Development And Preliminary Application Of Immunochromatographic Strips For Rapid Detection Of Antibodies Against M. Bovis

Mycobacterium bovis results in bovine tuberculosis (bTB) in a range of animal species and humans. It is considered as B type infectious disease by world organization for animal health (Office Intemational Des Epizooties, OIE) and considered as two type infectious disease in our country. Investigation of tuberculosis pathogens showed that M. bovis is one of important pathogens of human tuberculosis. Therefore, the expert in World Health Organization (WHO) committee once said that tuberculosis can not be controlled unless bovine tuberculosis is eradicated.Due to the lack of an effective vaccine and medicine that could prevent and treat bovine tuberculosis, many countries adopt the "test-and-slaugter" policy. So an accurate and timely diagnostic method is very important for preventing and controlling bTB. For the detection of bTB, tuberculin skin test (TST) is the only method recommended by OIE. Tuberculin is also called purified protein derivatives (PPD) which is a protein mixture that has complex components. PPD includes many antigens shared with other Mycobacterium species besides specific antigens of M. bovis. Hence, the PPD skin test can not avoid the cross reaction with othermycobacterial. Moreover the method propably provides false positive results when there is serious tuberculosis or immunosuppressive conditions exist. Therefore, to develop a specific and sensitive diagnostic antigen and diagnostic method for bTB is an urgent task for bTB control.As an intracellular parasite, M. bovis exhibits large individual differences in antibody reactivity and has a wide antibody reactivity spectrum. MPB70,MPB83,CFP-10 and ESAT-6 are specific and secreted antigens of M. bovis and infected animal can produce their antibodies. Single antigen can not reflect the antibody level and infection status in vivo. It has been confirmed that a mixture of antigens of M. bovis may dramatically increase the sensitivity of serologic diagnostic methods. However, for such the "cocktail" antigens, their expression and purification must be carried out individually, which takes much time and effort. This may lead to the problem in terms of the ratio of various antigens, which may affect the sensitivity and specificity of the diagnostic methods.In the present study, four highly specific antigen genes, namely, mpb70, mpb83, cfp10 and esat-6 were recombined by spliced overlap extension technology, and the fusion protein rM70-83-C10-E6 was efficiently expressed, in order to provide a sensitive and specific diagnostic antigen for diagnosis of bTB.Gold-Immunochromatographic assay (GICA) is a new immunological detecting technology since the early 1980s. It not only has good specificity and sensitivity, but also is cheap, simple and rapid. MPB83 and MPB70 were proved to be specific antigens secreted by bTB. Their amino acid sequences have 63% of identity. Analysis of MAbs against MPB70 and MPB83 showed that most MAbs shared epitopes on these molecules.In the present study, strips were prepared with the principle of GICA with the two recombinant M. bovis antigens MPB83 and MPB70, in order to develop a sensitive, specific, rapid and practical method to detect antibodies against M. bovis for diagnosis of bTB. The research included:1. Cloning and expression mpb70,mpb83,cfp-10 and esat-6 of M. bovisWe cloned the coding region of MPB70,MPB83,CFP-10 and ESAT-6 gene for M.bovis, introduce them into the prokaryotic expression vector, then expressed and purifed the fusion protein. The properties of fusion protein were evaluated. ELISA shows that the protein could be recognized by the positive antiserum to M. bovis and mono-specific antisera to antigens of MPB70,MPB83 and CE (cfp10-esat6) respectively. Western blot analysis shows that the protein could be identified only by the antiserum to M. bovis but not the sera against.M, paratuberculosis, brucellosis, babesia and infectious bovine rhinotracheitis virus. Heat treatment test demonstrates that the fusion protein is heat stable at 60℃for 1h. It is concluded that the fusion protein has antigenicity and is heat-stable. It will be a hopeful antigen used in M. bovis diagosis.2. Establishment of colloidal-gold rapid detection testStrips were prepared with the principle of GICA with the two recombinant M. bovis antigens MPB83 and MPB70. MPB83 was labeled by colloidal gold and MPB70 was used as the test capture reagent to detect the corresponding antibodies. The highest sensitivity was found with strips prepared by 40 nm particles of colloidal gold. The optimal labeling pH of colloidal gold solution was 6.0. The best amount of MPB83 for colloidal gold labelling was 6.5μg per ml. The optimal concentrations of capture reagent MPB70 and control coating protein anti-MPB83 IgG was 3.0mg/mL and 2.5mg/mL, respectively. The strips did not react with positive serum of other unrelated bovine diseases displaying a high specificity. Comparison test showed that the strips had a higher sensitivity than a commercial MPB70 test strips from Korea.3. Clinical application of colloidal-gold rapid detection stripsA batch of colloidal gold test strips was manufactured to detect clinical samples, in parallel with isolation of M. bovis, TST and test strips from Korea. The agreement ratios were 85%, 79.73%% and 98.75% respectively between this strip and M. boris isolation, TST, and the Korea produced MPB70 strip. This novel colloidal gold test strip for bTB antibody detection is sensitive, specific, convenient to be used, rapid to get results. It would posses a good prospect in bTB detection used alone or as a supplementary diagnostic method for TST in bTB eradication program.