High occupancy binding of antigenic peptides to purified, immunoadsorbed H-2Db beta 2m molecules.
In an effort to examine the peptide binding properties of purified class I MHC molecules, we have developed a solid phase, radiolabeled peptide binding assay based on the use of H-2Db molecules bound to agarose beads via heavy chain-specific mAb. Using purified Db beta 2m, recovered from RMA-S cells and bound to immunoadsorbent beads through either alpha 1 or alpha 3 region specific antibodies, complete occupancy of these molecules could be achieved with 125I-Y366-374 influenza nucleoprotein peptide (Kd 10(-7) M). Approximately 12% of the Db beta 2m dimers recovered from RMA cells could be occupied by this influenza nucleoprotein peptide under the same conditions. When free Db heavy chains were isolated from beta 2m negative R1E.Db cells by bead-bound alpha 3-region specific antibody (28-14-8S) and were incubated with human beta 2m, high affinity (Kd 10(-8) M) binding sites were created for the 125I-Y367-374 influenza nucleoprotein peptide. In addition to demonstrating that a significant fraction of the heavy chains present in R1E.Db cells are in a beta 2m-reactive form, the R1E.Db cells provide an alternate approach to that of RMA-S derived Db beta 2m empties for the creation of homogeneous complexes of Db, beta 2m, and antigenic peptide. We anticipate that these bead-bound empty and defined peptide-class I complexes may be useful in the further study of class I MHC target structure formation and recognition.
Burshtyn
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Department of Immunology, University of Toronto, Canada.
Barber
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BH
D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral / Q000378:metabolism*; D015683:Gene Products, gag / Q000276:immunology; D006183:H-2 Antigens / Q000378:metabolism*; D015497:HIV-1 / Q000276:immunology; D006801:Humans; D066298:In Vitro Techniques; D009980:Influenza A virus / Q000276:immunology; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding; D014721:Vesicular stomatitis Indiana virus / Q000276:immunology; D014758:Viral Core Proteins / Q000737:chemistry / Q000276:immunology*; D001613:beta 2-Microglobulin / Q000378:metabolism
D000956:Antigens, Viral; D015683:Gene Products, gag; D006183:H-2 Antigens; D046911:Macromolecular Substances; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D010455:Peptides; D014758:Viral Core Proteins; D001613:beta 2-Microglobulin