Human T cell leukemia antigens on the cell membranes: purification molecular characterization, and preparation of specific antisera. (1981)

Abstract Human T cell leukemia-associated (HTL) antigens were isolated from cell membranes of 2 T leukemia cell lines, MOLT-4 and JM, without using radiolabeling. In each series of experiments, cells were mechanically disrupted and subjected to differential centrifugation, which yielded a cell membrane preparation. The membrane proteins, including HTL antigens, were solubilized from the membrane preparation by sodium deoxycholate treatment and were fractionated by affinity chromatography on a Lens culinaris lectin column. All HTL antigen activity was found in the lectin-bound (glycoprotein) fraction for both MOLT-4 and JM. The specific HTL antigen activity in this glycoprotein fraction of MOLT-4 was found to be increased over 90-fold as compared to MOLT-4 cells. The glycoproteins of MOLT-4 and JM were fractionated by gel filtration on a AcA 34 column, and 2 HTL-antigen peaks, 1 major and 1 minor, were detected for both MOLT-4 and JM. MOLT-4 glycoproteins that were contained in the major HTL-antigen peak fractions were further purfied by a novel system of passive immunoaffinity chromatography. The yield of the purified HTL antigen preparation was approximately 1 mg per 5.5 × 1010 MOLT-4 cells. The immunochemical properties of this HTL antigen preparation, termed pHTL antigen preparation, were characterized before and after being radiolabeled. Analysis of the non-radiolabeled pHTL antigen preparation by sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE) under both reducing and nonreducing conditions showed 3 glycoprotein components with approximate m.w. of 100,000, 83,000 and 38,000. In addition a very weak band of 54,000 m.w. component was seen. The 38,000 m.w. component appears to be a degradation product of the 100,000 m.w. component. Radiolabeling of the pHTL antigen preparation by either the chloramine-T or the I0D0-GEN method resulted in labeling mainly the 100,000 m.w. component. Increased radiolabeling of other components was achieved either by Bolton-Hunter reagent or by SDS treatment of the antigen preparation followed by chloramine-T radioiodination. SDS-PAGE of the immunoprecipitate obtained by reacting the radiolabeled pHTL antigen preparation with the IgG of anti-HTL antiserum revealed a single major component of 100,000 m.w. along with minor components of 54,000 and 38,000 m.w. This anti-HTL antiserum had been prepared by extensive absorption of rabbit anti-(MOLT-4) antiserum. No reaction was observed between the pHTL antigen components and the 2 reported monoclonal anti-HTL antibodies, OKT6 and NA1 /34. It is important to note that immunization of rabbits with a small amount of the non-radiolabeled pHTL antigen preparation yielded antisera that showed strong specificity for leukemia cells without any absorption. These anti-pHTL antisera were found to react only with the 100,000 m.w. component. Thus, we isolated, without using radiolabeling, milligram quantities of a human leukemia-associated cell membrane antigen preparation that contains a single immunodominant glycoprotein of 100,000 m.w. composed of a single polypeptide chain. This 100,000 m.w. glycoprotein together with the 54,000 m.w. glycoprotein are the first reported human leukemia-associated cell membrane antigens that were identified as isolated proteins without the aid of radiolabel markers.