ISOLATION OF A 33-KDA PROTEIN ANTIGEN FROM DELIPIDIFIED MYCOBACTERIUM-TUBERCULOSIS H(37)RV

A 33-kDa protein (TB33) was isolated from a delipidated cell sonicate (CS) of Mycobacterium tuberculosis H(37)Rv (grown in Middlebrook 7H9 b roth supplemented with glucose) using immobilized metal affinity chrom atography (IMAC) on a nickel-nitrilotriacetic acid (Ni-NTA) column. TB 33 could not be isolated from the culture filtrate (CF) of M. tubercul osis H(37)Rv using Ni-NTA. TB33 was recognized by monoclonal antibodie s (mAb) known to react with proteins of M. tuberculosis with a molecul ar mass of 33/34 kDa; namely, mAb F126-5, F67-1 and F126-2. The N-term inal amino acid sequence of TB33 was found to be aa-Xaa-Thr-Pro-Ala-As p-Val-Ser/Cys-Asn-Val-Ala-Ile and thus, shows identity with the N-term inal of antigen 84 of M. tuberculosis except for two mismatches. Antib odies to TB33 could be raised in mice by administering four injections of TB33 (40 mu g total protein). Sera from tuberculosis patients reac ted with TB33, while those from normal healthy individuals did not.