A sensitive method to detect defined peptide among those eluted from murine MHC class II molecules.
We developed a sensitive competitive inhibition radioimmunoassay able to trace pmoles of a defined peptide eluted from major histocompatibility complex (MHC) class II molecules that were subsequently fractionated by RP-HPLC. In this assay we used a model synthetic peptide corresponding to amino acid residues 110-120 from the hemagglutinin (HA) of PR8 influenza virus, and affinity purified rabbit antibodies specific for this peptide. The HA110-120 peptide binds to I-Ed class II molecules on the surface of APCs and is recognized by specific CD4+ T helper cells. 2PK3 B lymphoma cells (H-2d) were pulsed with HA110-120 peptide or PR8 virus, lysed, the MHC class II molecules extracted, and bound peptides eluted. After separation by RP-HPLC, the fractions were tested for inhibition of the binding of rabbit anti-HA110-120 antibodies to peptide coated microtiter plates. A significant inhibitory activity was observed with one peak when the cells were pulsed with HA110-120 peptide and two peaks when pulsed with PR8 virus. The inhibitory activity was correlated with the presence of HA110-120 peptide as demonstrated by peptide sequencing. The assay is reproducible and sensitive to 1 pmol of antigenic peptide. This assay can be useful to identify microbial peptides with defined structure and antigenicity among the multiple peptides bound to class II molecules.
Brumeanu
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Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029.
Kohanski
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Bona
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Zaghouani
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D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive / Q000276:immunology; D002851:Chromatography, High Pressure Liquid; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral / Q000276:immunology*; D000949:Histocompatibility Antigens Class II / Q000276:immunology*; D008213:Lymphocyte Activation / Q000276:immunology; D008285:Major Histocompatibility Complex / Q000276:immunology*; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology*; D011817:Rabbits; D011863:Radioimmunoassay / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000276:immunology
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D000949:Histocompatibility Antigens Class II; D010455:Peptides