Two nanobody-based immunoassays to differentiate antibodies against genotype 1 and 2 porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) infection causes significant economic loss to the global pig industry. Genotype 1 and 2 PRRSV (PRRSV-1 and -2) infections have been reported in China, Europe and America. For accurate prevention, nanobodies were first used as diagnostic reagents for PRRSV typing. In this study three nanobodies targeting both PRRSV-1 and -2, two targeting PRRSV-1 and three targeting PRRSV-2, were screened and produced. To develop two competitive ELISAs (cELISAs), the g1-2-PRRSV-Nb3-HRP nanobody was chosen for the g1-2-cELISA, to detect common antibodies against PRRSV-1 and -2, and the g1-PRRSV-Nb136-HRP nanobody was chosen for the g1-cELISA, to detect anti-PRRSV-1 antibodies. The two cELISAs were developed using PRRSV-1-N protein as coating antigen, and the amounts for both were 100 ng/well. The optimized dilution of testing pig sera was 1:20, the optimized reaction times were 30 min, and the colorimetric reaction times were 15 min. Then, the cut-off values of the g1-2-cELISA and g1-cELISA were 26.6% and 35.6%, respectively. Both of them have high sensitivity, strong specificity, good repeatability, and stability. In addition, for the 1534 clinical pig sera, an agreement rate of 99.02% (Kappa values = 0.97) was determined between the g1-2-cELISA and the commercial IDEXX ELISA kit. For the g1-cELSIA, it can specifically detect anti-PRRSV-1 antibodies in the clinical pig sera. Importantly, combining two nanobody-based cELISAs can differentially detect antibodies against PRRSV-1 and -2.