Development of an indirect ELISA method based on the VP4 protein for detection antibody against duck hepatitis A
Juan Li1, Yingying Cao1, Mingshu Wang1
1Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, PR China; Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, PR China; Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan, 611130, PR China.
Abstract:
In Picornavirus, the VP4 gene is located inside the viral capsid, but the antibodies it produces have neutralizing activity. At the same time, we know that the VP4 gene is relatively conserved among the four structural proteins, which makes the usage VP4 protein-based antigen potentially meaningful. Therefore, we used purified duck hepatitis A virus type 1 (DHVA-1) recombinant VP4 protein as the coating antigen to establish an indirect method. The optimal antigen, serum and enzyme-labeled antibody dilutions were 1:400 (3.375 μg/mL), 1:80 and 1:1600, respectively. The optimal blocking buffer was 1% skimmed milk powder. The cutoff value was determined to be 0.203, and the analytical sensitivity was 1:1600. The established ELISA method has good specificity, repeatability and sensitivity. It has a high coincidence rate of 70.8 % with the DHVA-1 as the coating antigen. So, it can be used for the detection of DHAV-1 serum antibodies.
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