Analysis of monoclonal antibodies reactive with human class II beta chains by two-dimensional electrophoresis and western blotting

Susan F. Radka, Carolyn E. Machamer, Peter Cresswell

Research output: Contribution to journalArticlepeer-review

Abstract

We have used the Western blotting technique to examine B lymphoblastoid cell line (B-LCL) membrane proteins separated by two-dimensional gel electrophoresis specifically to analyze the binding patterns of monoclonal antibodies to separated HLA class II antigen beta (β) subunits. The B-LCL LG-10 (homozygous for DR7), in which at least two sets of class II molecules can be distinguished on the basis of different electrophoretic mobilities, was examined with five monoclonal antibodies which detect monomorphic determinants. Four of the antibodies reacted with only DR β subunits, while one antibody, XD5.A11, reacted with DR and with additional β chains. Examination of two polymorphic monoclonal antibodies, SFR3-DR5, specific for HLA-DR5, and SFR3-PI.1, which reacts with a determinant absent from DR3 and DR7 homozygous lines, showed that both bind β subunits from Swei, a DR5 homozygous line. Purification of a subpopulation of Swei class II molecules using an SFR3-PI.1 affinity column showed that the determinants recognized by SFR3-DR5, SFR3-PI.1, and a monomorphic monoclonal antibody reactive only with HLA-DR β subunits of LG-10, reacted with identical β subunits. Additional class II antigen subunits reactive with XD5.A11 were nonreactive with the polymorphic antibodies and the HLA-DR-specific monomorphic monoclonal antibody.

Original languageEnglish (US)
Pages (from-to)177-188
Number of pages12
JournalHuman Immunology
Volume10
Issue number3
DOIs
StatePublished - Jul 1984
Externally publishedYes

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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