies-based immuno-diagnostic assay, Avidin-Biotin (AB) recombinant nucleoprotein competitive ELISA (ABrC-ELISA) for the detection of PPRV antibodies in the sheep and goats. The PPRV N-terminal immunogenic region (1-266 aa) of nucleoprotein (NPN) coding sequence was amplified and cloned into the pETite vector. The rPPRV-NPN with a molecular weight of ∼ 30 kDa was expressed in E. coli, purified, and characterized by SDS-PAGE and immunoblot using standard PPRV specific sera. The Ni-NTA affinity-purified rPPRV-NPN as coating antigen and its hyperimmune serum as competitive antibodies raised in guinea pigs were evaluated as diagnostic reagents in ABrC-ELISA using the known standard panel of sera. The threshold (cut-off) Percentage Inhibition (PI) value was determined as 45 (mean ± 3 SD) based on the reactivity of the known sheep and goats sera to PPRV antibodies [negative (n = 140) and positive (n = 98)] and the assay had a sensitivity of 97 % (95 % Confidence Interval (CI): 91.3-99.4 %) and specificity of 100 % (95 % CI: 97.4-100 %) with an excellent Area under curve (AUC) of 0.997 (95 % CI: 0.99-1.0). On evaluation of diagnostic performance of the assay using the sheep and goats sera (n = 391) from vaccinated, infected, and non-vaccinated animals, the ABrC-ELISA showed the relative diagnostic sensitivity of 95.88 % (95 % CI: 92.56-98.01 %) & 98.77 % (95 % CI: 96.43-99.74 %) and diagnostic specificity of 97.97 % (95 % CI: 94.19-99.58 %) & 90.54 % (95 % CI: 84.64-94.73 %) against indigenous PPR competitive ELISA kit & IDvet Screen® PPR Competition kit, respectively. The study showed that ABrC-ELISA is rapid, sensitive, and specific and can be a better alternative assay for the detection of the PPRV antibodies in the sera of small ruminants for serosurveillance / seromonitoring of PPR not only at the eradication and post-eradication phases in the disease-controlled endemic countries but also in the PPR non-endemic countries.

Balamurugan V ，Varghese B ，SowjanyaKumari S ，Vinod Kumar K ，Muthuchelvan D ，Nagalingam M ，Hemadri D ，Roy P ，Shome BR ... -  《-》

Comparative diagnostic efficacy of Avidin-Biotin recombinant nucleoprotein competitive ELISA for serosurveillance and monitoring of peste des petits ruminants in sheep and goats.

In this study extensive evaluation of Avidin-Biotin recombinant nucleoprotein competitive ELISA (ABrC-ELISA) was carried out by mass screening of a large number of sera to make use of this assay for serosurveillance and seromonitoring of peste des petits ruminants (PPR) in sheep and goats to evaluate its diagnostic efficacy value and strengthen findings associated with the assay. The recombinant PPR virus (PPRV) nucleoprotein was over-expressed in E. coli, Ni-NTA affinity-purified, and characterized and used as coating diagnostic antigen in ABrC-ELISA, and evaluated using the field sera from animals. On evaluation of the diagnostic performance or efficacy of this assay using the pre-vaccinated and post-vaccinated sera of sheep and goats (n = 1437), the ABrC-ELISA showed a relative diagnostic sensitivity of 87.2% (95% CI: 84.1-90%) and diagnostic specificity of 92.0% (95% CI: 90-93.7%), against well-established existing indigenous H protein-specific PPR competitive ELISA kit with an accuracy of 90.1% (95% CI: 88.5-91.7%) and good or substantial agreement of Cohen's Kappa value of 0.79 ± 0.017 SE (95% CI: 0.76 to 0.82). These findings suggest that the ABrC-ELISA is a potential additional diagnostic tool of a rapid, sensitive, and specific assay for the detection of the PPRV nucleoprotein antibodies in sera of sheep and goats. This PPR Ab Chek kit can be used extensively under field conditions for serosurveillance, and seromonitoring of PPR in sheep and goats at the eradication /post-eradication phase in disease-controlled countries or PPR non-enzootic countries.

Balamurugan V ，Bokade PP ，Kumar KV ，SowjanyaKumari S ，Nagalingam M ，Hemadri D ，Shome BR ... -  《-》

Avidin-Biotin recombinant antigen capture ELISA for the detection of peste des petits ruminants virus in the clinical specimens of sheep and goats.

This study describes the development of Avidin-Biotin recombinant Antigen Capture ELISA (ABrAC ELISA) for the detection of the peste des petits ruminants virus (PPRV) antigens in the clinical specimens of sheep and goats. The assay uses the truncated recombinant PPRV N-terminal immunogenic region of nucleoprotein (rPPRV-NPN) as a reference positive antigen and its polyclonal antibodies as capture/detective antibodies and the rabbit PPRV polyclonal antibodies as coating antibodies. The cut-off value was determined as double times the mean reactivity of blank control based on the reactivity of the PPR confirmed negative and positive control panel samples. On assessing the specificity with the related differential diagnosis of the disease-causing viruses and bacteria, the assay showed specific detective reactivity to PPRV. Further, on evaluation using clinical specimens (n-274) of sheep and goats, the assay showed that the relative diagnostic sensitivity of 86.49 % (95 % confidence interval (CI): 71.23-95.46 %) and diagnostic specificity of 96.20 % (95 % CI: 92.91-98.25 %) against PPRV nucleoprotein-specific monoclonal antibody-based sandwich-ELISA (PPR s-ELISA) kit, with an accuracy of 94.89 % (95 % CI: 91.58-97.18 %) and Cohen's Kappa value of 0.791 + 0.055 SE (95 % CI: 0.68-0.90) with substantial agreements. The ABrAC-ELISA is an alternative method of an immunoassay for the rapid, sensitive, and specific detection of the PPRV antigens m the clinical specimens of sheep and goats for surveillance or diagnosis of PPR. This study also shows that the rPPRV-NPN and its specific polyclonal antibodies could be the sustainable source of safe diagnostic reagents without the need to handle the infectious virus during the eradication and post-eradication phases in endemic countries like India or PPR non-endemic countries.

Balamurugan V ，Varghese B ，Sowjanya Kumari S ，Vinod Kumar K ，Muthuchelvan D ，Nagalingam M ，Roy P ... -  《-》

Potential diagnostic application of the baculovirus-expressed recombinant truncated nucleocapsid protein of peste des petits ruminants virus in ELISA.

The study describes the expression of recombinant truncated nucleocapsid protein (NP) of peste des petits ruminants (PPR) virus in the baculovirus system (PPRV-rBNP) and its potential application as a diagnostic antigen in ELISA for diagnosis of PPR in sheep and goats. The PPRV N-terminal immunogenic region (1-266 aa) of the NP coding sequence was amplified and cloned into the pFastBac HT A vector. The PPRV-rBNP with a molecular weight of ∼30 kDa was expressed in an insect cell system using generated recombinant baculovirus through Bac-to-Bac® Baculovirus Expression System. The crude PPRV-rBNP or Ni-NTA affinity-purified NP was characterized by SDS-PAGE and immunoblot using standard PPRV-specific sera. The PPRV-rBNP reacted well with PPRV anti-N specific monoclonal and polyclonal antibodies and PPRV-specific antiserum, suggesting that the expressed PPRV-rBNP is in its native form. The crude PPRV-rBNP as a diagnostic antigen was evaluated either as a coating antigen or standard positive control antigen in the Avidin-Biotin ELISA using the known standard panel reagents. The results showed that the expressed PPRV-rBNP can be an alternative diagnostic antigen to E. coli expressed recombinant PPRV-NPN and the utility of PPRV-rBNP avoids the need to use live PPRV antigen in the diagnostic ELISA. Hence, this allows scope in the future for large-scale field application of the recombinant antigen-based assays for diagnosis/surveillance and monitoring of PPR at the eradication as well as post-eradication phases in endemic countries or PPR non-endemic countries.

SowjanyaKumari S ，Bokade PP ，Kumar KV ，Bharath V ，Shome BR ，Balamurugan V ... -  《-》

Development of a competitive ELISA for detecting antibodies to the peste des petits ruminants virus using a recombinant nucleoprotein.

Libeau G ，Préhaud C ，Lancelot R ，Colas F ，Guerre L ，Bishop DH ，Diallo A ... -  《RESEARCH IN VETERINARY SCIENCE》