title: An enhanced and scalable process for the purification of SIV Gag-specific MHC tetramer.
abstract: A recently developed method for the identification and quantitation of antigen-specific T lymphocytes involves the use of complexes of biotinylated major histocompatibility complex (MHC) and avidin conjugated to a fluorescent reporter group. This complex, dubbed the "tetramer," binds to antigen-specific T lymphocytes in vitro, which can then be sorted and counted by fluorescence-activated flow cytometry to measure immune response. Our research has focused on developing the purification process for preparing tetramer reagent. Our goal was to reengineer a published lab-scale purification process to reduce the number of processing steps and to make the process scalable. In our reengineered process, recombinant MHC alpha chain is isolated from Escherichia coli as inclusion bodies by tangential flow filtration. The purified MHC alpha chain is refolded with beta-2-microglobulin and the target peptide antigen to form the class I MHC. The resulting MHC is purified by hydrophobic interaction chromatography (HIC) and biotinylated enzymatically, and the biotinylated MHC is purified by a second HIC step. The tetramer is prepared by mixing biotinylated MHC with an avidin-fluorophore conjugate. The tetramer is further purified to remove any excess MHC or avidin components. Analysis by flow cytometry confirmed that the tetramers generated by this new process gave bright staining and specific binding to CD3+/CD8+ cells of vaccinated monkeys and led to results that were equivalent to those generated with tetramer produced by the original process.
authors.affiliation: Department of Virus & Cell Biology, Merck Research Laboratories, West Point, Pennsylvania 19486, USA.
authors.forename: K M
authors.initials: KM
authors.lastname: Grimm
authors.forename: W L
authors.initials: WL
authors.lastname: Trigona
authors.forename: G J
authors.initials: GJ
authors.lastname: Heidecker
authors.forename: J G
authors.initials: JG
authors.lastname: Joyce
authors.forename: T M
authors.initials: TM
authors.lastname: Fu
authors.forename: J W
authors.initials: JW
authors.lastname: Shiver
authors.forename: P M
authors.initials: PM
authors.lastname: Keller
authors.forename: J C
authors.initials: JC
authors.lastname: Cook
mesh_terms: D000818:Animals; D001710:Biotin / Q000737:chemistry / Q000302:isolation & purification; D019921:Biotinylation; D002469:Cell Separation / Q000379:methods; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry / Q000302:isolation & purification; D015683:Gene Products, gag / Q000276:immunology*; D015395:Histocompatibility Antigens Class I / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D002479:Inclusion Bodies; D008253:Macaca mulatta; D046911:Macromolecular Substances; D008285:Major Histocompatibility Complex; D017510:Protein Folding; D011994:Recombinant Proteins / Q000302:isolation & purification; D018449:SAIDS Vaccines / Q000008:administration & dosage / Q000276:immunology; D015302:Simian Immunodeficiency Virus / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology*; D001613:beta 2-Microglobulin / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification
chemical_list: D005456:Fluorescent Dyes; D015683:Gene Products, gag; D015395:Histocompatibility Antigens Class I; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D018449:SAIDS Vaccines; D001613:beta 2-Microglobulin; D001710:Biotin