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. 2020 Dec;13(12):2587-2595.
doi: 10.14202/vetworld.2020.2587-2595. Epub 2020 Dec 5.

Development of recombinant nucleocapsid protein-based indirect enzyme-linked immunosorbent assay for sero-survey of porcine reproductive and respiratory syndrome

S Phani Kashyap  1   2 , Jagadish Hiremath  1 , S Vinutha  1   2 , Sharanagouda S Patil  1 , Kuralayanapalya P Suresh  1 , Parimal Roy  1 , Divakar Hemadri  1
Affiliations

Development of recombinant nucleocapsid protein-based indirect enzyme-linked immunosorbent assay for sero-survey of porcine reproductive and respiratory syndrome

S Phani Kashyap et al. Vet World. 2020 Dec.

Abstract

Background and aim: Porcine reproductive and respiratory syndrome (PRRS) is a disease endemic in many countries and is of economic importance. India was free from PRRS until the first outbreak was reported from a North-East Indian state in 2013. Since then, disease outbreaks have been reported from North-East India and the pilot study conducted earlier showed that it is gradually spreading to the rest of India. Considering there are no locally developed population screening tests available for PRRS and imported diagnostic/screening tests are expensive, the present study was aimed at developing recombinant nucleocapsid (rN) protein-based indirect enzyme-linked immunosorbent assay (iELISA).

Materials and methods: The rN protein of PRRS virus (PRRSV) was produced following standard cloning, expression, and purification procedures. Using this antigen, iELISA was optimized for the detection of serum antibodies to PRRSV. The sensitivity and specificity of the test were assessed by comparing it with a commercial PRRSV antibody detection kit.

Result: A total of 745 serum samples from ten different states of India were screened using the developed iELISA. The iELISA had a relative specificity of 76.18% and sensitivity of 82.61% compared to the commercial ELISA (Priocheck PRRSV ELISA kit, Thermo Fisher Scientific, USA).

Conclusion: The iELISA, which deployed rN protein from Indian PRRSV, was found to be suitable in the serological survey and may be a useful tool in future disease surveillance programs.

Keywords: indirect enzyme-linked immunosorbent assay; nucleocapsid protein; porcine reproductive and respiratory syndrome; sensitivity; specificity.

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Figures

Figure-1
Figure-1
Porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein clone construct with the pET28a vector. The figure describes the full length of nucleocapsid protein PRRSV genotype 2 with hexahistidine tag at N and C terminals.
Figure-2
Figure-2
Analysis of expression of porcine reproductive and respiratory syndrome virus nucleocapsid protein by sodium dodecyl sulphate–polyacrylamide gel electrophoresis. M, Molecular weight protein marker, 1=Untransformed Escherichia coli lysate, 2=Uninduced E. coli lysate at 1 h, 3=Uninduced E. coli lysate at 3 h, 4=Induced E. coli cell lysate showing expressed rN protein (~19 kDa).
Figure-3
Figure-3
Western blot analysis of expressed recombinant nucleocapsid protein showing reactivity with (a) horseradish peroxidase-conjugated anti-His-tag monoclonal antibody (lane 2) and (b) porcine reproductive and respiratory syndrome virus positive pig serum (lane 2). Lane-1 is having protein marker in both the figures.
Figure-4
Figure-4
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis of affinity chromatography purified recombinant nucleocapsid protein. Lane-1=Protein molecular weight markers, Lane-2=Purified recombinant nucleocapsid protein.
Figure-5
Figure-5
Bland-Altman plot depicting the level of agreement between the commercial enzyme-linked immunosorbent assay (ELISA) and recombinant nucleocapsid protein indirect ELISA based on the normalized optical density (percent positive) values.
Figure-6
Figure-6
Receiver operating characteristics curve for recombinant nucleocapsid protein indirect enzyme-linked immunosorbent assay. The gray dotted lines indicate 95% confidence limits.
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