Indirect enzyme-linked immuno sorbent assay (ELISA) detection method for Haemophilus parasuis antibody based on TbpA protein

Abstract:

[Background] Haemophilus parasuis (HPS) is the pathogen of Gl?sser’s disease. Antibiotic therapy and vaccination are not obvious for the prevention and control of the disease, it is particularly important to establish a rapid and accurate antibody detection method. [Objective] Using the expression and purified transferrin-binding protein (TbpA) of HPS to establish an indirect enzyme-linked immuno sorbent assay (ELISA) method for detecting HPS antibodies. [Methods] The tbpA gene of HPS was cloned and connected pET-SUMO prokaryotic expression vector. After identification by PCR, double enzyme digestion and sequencing, the positive recombinant plasmid was transformed into the receptor bacteria Escherichia coli Rosetta(DE3), and the expression was induced by IPTG, and the expression products were identified by SDS-PAGE and Western Blot. Using purified TbpA as coating antigen, the indirect ELISA method for detecting HPS antibody was established through the optimization of a series of reaction conditions, and its clinical application and evaluation were carried out. [Results] The optimum reaction conditions of this method were as follows: 5 μg/mL, 4 °C overnight concentration of coated antigen; 5% skim milk powder sealed at 37 °C for 2 h; The dilution of serum was 1:1 600, 37 °C incubation 45 min; The dilution of HRP was 1:5 000, 37 °C action 30 min; The optimal reaction time of TMB was 5 min. This method can specifically detect the HPS antibody. The positive serum is still positive after 1:12 800 dilution, but no cross-reactivity with other porcine pathogen positive serum. the coefficient of variation between in the batch and batches all less than 6%. The total coincidence rates of this method were 90.00%, 86.67% and 90.00% compared with the commercial ELISA kit and Western Blot, whole bacterial for indirect ELISA, among which the positive coincidence rates were 90.38%, 88.46%, 92.00%, and the negative coincidence rates were 87.50%, 75.00% and 80.00%, respectively. The positive rate of immune antibody was 80%, and the positive rate of infection antibody was 19.50%. [Conclusion] This study based on the recombinant TbpA established indirect ELISA detection method for HPS antibodies with good specificity, sensitivity and repeatability, as well as the reliability of clinical applications, provides a technical means for HPS immune surveillance and epidemiological investigation.

Key words:Haemophilus parasuis, transferrin-binding proteins A, indirect ELISA, antibody detection, establishment and application