Development of a Double-Antibody Sandwich ELISA Based on a Monoclonal Antibody against the Viral NS1 Protein for the Detection of Chicken Parvovirus

Abstract

Chicken parvovirus (ChPV) infection can cause runting-stunting syndrome (RSS) in chickens. There is currently no commercially available vaccine for controlling ChPV, and ChPV infection in chickens is widespread globally. The rapid detection of ChPV is crucial for promptly capturing epidemiological data on ChPV. Two monoclonal antibodies (mAbs), 1B12 and 2B2, against the ChPV NS1 protein were generated. A double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for detecting ChPV based on the mAb 1B12 and an anti-chicken polyclonal antibody against the ChPV NS1 protein. The detection limit for the ChPV recombinant pET32a-NS1 protein was approximately 31.2 ng/mL. A total of 192 throat and cloaca swab samples were analyzed for ChPV by the established DAS-ELISA and nested PCR methods. The concordance rate between the DAS-ELISA and the nested PCR method was 89.1%. The DAS-ELISA can detect the ChPV antigen without any cross-reaction with FAdV-4, FAdV-1, NDV, AIV, MS, CIAV, aMPV, EDSV, IBV, or AGV2. The method also has high repeatability, with a coefficient of variation (CV) of less than 5%. These findings indicate that the DAS-ELISA exhibits high accuracy, good sensitivity, and specificity, making it suitable for viral detection, field surveillance, and epidemiological studies.

Keywords: DAS-ELISA; NS1 protein; chicken parvovirus; monoclonal antibodies.

Figure 1

The reactivity of the monoclonal antibodies: (a). Western blot analysis of the mAbs. mAb 1B12 (lane 1, lane 2, and lane 3) reacted with the 104 kDa recombinant pET32a-NS1 protein; mAb 2B2 (lane 4 and lane 5) reacted with the 104 kDa recombinant pET32a-NS1 protein. The mAbs 1B12 and 2B2 (lane 6 and lane 7) reacted with the 79 kDa NS1 protein expressed in LMH cells; (b). IFA was performed on LMH cells transfected with the ChPV infectious plasmid pBluescript II SK(+)-ChPV. Scale bars, 100 µm.

Figure 2

Determination of the limit of detection of the NS1-DAS-ELISA using the recombinant pET32a-NS1 protein antigen. The cut-off value was 0.139.