Development of blocking ELISA for detection neutralizing antibodies against pseudorabies virus

Pseudorabies caused by pseudorabies virus (PRV), is characterized by fever, reproductive disorders, and neurological symptoms in pigs. The viral glycoprotein D (gD) of PRV plays a key role in mediating host cell entry and inducing neutralizing antibody production. In this study, A BHK cell line capable of stably secreting and expressing the gD-Fc fusion protein was successfully established and subsequently adapted to suspension culture. The target protein present in the supernatant was purified and used for subsequent mouse immunization. Two monoclonal antibodies (mAbs), 1B5 and 1H6, were successfully generated and identified by indirect enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence assay (IFA), which specifically recognized two conformation-dependent epitopes of the gD protein of PRV. Functional characterization revealed that both mAbs exhibited neutralizing activity, with IC₅₀ values of 38.01 μg/mL and 4.86 μg/mL, respectively, and their binding can be effectively blocked by PRV-positive sera. Moreover, two epitopes recognized by the both mAbs demonstrate a high level of conservation across common PRV isolates, including HLJ8, HeN1, SC and Bartha strains. Based on these properties, a blocking ELISA (bELISA) method was established to detect the level of PRV neutralizing antibodies. This assay exhibits high specificity and sensitivity comparable to the virus neutralization test (VNT), detecting PRV-positive sera at dilutions up to 1:512. Testing of 115 clinical pig serum samples showed a high agreement rate of 93.9% between bELISA and VNT. Overall, this bELISA provides a reliable and practical tool for evaluation of PRV vaccine-induced immunity.