Variables influencing cell binding assays for antibody binding kinetics

Owing to growing interest in the use of cell binding assays for quality testing of radiolabelled antibodies in radioimmunoscintigraphy, factors in the assay which might affect final outcome were evaluated in order to accumulate information for effective and reliable assay design. For unmodified antibody, magnitude of binding at certain antibody concentrations changed with alteration in number of target cells attached to the microtitre plate. The level of signal detection could either be enhanced or suppressed by changing the dilution of the enzyme-linked tester antibody as in the case of the enzyme-linked immunosorbent assay (ELISA). Accordingly, changes in either assay factor resulted in a shift in the slope of the binding curve even when the immunoreactivity of the antibody remained unchanged. A conventional technique of single cell preparation by trypsin-EDTA was investigated for the trypsin action on tumour cell antigenicity. No adverse effects could be observed if dissociated cells were incubated for 24 hours before the assay. Variation in number of target cells seemed to be a key factor in determining the power of the assay to reveal changes in the quality of the radiolabelled antibody. Our findings suggested the need to standardize factors such as the size of the seed, determination of plating efficiency and the time for culture in order to assure interassay reproducibility.