Separation of Mycobacterial Soluble Antigens by Isoelectric Focusing

Abstract

Ion-exchange chromatography and gel filtration have been reported to yield partial separation of mycobacterial antigens. These procedures were used in combination with isoelectric focusing in an attempt to purify antigens of Mycobacterium tuberculosis strain H37Ra. The fractionating action of isoelectric focusing is dependent upon differences in the isoelectric points of the proteins to be separated. Culture filtrate of M. tuberculosis H37Ra was chromatographed on Sephadex G-200. This resulted in two widely separated peaks. The first peak, presumably containing high-molecular-weight substances, was then fractionated on a diethylaminoethyl Sephadex anion-exchange column. Three peaks were collected, and each was subjected to isoelectric focusing. Each peak was further separated into two or more fractions. The serological reactivity of each fraction was determined by immunodiffusion and immunoelectrophoresis. Sensitized guinea pigs were also skin-tested with the fractions. Two of the fractions contained only a single precipitinogen. One fraction contained two precipitinogens. A fourth fraction contained three precipitinogens and was also the only fraction to display sensitin activity. Four of the fractions were inactive either as precipitinogens or sensitins. The results suggest that the methods described are useful for the separation of mycobacterial antigens.

Characterization and Comparison of Mycobacterial Antigens by Two-Dimensional Polyacrylamide Gel Electrophoresis

“…This observation is further supported by the fact that at least 85%O of the mycobacterial constituents have acidic isoelectric points (pI) between pH 4 and 5 (Wright, to be published). These charge characteristics plus the complexity of the cell filtrates and cell extracts as described in the previous paper (2) and so well depicted by electroprotein mapping illustrated in this report are undoubtedly the reasons that the technique of isoelectrifocusing will have little success in the initial fractionation of crude mycobacterial cell preparations (8). Even if stable, narrow pH gradients can be established, several components will have the same pI and, therefore, will be detected together as a single band (5), whereas in pore gradient electrophoresis, molecules with similar molecular size will continue to be separated as a result of variations in specific density and shape of the molecules (5, 9, 7).…”

Mycobacterial antigens: a review of their isolation, chemistry, and immunological properties.

“…The combination of gel filtration with Sephadex G-200, DEAE-Sephadex ion-exchange chromatography, and isoelectric focusing was used to fractionate culture filtrate of M. tuberculosis by Moulton et al (146). They obtained nine fractions, four of which had precipitinogens when studied by immunodiffusion with rabbit antisera, two giving only single lines.…”

Chemical, physical, and immunological characteristics of purified protein derivatives from Mycobacterium bovis

“…Ammonium sulfate or trichloroacetic acid are the most common precipi tating agents employed, although many other fractionation methods have been used experimentally (2,4,15,26,41,51,78). Commercial tuber culin has generally been prepared from M. tuberculosis, the human type of tubercle bacillus, although other tuberculins have been prepared from M. bovis, Mycobacterium avium, and many other mycobacteria.…”

“…tuberculosis grown on a synthetic medium containing either asparagine or ammonium glutamate as the primary source of nitrogen (5,16 Since Koch's time, many investigators have made attempts to improve and purify the active components present in tuberculin (10,13,15,26,38,39,41,43,45,48,51,53,(76)(77)(78). The main constituents of tuber culin are proteins, peptides, carbohydrates, and nucleic acids.…”