title: A high throughput MHC II binding assay for quantitative analysis of peptide epitopes.
abstract: Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design(1,2). Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols(1,3-5). Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.
authors.affiliation: Thayer School of Engineering, Dartmouth College.
authors.forename: Regina
authors.initials: R
authors.lastname: Salvat
authors.affiliation: Institute for Immunology and Informatics, University of Rhode Island.
authors.forename: Leonard
authors.initials: L
authors.lastname: Moise
authors.affiliation: Department of Computer Science, Dartmouth College.
authors.forename: Chris
authors.initials: C
authors.lastname: Bailey-Kellogg
authors.affiliation: Thayer School of Engineering, Dartmouth College; Karl.E.Griswold@dartmouth.edu.
authors.forename: Karl E
authors.initials: KE
authors.lastname: Griswold
mesh_terms: D000483:Alleles; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation / Q000379:methods; D000939:Epitopes / Q000032:analysis* / Q000276:immunology; D006681:HLA-D Antigens / Q000235:genetics / Q000276:immunology*; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D010455:Peptides / Q000276:immunology*
chemical_list: D000939:Epitopes; D006681:HLA-D Antigens; D010455:Peptides