An enhanced and scalable process for the purification of SIV gag-specific MHC tetramer

A recently developed method for the identification and quantitation of anti gen-specific T lymphocytes involves the use of complexes of biotinylated ma jor histocompatibility complex (MHC) and avidin conjugated to a fluorescent reporter group. This complex, dubbed the "tetramer," binds to antigen-spec ific T lymphocytes in vitro, which can then be sorted and counted by fluore scence-activated flow cytometry to measure immune response. Our research ha s focused on developing the purification process for preparing tetramer rea gent. 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 pur ified 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 (MC) 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 compone nts. Analysis by flow cytometry confirmed that the tetramers generated by t his new process gave bright staining and specific binding to CD3+/CD8+ cell s of vaccinated monkeys and led to results that were equivalent to those ge nerated with tetramer produced by the original process. (C) 2001 Academic P ress.