Polystyrene beads coated with antibodies directed to HLA class I intracytoplasmic domain: the use in quantitative measurement of peptide-HLA class I binding by flow cytometry.
Protein-reactive, conformation-independent anti-peptide antibodies were raised in rabbits against a C-terminal sequence SDSAQGSDVSLA, common to most HLA-A and -B locus products. Antibodies were coupled to 4.5-microm polystyrene beads through the Fc portion by the use of protein A. The antibody-coupled beads showed a high capacity to bind HLA-A and -B proteins as well as their alpha chains by the intracytoplasmic domain, keeping the extracellular domains solvent exposed. The density of HLA class I proteins bound on the beads was approximately the same as that on cultured B cells. The antibody beads made it possible to quantitate peptide-HLA class I binding, i.e., in vitro HLA class I assembly by flow cytometry. The assembly rate determined by the provisionally called flow cytometric HLA class I assay was 15%-19% for the reassembly of dissociated HLA class I proteins with the released selfpeptides. With single synthetic peptides, the highest rate so far obtained was 6.5%. The assay specificity and reproducibility were satisfactory.
Chersi
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A
Istituto Regina Elena for Cancer Research, Rome, Italy. biochimica@ipo.it
Rosano
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L
Tanigaki
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N
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification / Q000378:metabolism*; D000918:Antibody Specificity; D001324:Autoantigens / Q000378:metabolism; D002460:Cell Line; D003593:Cytoplasm / Q000276:immunology*; D005434:Flow Cytometry / Q000379:methods; D006680:HLA Antigens / Q000276:immunology* / Q000378:metabolism; D015395:Histocompatibility Antigens Class I / Q000276:immunology* / Q000378:metabolism; D007279:Injections, Subcutaneous; D008863:Microspheres; D010446:Peptide Fragments / Q000008:administration & dosage / Q000138:chemical synthesis / Q000276:immunology* / Q000378:metabolism*; D011137:Polystyrenes / Q000276:immunology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D015203:Reproducibility of Results; D013205:Staphylococcal Protein A
D000906:Antibodies; D001324:Autoantigens; D006680:HLA Antigens; D015395:Histocompatibility Antigens Class I; D010446:Peptide Fragments; D011137:Polystyrenes; D013205:Staphylococcal Protein A