An enzyme immunoassay for cell proliferation using monoclonal antibodies directed against a cell proliferation-associated antigen.

Abstract

An enzyme immunoassay (EIA) for assessment of cell activation and proliferation was developed by the use of monoclonal antibodies (MoAb) directed against a ubiquitous growth-associated antigen, gp125. Test cells distributed in a microtest plate were labeled with anti-rat gp125 MoAb, B3, for rat cells or anti-human gp125 MoAb, HBJ127, for human cells and subsequently with rabbit anti-mouse immunoglobulins. The cell-bound antibody was assessed by a colorimetric enzyme assay using horse-radish peroxidase-modified protein A and 2,2'-azinobis(3-ethylbenzthiazoline sulfonic acid). To terminate the reaction and to make the reaction mixture transparent, sodium dodecyl sulfate (SDS) was added to the mixture. The intensity of the developed color was measured by use of a multiwell scanning spectrometer. The titration curves obtained by the EIA for cells were practically similar to those obtained by the conventional 3H-thymidine (Tdr) uptake method, and the appropriate cell number to be used for the assay was indicated to be 3 to 50 x 10(3) cells per well. This method was applicable not only for quantitation of cell number of growing cells but also for measuring mitogenic responses of lymphocytes as revealed by the data obtained from Con A stimulation of lymphocytes. These results indicate that this new EIA method using anti-gp125 antibodies is useful for quantitative assays of cell growth and cell activation in the research fields of oncology and immunology.