Double antibody sandwich ELISA based on rabbit monoclonal antibody targeting gD protein for the detection of bovine herpesvirus 1.

Bovine herpesvirus 1 (BHV-1) is a highly contagious and latent virus that induces various diseases in the respiratory and reproductive systems. It is widespread in numerous countries, including China, and has a high positive detection rate, causing significant economic losses to global cattle industry. Timely and precise diagnosis is essential for effective preventative and control strategies. This study constructed a rabbit phage single chain fragment variable (scFv) display library with 7.14 × 10¹⁰ cfu/mL based on BHV-1 gD protein expressed in a prokaryotic system. Following three rounds of biopanning, three high-affinity scFv targeting the gD protein were obtained, and CHO-K1 cells were employed to express three high-affinity secreted rabbit monoclonal antibodies (RmAb). Double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was established with D3 as the capture antibody and D2 as the detection antibody. The findings indicated that optimal reaction conditions were: 3 % BSA blocking, 90 min antigen incubation time, 15 min color development time, and a cutoff value of 0.8525. The specificity test demonstrated that the method exclusively responded with BHV-1, exhibiting no cross-reactivity with other bovine-related viruses. Additionally, the coefficients of variation between Intra-batch and Inter-batch were below 5 %, indicating good stability and reliability. This study is the first application of RmAb in developing a detection method for BHV-1, aimed at improving the specificity and sensitivity of the method, thereby offering robust scientific technical assistance for the epidemiological surveillance and prevention of this disease.