Isolation of a human B lymphocyte membrane protein with Ia-like properties

Abstract

A rapid method for isolation of a major surface membrane glycoprotein from whole, unfractionated cultured human B lymphoblasts is described. After detergent solubilization the method uses gel filtration followed by affinity chromatography on Sepharose Con A and then alkaline acrylamide gel electrophoresis. Specific high-titre, rabbit antisera to the isolated protein reacted with cultured and normal peripheral blood B lymphocytes, as well as peritoneal macrophages from a renal dialysis patient. The antisera selectively inhibited the mixed lymphocyte reaction at high dilution. The protein reacted with a heterologous antiserum to HL-B antigens and contained subunits of MW 33 000 and 27 000. Resolution of the subunits, however, required a discontinuous SDS gel system. These properties indicate its similarity to murine Ia antigens. The protein was not associated with beta 2 microglobulin and showed no structural or antigenic similarity to the major erythocyte glycoprotein, glycophorin. Antisera to the protein failed to precipitate surface-radiodinated components from similarily treated extracts of cultured human T lymphoblasts. This method now makes available a reference membrane glycoprotein from a differentiated, nucleated human cell in sufficient purity and quantity for kinetic and biosynthetic studies.

Changes in HLA‐DR antigen expression on cultured human melanoma cells during theophylline treatment

“…Their derivation and conditions of culture have been previously described (Liao et al 1975, Liao et al 1979. Rabbit anti-HLA-DR antiserum which recognizes backbone specificities on DR molecules was prepared in the laboratory of one of the authors (L. M. Jerry), by immunizing a rabbit with purified human B lymphocyte membrane glycoprotein followed by absorption with T lymphoid cells (Jurkat) (Sullivan et al 1979 formed as previously described ). The indicator sheep red blood cells (SRBC) used to trace the rabbit anti-HLA-DR antiserum were pre-sensitized by rabbit anti-SRBC antibody followed by sheep anti-rabbit IgG antibody.…”