The Development And Preliminary Application Of ELISA Method Used For TB Detection With Antibodies As The Targets

Tuberculosis is a chronic infectious diseases caused by Mycobacterium tuberculosis, with a high mortality rate. It not only restricts the healthy development of animal husbandry, but also poses a great threat to human public health. In recent years, the incidence of the disease increased year by year. It was reported that about one-third of the world’s population is infected with Mycobacterium tuberculosis every year, while nearly 130 million people died in tuberculosis. Region of difference(RD) between Mycobacterium tuberculosis and the vaccine strain M. bovis bacille Calmette-Guérin(BCG) are hot spots of screening specific antigen used in tuberculosis diagnosis and vaccine development.RD2 and RD11 are absent in BCG during generation. The virulence of BCG is further attenuated due to the loss of RD2 and RD11.As the gene of RD2 and RD11 from Mycobacterium tuberculosis,rv1981 c and ppe59 are closely related to virulence and immunogenicity of Mycobacterium tuberculosis and also considered with potential valuable to the detection and treatment of tuberculosis.In the paper, the prokaryotic expression vector of rv1981 c and ppe59 from Mycobacterium tuberculosis with histidine-tag were established. In E.coli, the protein were efficiently expressed and purified Ni affinity chromatography. Then the purified protein was used as envelope antigen, and TB patients serum specific antibody was determined by indirect ELISA detection method.The results showed that the rv1981 c and ppe59 of M.tuberculosis gene are successfully amplified. At the same time, the recombinant plasmid of p ET28a-rv1981 c and p ET28a-ppe59 were established.The protein of Rv1981 c and PPE59 were successfully expressed. Their relative molecular mass are 40.1k D and 23.4k D respectively.Expressed products were purified by Ni-affinity chromatography column to obtain the fusion protein of a higher purity. These two kinds of protein showed good reactogenicity through reacted with monoclonal anti-HIS. Rv1981 c and PPE59 protein were used as coating antigen,to establish the optimal coating concentration of Rv1981c-ELISA and PPE59-ELISA was 10μg/m L, and the optimal serum dilution was 1:800,while the enzyme-labeled secondary antibody working concentration was 1: 5000, the optimal substrate reaction time was 15 min. By using the detection method of PPD-ELISA,Rv1981c-ELISA and PPE59-ELISA,Liaoyuan thoracic examination confirmed 99 cases serum of TB,30 patients with non-tuberculous respiratory disease patients serum and 50 healthy human serum respectively. The sensitivity and specificity of three methods results showed that the sensitivity of PPD-ELISA was 65.6%,and the specificity of PPD-ELISA was 90%;The sensitivity of Rv1981c-ELISA was 57.5% and the specificity of Rv1981c-ELISA was 82%; The sensitivity of PPE59-ELISA was 63.6% and the specificity of PPE59-ELISA was 88%;Both the sensitivity and specificity of Rv1981c-ELISA and PPE59-ELISA were lower than the PPD-ELISA, but they still had good sensitivity and specificity.Therefore, the two antigens not only can be used as serological diagnosis of tuberculosis candidate antigens in serological diagnosis of tuberculosis, but also have a certain value.