A lactate dehydrogenase (LDH)-based immunoassay for detection of cell surface antigens and its application to the study of MHC class I-binding peptides.
A lactate dehydrogenase (LDH)-based immunoassay, referred to as CPEIA (cell panning enzyme immunoassay), has been developed for the detection of cell-surface antigens. CPEIA is similar to a panning assay, in that it is based on the capture of cells bearing an antigen of interest by means of an antibody immobilized to a 96-well microtiter plate. Attachment of the cells is then measured by addition of a substrate for the intracellular enzyme lactate dehydrogenase. The substrate solution also contains the nonionic detergent Triton X-100 to lyse the cells and release LDH, which converts the substrate p-iodonitrotetrazolium violet (INT) from yellow to red. The intensity of the color resulting from the LDH-catalyzed reaction is proportional to the number of cells bound to the plate. The procedure does not require fixation of the cells, centrifugation, and blocking steps, resulting in a more convenient assay. CPEIA has been used for the detection of MHC class I antigens and other molecules on the surfaces of mouse cell lines and concanavalin A (ConA)-stimulated T lymphocytes. In addition, the assay has been used to detect peptide binding to Db and Kb MHC class I molecules on the surface of the mutant cell line RMA-S. The half-maximal responses for peptide-MHC class I interactions at different peptide concentrations can be determined with the assay, allowing the apparent dissociation constants to be calculated.
Sigal
L J
LJ
University of Nebraska, Lincoln 68588.
Berens
S
S
Wylie
D
D
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000954:Antigens, Surface / Q000032:analysis*; D000956:Antigens, Viral / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D006183:H-2 Antigens / Q000032:analysis* / Q000378:metabolism; D007124:Immunoenzyme Techniques; D066298:In Vitro Techniques; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008213:Lymphocyte Activation; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology* / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D000956:Antigens, Viral; D006183:H-2 Antigens; D010455:Peptides; D007770:L-Lactate Dehydrogenase