Development of an indirect enzyme-linked immunosorbent assay based on the nucleocapsid protein of bovine parainfluenza virus type 3

Abstract

Purpose: Bovine parainfluenza virus type 3 (BPIV3) is an important pathogen in the bovine respiratory disease complex. The purpose of this study was to develop an efficient and rapid serological detection method, an indirect enzyme-linked immunosorbent assay (iELISA), for BPIV3.

Methods: The full-length N gene of BPIV3 was amplified from reverse-transcribed BPIV3 cDNA and ligated into the pET-28a prokaryotic expression vector. The recombinant target N protein was correctly expressed. After purification, the target N protein was used to immunize BALB/c mice to prepare polyclonal antibodies. After experimental optimization, an iELISA for detecting antibodies against the BPIV3 N protein was established using purified protein and prepared polyclonal antibodies.

Results: The recombinant N protein was efficiently expressed after induction with isopropyl β-D-1-thiogalactopyranoside at 32°C for 16 h. Immunization of BALB/c mice with the purified recombinant N protein elicited polyclonal antibodies that specifically reacted with BPIV3, with an antibody titer of 1:256000. The optimized iELISA conditions were as follows. Microtiter plates were coated with antigen at 5× 10^-2 μg/mL, incubated overnight at 4 °C, and blocked with 1% bovine serum albumin for 2 h at 37 °C. Subsequently, plates were incubated with primary polyclonal antibodies at a working dilution of 1:8000 and horseradish peroxidase-conjugated secondary antibodies at a dilution of 1:8000 for 1 h at 37 °C. Finally, the colorimetric reaction was allowed to proceed for 10 min. Specificity tests showed that this method had no serological cross-reaction with other pathogens. Both intra-assay and inter-assay coefficients of variation were lower than 10%. Positive signals were still detected when the clinical-positive BPIV3 serum was diluted 1:6400. The concordance rate between this method and the virus neutralization test for clinical serum samples was 95.8%. A total of 176 cattle serum samples were tested using this iELISA method, yielding a serum antibody positivity rate of 94.9%.

Conclusion: The N protein polyclonal antibody prepared in this study provides an important biological basis for research on the mechanism of BPIV3 infection. The establishment of an iELISA can enable the rapid screening of clinical samples, thereby providing reliable technical support for the epidemiological investigation of BPIV3.

Keywords: bovine parainfluenza virus type 3; indirect enzyme-linked immunosorbent assay; nucleocapsid protein; polyclonal antibody; serological detection.