A sandwich type enzyme-linked immunosorbent assay for proliferating cell nuclear antigen (PCNA)/cyclin using...

Three hybridomas producing monoclonal antibodies to proliferating cell nuclear antigen (PCNA)/cyclin were newly derived. The specificity of established monoclonal antibodies to PCNA, TOB7, TO17 and TO30, was compared with that of the previously reported PCNA-specific monoclonal antibodies, 19A2 and 19F4. TOB7, TO17 and TO30 reacted with purified PCNA in an enzyme-linked immunosorbent assay (ELISA). A 34 kDa PCNA polypeptide was noted by immunoblotting, the same polypeptide as recognized by 19A2 and 19F4. Epitopes recognized by all those monoclonal antibodies to PCNA were analyzed by competitive inhibition tests using ELISA. The results of those experiments suggested that the epitope recognized by TO17 and TO30 was almost identical to that of 19A2 and closely related to that of 19F4, but different from that of TOB7. Based on those results, a sandwich type ELISA for detection of PCNA was developed using TO17 and TOB7. This system was applied to measure the concentration of PCNA in normal peripheral blood lymphocytes (PBL) before and after stimulation by phytohemagglutinin (PHA), and in tissue extracts from rabbit kidney and thymus (RKE and RTE, respectively). Before mitogenic stimulation, the PCNA concentration in PBL extracts was less than 6 ng/ml (cell concentration 2.5 x 10(7)/ml), but at 48 h after PHA stimulation, when more than 70% of cells were in S phase, its concentration became 1280 ng/ml. RTE which contained many proliferative lymphocytes had much higher concentration of PCNA than RKE. Those results suggested that sandwich type ELISA using TO17 and TOB7 was useful as the quantitative assay to detect blast-transformation and proliferation of cells in vivo.