Mycobacterium Tuberculosis Antigens Quantitative Detection Technology And Preliminary Evaluation

Tuberculosis (TB) remains a major challenge to global health. There were an estimated8.6million incident cases in2012and, of1.3million TB deaths,0.3million were in HIV-positive people.It is estimated that the incidence of tuberculosis worldwide and the number of cases attributable to coexisting human immunodeficiency virus (HIV) infection will increase substantially during the next decade. Most of this burden occurs among the low-income countries of the world, particularly those in South East Asia and sub-Saharan Africa.In high-burden settings, the cornerstone of TB diagnosis remains sputum smear microscopy. The sensitivity in routine clinical practice varies between35and80%in HIV-negative patients, but drops to as low as20%among HIV-infected patients. Furthermore, in the latter group, sputum scarcity is common increasing the need for procedures such as sputum induction or bronchoscopy for sample acquisition. Conventional sputum microscopy is also of limited usefulness in children and those with extrapulmonary TB. Mycobacterial cultures take several weeks to provide results, are expensive, require specialized laboratory facilities, and remain unavailable in most high-burden settings. Newer quantitative T-cell assays [interferon-gamma release assay (IGRAs)] have little current utility in high-burden settings and cannot distinguish latent from active TB. Nucleic-acid amplification tests (NAATs) have limited specificity and are not widely available in high-burden countries. The detection techniques and products of diagnosis of tuberculosis still need much cheaper, faster and populizer.Most assays developed so far are based on the detection of specific circulating antibodies. The serodiagnosis of tuberculosis has been the subject of investigation for a long time, but we still lack a test with widespread clinical utility. Detection of antigen almost certainly confirms the presence of active disease. In contrast, detection of antibody does not always indicate active disease as the antibody response can be detected even six months after clearance of the infection. More efforts should be directed toward developing assays based on the detection of antigens in body fluids. Such tests could be useful for the diagnosis and follow-up of patients during treatment. Because TB is highly contagious during the active stage of the disease, a rapid and specific detection of the disease is therefore vital to contain the spread of TB and at the same time will help physicians to initiate proper and timely treatment, which in turn can reduce the chance of bacterial evolution towards MDR and XDR strains.Since1983, Sada first establish the double antibody sandwich enzyme-linked immunosorbent assay (sELISA) to examine tuberculosis antigen in CSF, scholars constantly detecting the Mycobacterium tuberculosis antigens in the sputum and cerebrospinal fluid by ELISA and latex agglutination test. Recently researches while prefer to use ELISA and colloidal gold test to detect the PPD derivatives and specific composition of the bacteria, such as the38kD antigen, MPT64, LAM in the serum, CSF and urine, which sensitivity and specificity were separately20%to94.0%and78.6%to100%. However, these antigens are relatively simple, and the correlation with humoral immunity has not yet been revealed, moreover, the sensitivity and specificity of the experiment were failed to make substantial progress. Thus, none of these tests could be widely used in detecting the mycobacterial antigens of active tuberculosis.In this study, prokaryotic recombinant plasmid pBVILl-38KD, pBVILl-ESAT6, pBVIL1-CFP10had been constructed and expressed in E.coli, induced by temperature and then purified proteins by ion exchange chromatography. Fusion protein IL1-38KD+ESAT6+CFP10and GST-38KD+ESAT6+CFP10were induced by temperature and isopropyl-beta-D-thiogalactopyranoside (IPTG) separately and then purified by ion exchange chromatography and affinity chromatography respectively. Proteins were used to immunize animals to obtain the high titer of monoclonal and polyclonal antibodies, which were purified by Protein G affinity chromatography, and the double antibody sandwich ELISA was established to purify antibody as coating antibody and detection antibody. On the basis of these mehods, the chemiluminescence test was established to do the quantitative detection of mycobacterium tuberculosis antigens and to evaluate the value of preliminary clinical.The study was divided into five parts.1) Mycobacterium tuberculosis38KD, ESAT6, CFP10genes were successfully amplified from the Mycobacterium tuberculosis H37Rv genomic DNA as template by PCR. By molecular cloning methods, we constructed Mycobacterium tuberculosis38KD, ESAT6, CFP10gene prokaryotic expression plasmid pBVIL1-38KD, pBVILl-ESAT6, pBVILl-CFP10. In prokaryotic expression system, the plasmid pBVIL1-38KD, pBVIL1-ESAT6, pBVILl-CFP10was induced by42℃water, which can effectively expressed about53kDa of IL1-38KD,21.3kDa of IL1-ESAT6,21.1kDa of IL1-CFP10fusion protein. The pBVILl-38KD, pBVIL1-ESAT6, pBVILl-CFP10fusion protein was purified and obtained by ion exchange chromatography. The activity of the purified protein were identified by indirect EILS A, showed that purified protein with good sensitivity and specificity.2) The fusion protein pBVIL1-38+6+10, PGEX-38+6+10have been successfully constructed in previous research, and cloned and expressed fusion protein on the basis of it. The concentration and purity of the fusion protein were high after purified by ion exchange chromatography and affinity chromatography. After identifying the activity of the fusion protein, compared with the single one, the sensitivity was improved and had a good specificity.3) The purified recombinant protein were used to immunized rabbits to obtain the rabbit against MTB-specific antigens (38KD, ESAT6, CFP10and fusion antigen38KD+ESAT6+CFP10) polyclonal antibody, which titer was1:32000,1:16000,1:32000,1:128000. The purified recombinant proteins were used to immunize BALB/c mice to obtain rats against MTB-specific antigen monoclonal antibody, the anti-38kD positive clones have four strains:BBA171, BFE624, BBG411, BEA831, which titers were1:256000.1:128000、:256000、1:128000; anti-ESAT6positive clones have one:BFF1024, which titer was1:512000; anti-CFP10positive clones have one:BBG1028, which titer was1:256000.4) This research set up double antibody sandwich ELISA by using monoclonal, polyclonal antibody as coating and labeled antibody. All reaction conditions were optimized and the best procedure was established:coating and labeled antibody are rabbit anti-38KD+ESAT6+CFP10and HRP-rabbit anti-38KD+ESAT6+CFP10; the concentration of coating antibody is0.25ug/ml; samples and diluent as50ul+50ul dilution; the concentration of labeled antibody is1:600. Meanwhile, this research established a standard curve(y=0.0218x+0.1387, R2=0.9933) of antigen ELISA to determine the minimum detection limit(1.11ng/ml). On the basis of MTB antigen ELISA, a method for detecting MTB antigen chemiluminescent ELISA and a standard curve(y=15583x+89992, R2=0.9962)of chemiluminescent ELISA were established to determine the minimum detection limit(0.46ng/ml). 5) By detecting the antigen concentration in serum of patients who received TB treatment at different times in using antigen chemiluminescent ELISA, MTB-specific antigens could gradually decrease along with the treatment time. By testing the tuberculosis-specific antigens in sputum culture supernatants of MTB, a higher compliance rate (92%) between antigen chemiluminescent ELISA and mycobacterial culture positive can be concluded, meanwhile, the antigen detection rate could reach27.11%in the supernatant of mycobacterial culture negative. With the quantitative analysis, the MTB specific antigens38KD, ESAT6, CFP10secretion mainly concentrated in0.75ng/ml-40ng/ml in the culturing time. The application of combine using MTB-specific antigen chemiluminescent quantitative detdction and commercial antibody detection kit revealed the complementary between antigen detection and antibody detection.In conclusion, the antigen chemiluminescent quantitative detdction established in this study could provide a reliable reference value in the diagnosis of tuberculosis. Antigen test could distinguish TB infection patients with past ones, and prove the clinical value in the treatment efficacy.