Isolation and Characterization of a 31 kDa Mycobacterial Antigen from Tuberculous Sera and its Identification with in vitro Released Culture Filtrate Antigen of Mtb H₃₇Ra Bacilli

Abstract

Antigens released in vivo are of considerable interest in the immunodiagnosis of infectious diseases. Circulating antigen was isolated from bacteriologically confirmed tuberculous sera by ammonium sulphate precipitation. The protein fraction between 36% and 75% ammonium sulphate was reactive with tuberculosis (TB) sera showing the presence of circulating tubercular antigen (CTA). Fractionation of CTA on ultrogel AcA 34 gel filtration column gave 3 protein fractions CTA1, CTA2 and CTA3. CTA2 showed maximum antigenic activity by sandwich enzyme-linked immunosorbent assay (ELISA). SDS-PAGE fractionation and seroreactivity studies showed the presence of highly reactive tubercular antigen in CTA2-7 protein fraction by sandwich ELISA. Further fractionation of CTA2-7 on cation exchange fast-protein liquid chromatography (FPLC) gave 4 antigenic fractions, of which CTA2-7D was seroreactive similar to 31 kDa antigen (ESAS-7F) isolated from in vitro culture medium. Furthermore, CTA2-7D could inhibit binding of in vitro released ESAS-7F to affinity purified antibodies in inhibition ELISA. CTA2-7D antigen may be used as a target antigen in confirming active tubercular infection. Biochemical characterization showed circulating antigen CTA2-7D to be a lipoglycoprotein is released in vivo. ESAS-7F as a glycoprotein is released in vitro culture.