Preparation Of Mycobacterium Tuberculosis Antigens And Its Application In The Diagnosis

Objectives Currently, there are a wide variety of TB detection methods. Immunologydetection methods mainly includes based on cell-mediated immunity and based onhumoral immunity. Based on cell-mediated immunity method detect skin tests andcytokine, and based on humoral immunity method detect antigen and antibody. In orderto acquire more specific and sensitive, the key part is to choose antigen, so choosing goodspecificity and high sensitivity antigen is very important for the TB testing. The subjectmainly study the application of MTB antigen in TB diagnosis.Methods According to the literature, the amino acid sequence of H37Rv RD1regionand Rv3452antigen were acquired in the TB databases Database (http://tb.lanl.gov/content/index）. Using Biosun software analyze RD1region and Rv3452antigenepitopes to screen the part of antigen epitope with higher peaks, and then to intercept thecorresponding amino acid sequences. Using TB databases online database analysishomology and the amino acid sequences of genotypes to determine the type-specificsections. Full-length and two advantages segments of Rv3452antigen were expressed.The vectors were constructed by molecular cloning techniques. After expression and Qcolumn purification, we successfully acquire seven antigens with better sensitivity andspecificity.Results Based on original test, using antigens with better sensitivity and specificityexpand the sample testing and groups. The sample have210cases to divide threegroups: the first group is smear and culture co-positive group (83cases）, the secondgroup is smear-negative and culture-positive group (48cases）, and the third group issmear and culture co-negative group (79cases）. The results of ELISA can analysisseven antigens:56.19%,65.7%,26.67%,58.57%,60.48%,70.85%and65.24%. Bybacteriology method, the detection rate of smear testing of same samples is39.52,culture detection rate is62.38%. Comparison results can obtain that detection results ofCFP-10, Rv3452and Rv3452-2are better than the detection results of culture, meanwhilethe detection results of seven antigens except ESAT-6antigen are better than thedetection results of smear. In order to understand the changing levels of IgG antibodies ofpatients in the treatment of the clinic, we selected88cases of serial serum of patients with active tuberculosis during the treatment. The88cases of TB patients that weretreated0month,2months,4months and6months were divided into three groups: thefirst group was smear and culture co-positive (34cases）, the second group wassmear-negative and culture-positive (32cases）, and the third group was smear andculture co-negative (22cases）. Using ELISA detect the changing levels of IgGantibodies. The results overall detection rate of TB patients were69.3%,73.8%,51.1%,84.1%,71.5%,68.1%and67.1%. Based on this results, we can get that the proportionof OD value decreased were47.5%,44.6%,40%,37.8%,31.7%,48.3%and45.7%.the proportion of OD value increased were27.8%,35.7%,40%,31.1%,39.6%,40%and30.5%. The proportion of constant OD values were24.6%,20%,20%,31.1%,28.5%,11.6%and23.7%. The results can express that the IgG antibodies decreasedoccupy a large proportion during the treatment of patients.Conclusions According to the results, we can infer that the detection rate of serology ishigher than bacteriology about TB antibodies detection in the premise of ensuring thepurity and specificity. Serological detection assays have more advantages, such as highsensitivity, fast, easy to operate, test specificity and low cost.