Specific in vitro binding of radiolabeled antibodies to cell surface antigens: interaction of antihuman thymocyte globulin with human target cells.
We have developed a highly sensitive and reproducible in vitro method to measure the rate and amount of binding of radiolabeled antibodies to target cells. We reduced non-specific binding of the antibodies to the target cells approx. 92% by removing protein aggregates from the antibody solutions. Using a specially designed cup that simultaneously allows washing and collection of target cells, cell loss was eliminated. This method was used to study the in vitro binding properties of the IgG fraction of anti-human thymocyte globulin (ATG) to nucleated human cells and to cells of other species. We found the initial rapid uptake of labeled ATG-IgG slowed with prolonged incubation. Incubation temperature and ATG concentration increased the rate of uptake. Sequential absorption studies indicated that initial uptake was due to rapidly binding antibodies. After these antibodies were removed, the rate of binding for antibodies that remained was several fold less than that of the antibodies removed by the initial absorption. Since temperature and the concentration of antibody and target cells can be rigidly controlled, this in vitro model system is ideally suited to quantify optimal conditions and kinetics of antibody binding to cell membrane antigens. Furthermore, the binding properties of antibody subpopulations in an antiserum may be determined by this technique. The maximum antibody binding capacity of various cell types can also be measured using the technique with a precision of +/- 14% on replicate determinations.
D000818:Animals; D000888:Antibodies, Anti-Idiotypic; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D000941:Antigens / Q000032:analysis*; D000961:Antilymphocyte Serum; D001666:Binding Sites, Antibody; D002462:Cell Membrane / Q000276:immunology*; D002478:Cells, Cultured; D002860:Chromium Radioisotopes; D000882:Haplorhini; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D007457:Iodine Radioisotopes; D008253:Macaca mulatta; D051379:Mice; D013601:T-Lymphocytes / Q000276:immunology*; D013696:Temperature; D013950:Thymus Gland / Q000276:immunology; D013997:Time Factors
D000888:Antibodies, Anti-Idiotypic; D000941:Antigens; D000961:Antilymphocyte Serum; D002860:Chromium Radioisotopes; D007074:Immunoglobulin G; D007457:Iodine Radioisotopes