Development and practical use of supersensitive assays for peptides with monoclonal antibodies

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Radioimmunoassay (RIA) is a major tool to Measure the hormone concentration in the basic research as well as in the clinical Practice. The detection limit of RIA is often around 10^<-15> mole/tube and the extraction is necessary for the measurement of the hormone level.Enzyme immunoassay (EIA) and immunoradiometric assay (IRMA) which use two antibodies for different epitopes have the higher detection limit, 10^<-18> mole/tube, than RIA. Therefore, these highly sensitive EIA and IRMA can measure the hormone level without extraction even in plasma. However, the development of antibodies for different epitopes of bioactive peptides with molecular weights less than 5, 000 daltons are very difficult. Therefore, no EIA and IRMA for peptide hormones (molecular weights<5, 000) are in practical use.The present study was designed to develop supersensitive assays for bioactive peptides with mopfoclonal antibodies for different epitopes. Using two monoclonal antibodies recognizing the ring structure or mid-portion of atrial natriuretic peptide (ANP) and its C-terminal part, respectively, we could develop highly sensitive assays, EIA and IRMA for ANP. The detection limits of the EIA and IRMA were 0.01 fmol/tube and 0.03 fmol/tube, respectively. We could also developed a highly sensitive IRMA for brain natriuretic peptide (BNP). Since these assay systems can measure plasma ANP and BNP levels without extraction, these will be useful tools for practical use in clinical medicine.