Microbiology Research Unit, Veterans Administration Hospital, and University of Utah College of Medicine, Salt Lake City, Utah 84113.
Infect Immun. 1971 Mar;3(3):378-84. doi: 10.1128/iai.3.3.378-384.1971.
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.
离子交换层析和凝胶过滤已被报道可部分分离分枝杆菌抗原。这些程序与等电聚焦联合使用，试图纯化结核分枝杆菌 H37Ra 株的抗原。等电聚焦的分馏作用取决于待分离蛋白质的等电点的差异。结核分枝杆菌 H37Ra 的培养滤液在 Sephadex G-200 上进行色谱分析。这导致了两个广泛分离的峰。第一个峰，推测含有高分子量物质，然后在 DEAE-葡聚糖阴离子交换柱上进行分级。收集了三个峰，并对每个峰进行了等电聚焦。每个峰进一步分为两个或更多的馏分。通过免疫扩散和免疫电泳测定每个馏分的血清学反应性。用这些馏分对致敏豚鼠进行皮肤试验。两个馏分仅含有一个沉淀原。一个馏分含有两个沉淀原。第四个馏分含有三个沉淀原，也是唯一显示致敏原活性的馏分。四个馏分既作为沉淀原也作为致敏原均无活性。结果表明，所描述的方法可用于分枝杆菌抗原的分离。
