Purification and Evaluation of 38 30 and 16kDa Antigens of Mycobacterium Tuberculosis for Rapid Diagnosis

Abstract

PURIFICATION AND CHARACTERIZATION OF 38kDa ANTIGEN: The 38kDa antigen was purified from M. tuberculosis H37Rv CFA by two dimensional electrophoresis, based on the combination of isoelectric focussing and preparatory electrophoresis. Asingle band of 38kDa in 1-D, resolved into 3 spots of very close pl values in the range of pH 5.0 in an analytical 2-D. MAb IT-23 (WHO Bank) that specifically reacts with 38kDa protein recognized this prote in in immunoblot. The conformational difference between the recombinant 38kDa (a-helix) and the 38kDa (f-sheet) purified in this study was established by circular dichroism (CD) studies. PURIFICATION AND CHARACTERIZATION OF 30kDa ANTIGEN: The 30kDa antigen, a component of the Antigen 85 complex of M. tuberculosis CFA was purified from CFA by preparatory HPLC using anion exchange column (QAE Sepharose), By analytical 2-D, 3 spots in the pl region of 4.0-4.5 are seen corresponding to antigen 85A, 85B and 85C. Three MAbs, IT-27, IT-44 and IT-49 specifically reacting with 30/31kDa recognized this purified protein. Antigen 85 complex was further separated into A, B and C components by passing through Hydrophobic interaction chromatography (Phenyl Sepharose column). PURIFICATION AND CHARACTERIZATION OF 16kDa ANTIGEN: The electroeluted 16kDa antigen from cell membrane bound proteins did not react in ELISA. So, the 16kDa antigen was purified from the cytosol antigens by gel filtration column. The existence of monomeric and oligomeric forms of the 16kDa protein, which is a characteristic feature reported for a-crystallin protein was demonstrated by RP HPLC. Circular dichroism studies revealed a and#946;-sheet structure for the purified 16kDa protein. Thus the 16kDa protein purified during this study shows similarity to the already reported and#945;-crystallin protein. The 16kDa gave a positive reaction with MAbs IT-1 and T-4 in the immunoblot. FUTURE SCOPE: CIC bound antibodies to specific antigens has to be evaluated among the extra pulmonary cases, where diagnosis is most difficult.