Usefulness of a small scale indirect 125I-labelled protein A binding assay for detection of monoclonal antibodies against hematopoietic cell surface antigens.

We describe a small-scale solid-phase indirect 125iodine protein A binding assay (IPA) and discuss its usefulness for detection of hematopoietic cell surface antigens and for screening hybridoma clones producing monoclonal antibodies (McAbs) against K562 cell surface antigens correlated with differentiation induced by 12-O-tetradecanoyl phorbol-13-acetate (TPA). This assay was performed in polystyrene Terasaki microtest plates instead of 96-well microtiter plates, using hematopoietic cells attached to the wells by air drying. This method facilitates washing procedures and reduces the radioactive waste. The specificity of this IPA method is as high as those of RIAs and ELISAs generally used. Titration curves of a McAb, My7, specific for CD13 on HL60 and TPA-treated HL60 (HL60-TPA) cells, were linear between the 10(-1) and 10(-4) dilutions. A difference in the CD13 expression between HL60 and HL60-TPA cells could be detected by both IPA and flow cytometry using My7 at 10(-1)-10(-3) dilutions. At the 10(-3) dilution, however, CD13 expressed on HL60 cells was detected by the IPA method but not by flow cytometry. These results show that the sensitivity of IPA is superior to that of flow cytometry. By screening hybridoma clones with this IPA method, we succeeded in producing three interesting and useful McAbs (21H73, 37G7 and 49C12) against K562 cell surface antigens whose expression was altered after the differentiation induced by TPA. We also demonstrated that the IPA method is suitable for cell surface marker analysis of leukemia cells freshly isolated from patients.