ASFV p30蛋白间接ELISA抗体检测方法的建立与分析软件开发应用OA

This study aimed to establish an indirect enzyme-linked immunosorbent assay(ELISA)based on the African swine fever virus(ASFV)p30 protein and to achieve automated analysis of test re-sults.Eukaryotically expressed no-Tag p30 protein was used as the solid-phase antigen.After optimizing ELISA reaction conditions using a checkerboard titration method,the assay's specificity and sensitivity were validated.Software for automated ELISA data analysis was coded in Python.The established indirect ELISA was then applied to detect ASFV antibodies in 700 serum samples collected from various swine populations in a city of Jiangsu Province in 2017 and from 2020 to 2024.The optimal conditions were deter-mined as follows:coating with 0.125 μg/mL antigen at 4℃ for 16 h,blocking with 10 g/L gelatin at 37℃for 2 h,incubation with serum diluted 1∶100 at 37℃ for 20 min,incubation with secondary antibody di-luted 1∶10 000 at 37℃ for 20 min,and color development in the dark at 27℃ for 8 min,with a cut-off value of 0.4.Compared to a commercial kit,the total coincidence rate of the developed method was 97.4％,with a sensitivity of 100％and a specificity of 96.4％.Results generated by the ELISA software were con-sistent with manual calculations.The overall positive rate for ASFV antibodies in the clinical serum sam-ples was 6.14％.All samples from 2017 and 2024 were negative,whereas positive samples were detected each year from 2020 to 2023,with annual positive rates ranging from a minimum of 1.61％to a maximum of 16.70％.The established method demonstrates high specificity and sensitivity,and the accompanying software provides reliable automated result analysis.This integrated approach offers a practical tool for clinical ASFV antibody detection,supporting rapid diagnosis and effective disease control.