The Chicken Erythrocyte-Specific MHC Antigen. Characterization and Purification of the B-G Antigen by Monoclonal Antibodies

Immunogenetics. 1987;25(6):373-82. doi: 10.1007/BF00396103.

Abstract

Mouse monoclonal antibodies with B-G antigen (major histocompatibility complex class IV) specificity were obtained after immunization with erythrocytes or partially purified B-G antigen. The specificities of the hybridoma antibodies were determined by precipitation of B-G antigens from 125I-labeled chicken erythrocyte membranes (CEM) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. The B-G antigen had an approximate molecular mass of 46-48 kd in reduced samples, depending on the haplotype, and in unreduced samples contained either dimers (85 kd), when labeled erythrocytes were the antigen source, or trimers (130 kd), when B-G was purified and precipitated from CEM. The B-G antigen was unglycosylated as studied by in vitro synthesis in the presence or absence of tunicamycin, binding experiments with lectin from Phaseolus limensis, and treatment of purified B-G antigen with Endoglycosidase-F or trifluoromethanesulfonic acid. Two-way sequential immunoprecipitation studies of erythrocyte membrane extracts with anti-B-G alloantisera and monoclonal antibodies revealed only one population of B-G molecules. Pulse-chase experiments have shown B-G to be synthesized as a monomer, with dimerization taking place after 20-30 min. No change in the monomer's molecular mass due to posttranslational modifications was revealed. The antigen was purified from detergent extract of CEM by affinity chromatography with a monoclonal antibody, and then reduced and alkylated and affinity-purified once more. Finally, reverse-phase chromatography resulted in a pure product. The B-G antigen was identified in the various fractions by rocket immunoelectrophoresis. The final product was more than 99% pure, as estimated by SDS-PAGE analysis followed by silver stain of proteins. The yield from the affinity chromatography step was 3-4 micrograms B-G/ml blood, calculated from Coomassie-stained SDS-PAGE of B-G using ovalbumin standards. The monoclonal antibodies were also used to identify the B-G (class IV) precipitation arc in crossed immunoelectrophoresis. No common precipitate with the B-F (class I) antigen was observed.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal / immunology*
  • Carbohydrates / analysis
  • Chickens / blood
  • Chickens / immunology*
  • Chromatography, Affinity
  • Epitopes / immunology
  • Erythrocytes / immunology*
  • Histocompatibility Antigens / biosynthesis
  • Histocompatibility Antigens / immunology
  • Histocompatibility Antigens / isolation & purification*
  • Immunoelectrophoresis, Two-Dimensional
  • Major Histocompatibility Complex
  • Molecular Weight

Substances

  • Antibodies, Monoclonal
  • Carbohydrates
  • Epitopes
  • Histocompatibility Antigens