A europium fluoroimmunoassay for measuring binding of antigen to class II MHC glycoproteins.
A dissociation-enhanced lanthanide fluoroimmunoassay employing europium-streptavidin and time-resolved fluorimetry was developed to measure binding of biotin-labeled peptides to class II MHC proteins. Binding of biotin-peptides as measured by this assay was saturable and inhibited in the presence of unlabeled peptide. Background fluorescence was minimal and there was a direct relationship between signal and biotin-peptide/class II complex concentration from 1.3 pmol to less than 1 fmol total class II. The sensitivity of the assay and the ability to selectively capture specific class II proteins from detergent lysates of cells with solid phase mAb made it possible to measure formation peptide/class II complexes in live APC cultured with biotin-labeled insulin. This assay is expected to be useful for routine measurement of peptide/class II binding and biochemical analysis of Ag processing events.
Tompkins
S M
SM
Department of Pathology, Emory University School of Medicine, Atlanta, GA.
Rota
P A
PA
Moore
J C
JC
Jensen
P E
PE
D000595:Amino Acid Sequence; D000937:Antigen-Antibody Reactions; D000938:Antigen-Presenting Cells / Q000276:immunology*; D001426:Bacterial Proteins / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D004306:Dose-Response Relationship, Immunologic; D005063:Europium; D015200:Fluoroimmunoassay / Q000379:methods*; D000949:Histocompatibility Antigens Class II / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D012680:Sensitivity and Specificity; D019809:Streptavidin; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology* / Q000494:pharmacology
D001426:Bacterial Proteins; D000949:Histocompatibility Antigens Class II; D007328:Insulin; D010455:Peptides; D014409:Tumor Necrosis Factor-alpha; D005063:Europium; D001710:Biotin; D019809:Streptavidin