Quantitation of the Pap reaction on single cells by absorption cytophotometry

The reaction product of the unlabeled antibody enzyme method was quantitated at the single cell level by absorption microphotometry. Attempts were made to demonstrate that the amount of dye deposited on individual cells can be taken as a measure of the number of antibody molecules bound. In an experiment with prefixed sheep erythrocytes loaded in excess with amboceptor a close relationship was observed between cell size and the amount of dye measured. Furthermore, the known differences in A1 antigen density on human A1 and A2 erythrocytes were satisfactorily reproduced with this quantitative approach. As regards T antigen density on human lymphocytes of different origin, the results presented tally with data in the literature. It is therefore concluded that the method is suitable for quantitating antibody amounts bound to individual cells.