Purification and characterization of class II histocompatibility antigens from a homozygous human B cell line.
Human class II histocompatibility antigens were purified from the Epstein-Barr virus-transformed human B lymphoblastoid cell line LG-2 by immunoaffinity chromatography. This is the first time all three subsets have been prepared as nonradioactive materials on a milligram scale. The yields of DR, DQ, and DP from 10 g of cells were approximately 12, 2, and 0.2 mg, respectively. Cross-contamination of the subsets was found to be less than 2% when assayed by measuring the binding of antigen-specific monoclonal antibodies to antigen immobilized on fixed erythrocytes. The three purified subsets were extensively characterized. They contained no detectable invariant chain. The three proteins were distinguished by their migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The denatured antigens were susceptible to partial removal of carbohydrate by endoglycosidase H and apparently complete removal of carbohydrate by endoglycosidase F. The isolated, denatured chains differed in their affinities for radiolabeled lectins, suggesting differences in carbohydrate structures. A water-soluble form of each antigen was prepared by a controlled papain digestion of the native antigen. Both native and denatured antigens were analyzed for their reactivities with a panel of class II antigen-specific monoclonal antibodies, allowing a precise definition of the specificities of the antibodies.
Gorga
J C
JC
Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138.
Horejsí
V
V
Johnson
D R
DR
Raghupathy
R
R
Strominger
J L
JL
D000478:Alkylation; D000911:Antibodies, Monoclonal; D001402:B-Lymphocytes / Q000276:immunology*; D002241:Carbohydrates / Q000032:analysis; D002460:Cell Line; D002472:Cell Transformation, Viral; D006681:HLA-D Antigens / Q000302:isolation & purification*; D004854:Herpesvirus 4, Human / Q000235:genetics; D006720:Homozygote; D006801:Humans; D046911:Macromolecular Substances; D008285:Major Histocompatibility Complex; D008970:Molecular Weight
D000911:Antibodies, Monoclonal; D002241:Carbohydrates; D006681:HLA-D Antigens; D046911:Macromolecular Substances