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The chicken erythrocyte-specific MHC antigen. Characterization and purification of the B-G antigen by monoclonal antibodies

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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 (1) in vitro synthesis in the presence or absence of tunicamycin, (2) binding experiments with lectin from Phaseolus limensis, and (3) 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 SDSPAGE analysis followed by silver stain of proteins. The yield from the affinity chromatography step was 3–4 μg 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.

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  1. Institute for Experimental Immunology, University of Copenhagen, Nørre Alle 71, DK-2100, Copenhagen Ø, Denmark

    Jan Salomonsen, Karsten Skjodt, Monna Crone & Morten Simonsen

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  1. Jan Salomonsen
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  2. Karsten Skjodt
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  3. Monna Crone
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  4. Morten Simonsen
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Salomonsen, J., Skjodt, K., Crone, M. et al. The chicken erythrocyte-specific MHC antigen. Characterization and purification of the B-G antigen by monoclonal antibodies. Immunogenetics 25, 373–382 (1987). https://doi.org/10.1007/BF00396103

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  • DOI: https://doi.org/10.1007/BF00396103

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