Abstract:

The aim of this study is to establish a rapid indirect CLIA antibody detection method for porcine pseudorabies virus gB protein, in order to provide technical support for the prevention and control of porcine pseudorabies disease and the evaluation of vaccine immunization level in large-scale pig herds. In this study, carboxylated magnetic beads were coupled with recombinant gB protein to form immunomagnetic beads, and the reaction conditions were optimized by using a fully automated chemiluminescence instrument; six different concentrations of standards were used to draw the standard curves; the negative and positive determination criteria were determined by drawing ROC curves; and the specificity, sensitivity, reproducibility, and compliance were evaluated after the preliminary establishment of the method. The results showed that the optimal pH for magnetic bead coupling was 6.0, the optimal concentration for protein coupling was 40 μg/mL, 10% BSA was the optimal sealant, the optimal dilution of enzyme-labeled secondary antibody was 1:20000, the serum reaction time was 5 min, the enzyme-labeled antibody reaction time was 10 min, and the pre-stimulation solution reaction time was 5 min. Finally, a standard curve with R²=0.9987 was drawn, and at the same time the judgment criteria were set as follows: ≥16.78 U was judged as positive, and <16.78 U was judged as negative. In the methodological evaluation, there was no cross-reactivity with the positive sera of 7 porcine pathogens, and the sensitivity was slightly higher than that of the commercial kits, with intra-batch and inter-batch coefficients of less than 10%, and the total compliance rate with the commercial ELISA kits was 98.9%. The indirect chemiluminescent antibody detection method for porcine pseudorabies virus gB protein established in this study can be used for epidemiological investigation of PR and assessment of vaccine immunization level, and provides theoretical reference for the development of subsequent kits.

Key words: porcine pseudorabies virus, recombinant gB protein, chemiluminescence, antibody, immunoassay, standard curve, ROC curve