A cytophotometric method to quantitate the binding of monoclonal antibodies to individual cells

A cytophotometric technique to quantitate binding of (monoclonal) antibodies to individual cells is described. This method is based on the detection of cell-surface antigens by immunoperoxidase staining procedures. The amount of reaction product of the peroxidase reaction with diaminobenzidine/H2O2 was quantitated by computerized scanning cytophotometry. The incubation period of peroxidase with the substrate required for an optimal cytophotometric determination of the reaction product proved to be 60 min. The reproducibility of individual absorbance measurements, the day-to-day reproducibility of the quantitation of monoclonal antibody binding, and the specificity and sensitivity of the detection of defined monoclonal antibodies, established the reliability of the present method. The sensitivity of the indirect immunoperoxidase procedure could be enhanced by using a biotin-avidin-peroxidase staining procedure.