Tb Serological Diagnostic Kit

Objective：To set up a standard enzyme-linked immunosorbent assay (ELISA)anddot immunofiltration assay (DIGFA) detection system based on screening ofMycobacterium tuberculosis (MTB) specific antigen. Detected the anti-tuberculosisantibody with these detection systems, and evaluated their value on the serodiagnosisof tuberculosis (TB)Methods:11kinds of MTB recombinant proteins were expressed and purified, andone kind of lipopolysaccharide (LPS) antigen were extracted and purified from MTB.The optimal conditions of ELISA including the concentration of coating antigen andthe dilutability of the second antibody were determined to establish standard ELISAdetection system. The anti-TB antibodies in470sera were detected by the ELISAdetection system, then the sensitivity and specificity of the detection system werecompared according to different cutoff values. The antigens with stronger specificity,higher sensitivity and better complementarity were selected to form an antigencombination, and its serodiagnostic value was evaluated. The DIGFA detectionsystem was developed with this antigen combination. The anti-TB antibodies in240sera were detected by the DIGFA detection system to evaluate its value on theserodiagnosis of TB. A commercial serological antibody detection kit TB-DOT fromShanghai Upper Bio-Tech Pharma Co., Ltd (Shanghai, China) was used as control.Result:1. Eleven kinds of TB recombinant proteins were sucessfully obtained throughNi2+affinity chromatography, and the purified LPS did not contain the residueof nucleic acid and protein.2. The optimal ELISA conditions were determined successfully as follow: thedilutability of the second antibody was1:10000, the concentrations of coatingprotein antigens were from1.6to6.3ug/ml, and the LPS antigen was0.16ug/ml. The sensitivities of single antigen were range from60.5%to82.9%,and the specificities were range from64.1%to92.7%with the cutoff valuemade by ROC curve. The sensitivities of single antigen were range from13.3%to40.5%, and the specificities were range from96.4%to99.5%with the x±3SD of healthy controls as the cutoff value. 3. The different antigen has some correlation and complementation each other, andcan not be substituted each other. The r38kD, MTB-LPS, r16kD, rMPT63antigens had stronger complementation. When these antigens were combined todetect the anti-TB antibody, the sensitivity increased from29.0%to65.2%, andthe specificity decreased from96.9%to92.2%.4. DIGFA detection system was developed using four antigens r38kD, MTB-LPS,r16kD and rMPT63. The results of240sera showed that there was nosignificant difference between the DIGFA detection system and TB-DOTcontrol kit in TB group and healthy group, but the DIGFA had higher specificitythan TB-DOT control kit in non-TB group.Conclusion:1. Twelve kinds of TB antigens were prepared successfully. ELISA and DIGFAdetection systems were set up using these antigens. The antigens had highersensitivities and lower specificities with the cutoff value made by ROC curvecompared with the x±3SD. Due to various antigens in sensitivity andspecificity are complementary to each other, the sensitivity would be improvedand the specificity would be reduced with the increases of antigen combination.Therefore, it is better to use x±3SD as cutoff value in the detection usingmultiple antigen combination.2. The DIGFA detection system had higher sensitivity compared to the conventionalacid-fast bacilli staining method, and had same sensitivity and better specificitycompared to TB-DOT control kit.