Separation of mycobacterial soluble antigens by isoelectric focusing

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.