The Development Of The Intracellular Mycobacteria ELISA Test Kit

Heparin-binding hemagglutinin(HBHA)is an adhesin secreted by mycobacteria.It is a glycoprotein produced by pathogenic mycobacteria and one of the important virulence factors for disseminated tuberculosis outside the lung.The application of HBHA plays a significant role in the diagnosis,treatment and prevention of M.intracellulare.In this paper,the constructed pET-32a-HBHA plasmid was transferred into BL21(DE3)and the HBHA protein was expressed under the inducer IPTG.The product was purified and concentrated on a histidine nickel column and identified by SDS-PAGE electrophoresis.HBHA protein was obtained,with the size of approximately 38 kDa,and the concentration was 1.6 mg/ml.Monoclonal antibody technology was applied to prepare purified HBHA monoclonal antibody against Mycobacterium tuberculosis HBHA using purified HBHA protein of M.intracellulare.Each of cell viability of three cells was 1:1600.Afterwards,a large number of antibodies with a titer of 2.56×10~5 were obtained.And HBHA monoclonal antibody was used as a coating antibody to establish an intracellular mycobacterial ELISA assay.The checkerboard method was used to optimize the detection conditions.At the same time,the best blocking solution was chosen.The specificity,sensitivity and accuracy of the test sample were evaluated.The evaluation results showed that this method did not cross-react with other antigens and had good results.Specificity,it can detect bacteria concentration of 200/ml,with good sensitivity.The detection accuracy was 93.3%,and was successfully assembled kit.The kit has important practical significance for the diagnosis and prevention of intracellular mycobacterial disease in livestock,and has a broad development prospect and a huge market demand.