Primary Studies Of ELISA Kits For Detection Specific Antibodies Or Antigens Of Mycobacterium Tuberculosis

Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis. According to the World Health Organization (WHO) statistics, one-third of the world's population were infected with Mycobacterium tuberculosis, causing over 3 million deaths annually. In China, the infection rate is 44.5%, the number of active tuberculosis patients is 4.51 million and 1.3 million deaths annually. Therefore, tuberculosis became one of the main infectious diseases which destroyed people's health. More and more scientists focus on the research of Mycobacterium tuberculosis. It is very important to find a convenient, fast, high sensibility and specificity diagnosis method to control the prevalence and spread of tuberculosis.Compare Mycobacterium tuberculosis genome to BCG genome with genome hybridization technique aider successfully sequenced the Mycobacterium tuberculosis genome in 1998, there are 16 Regions of Difference(including RD1-RD16)between BCG and Mycobacterium tuberculosis, the RD1 is only present in pathogenic Mycobacterium tuberculosis including Mycobacterium hominis , Mycobacterium boris, Mycobacterium africanum and atypical Mycobacterium, but is deleted in BCG and other none pathogenic Mycobacterium tuberclosis. The proteins that encoded by the genes of RD1 are attractted widespread attention in the mycobacterium tuberculosis vaccine development or the diagnosis aspect. Coding genes of CFP10, ESAT6 and PPE68 all exist in RD1, because of their intense immunogenicity, they can be used for the specific reagents to detect Mycobacterium tuberculosis. But It find that the sensibility is not very high in the diagnosis when using single protein, at present, researchers consider using two or more specificity protein unions are advantageous to enhancement sensitivity in the diagnosis.In the study, the prokaryotic recombinant plasmid pGcfp10, pGesat6 , pGcfp10-esat6 as well as pGesat6-ppe68 had been constructed and expressed in E.coli induced by isopropyl- beta-D-thiogalactopyranoside(IPTG), then the purified proteins were obtained which purified by GST purified kit and used to immunize rabits to obtain the high titer of polyclonal antibodies. In order to provide a method which has good specificity, sensitivity as well as in clinical practice and technical support for prevention and control of MTB in China, we take tuberculin purification protein derivative (PPD) as the reference to set up PPD-ELISA method, and also set up indirect ELISA methods which use ESAT6, CFP10-ESAT6, ESAT6-PPE68 as coating antigens to detect specific antibodies of tuberculosis patients. In order to diagnose the tuberculosis present infection, we set up double-antibody sandwich ELISA method which use polyclonal antibody as coating antibody to detect specific antigen of tuberculosis patients and provide new technical for early diagnosis, early treatment in tuberculosis.The study was divided into five parts.In the first part; the cfp10 gene of 303 bp and the esat6 gene of 288 bp were amplified from Mycobacterium tuberculosis DNA genome of H37Rv by PCR and directed cloned into expression plasmid pGEX-4T-1 to construct the prokaryotic recombinant plasmid pGcfp10 and pGesat6 , furthermore, the GST-CFP10 fusion protein of 36 kDa and GST-ESAT6 fusion protein of 32 kDa in size were expressed in prokaryotic cell efficiently as expected after induced by IPTG, then, the GST-ESAT6 fusion protein was purified with GST purified kit.In the second part, a fusion gene was constructed by splicing cfp10 gene and esat6 gene with GeneSOEing techniques and cloned into pGEX-4T-1 plasmid to construct the pmkaryotic expression recombinant plasmid pGcfp10-esat6, then, the CFP10-ESAT6 fusion protein of approximately 42 kDa in size was expressed in prokaryotic cell after induced by IPTG and purified with GST purified kit.In the third part, a fusion gene was constructed by splicing esat6 gene and ppe68 gene with GeneSOEing techniques and cloned into pGEX-4T-1 plasmid to construct the prokaryotic expression recombinant plasmid pGesat6-ppe68, the GST- ESAT6-PPE68 fusion protein of approximately 69 kDa in size was expressed in prokaryotic cell after induced by IPTG , then, purified with GST purified kit.In the fourth part, the E. coli containing the recombinant plasmids pGesat6, pGcfp10-esat6 and pGesat6-ppe68 were induced by IPTG, then, the fusion proteins were efficiently expressed and massive highly purified fusion proteins were obtained by GST purified kit. Furthermore, some of the purified fusion proteins were used to immunize rabits to obtain the high titer of polyclonal antibodies.In the fifth part, we set up optitmized PPD-ELISA procedure which use PPD as coating antigen, according to experiments at diluents for serum, blocking buffer, incubation time for enzyme-substrate reaction and the method of result interpretation. Similarly, we set up indirect ELISA methods using ESAT6, CFP10-ESAT6, ESAT6-PPE68 which all are specific to Mycobacterium tuberculosis as coating antigens to detect the specific antibodies of tuberculosis patients. Compare to PPD-ELISA, we think the methods that set up above have higher specificity. It will be advantageous to improve the accuracy of diagnosis for tuberculosis when using the methods to detect the patients again who were certified positive with PPD-ELISA. From comparison, we find the sensitivity of two ELISA methods based on coating with fusion proteins are higher than the sensitivity of single protein ESAT6 based ELISA, two ELISA methods based on coating with fusion proteins have the similar sensitivity, the specificity of CFP10-ESAT6-ELISA is higher than the specificity of ESAT6-PPE68-ELISA. the method CFP10-ESAT6-ELISA is the best. PPE68 is a specific antigen which has high value of research for tuberculosis diagnosis .We also set up double-antibody sandwich ELISA method which use polyclonal antibody as coating antibody to detect specific antigen of Mycobacteriurn tuberculosis . The results displaied that the three kinds of methods have low sensitivity(12%, 18%, 19% respectively)but high specificity(approximately 100%), therefore, we consider the ELISA method that detecting specific antigen of Mycobacterium tuberculosis is a workable new technique for diagnosing the tuberculosis present infection.In conclusion, the specific antigens-based MTB ELISA methods have high sensitivity and specificity, therefore, they have potential to be further developed to be tuberculosis diagnostic kits, which will meet the needs of Mycobacterium tuberculosis detection in clinic practice and provide technical support for the prevention and control of tuberculosis in China. Moreover, the ELISA methods which detect the specific antigens of Mycobacterium tuberculosis can differentiate tuberculosis present infection from past infection and evaluate the treatment efficiency, so they also have high value of research and development for tuberculosis diagnosis.