A system for preparation, detection and identification of a precipitinogen (eta) of strains of Mycobacterium tuberculosis, that is not detectable in strains of M. bovis.

: A precipitinogen (designated eta: eta), which was undetectable in BCG and the Ravenal strain of Mycobacterium bovis, was detected in sonic extracts of the H37Rv and H37Ra strains of M. tuberculosis by agar gel immunodiffusion. Since precipitinogen eta was very labile in cell extracts and crude preparations, reproducible methods for preparing and detecting the precipitinogen eta were investigated. Two conditions were found necessary for a stable preparation: (1) deferration of all materials to which eta precipitinogen was exposed throughout procedures and (2) use of phosphate buffer of over pH 7.5 and over 0.07 M for solubilizing eta. Two methods were effective and useful for purification of precipitinogen eta: (1) Salting-out using deferrated ammonium sulfate (35-40% saturation) and (2) gel permeation chromatography on Sephadex G-200 or Bio-gel P-300 that had been thoroughly washed with chelating agents. The purified eta prepared by a combination of these two methods, retained activity on storage in the frozen state for several years or on incubation at 40 C for 30 min. A system was developed for detection and identification of eta using stable, purified eta as a reference. eta Precipitinogen was heat-labile (60 C for 10 min), and behaved as an acidic protein with a molecular weight of 230,000 or approximately 260,000 daltons.