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

放射免疫分析(RIA)是基础研究和临床实践中测量激素浓度的主要工具。RIA的检测下限通常在10~(-15)摩尔/管左右，提取是测定激素水平所必需的。酶免疫分析(EIA)和免疫放射分析(IRMA)使用针对不同表位的两种抗体，其检测下限比RIA更高，为10~(-18)摩尔/管。因此，这些高灵敏度的EIA和IRMA即使在血浆中也可以在不提取的情况下测量激素水平。然而，针对分子量小于5000道尔顿的不同表位生物活性多肽的抗体的开发是非常困难的。因此，对于多肽激素(分子量为5000)，没有EIA和IRMA的实际应用。本研究旨在建立针对不同表位的单克隆抗体用于生物活性多肽的超灵敏检测。利用识别心钠素(ANP)环状结构或中间部分及其C末端部分的两种单抗，可建立高灵敏度的ANP检测方法EIA和IRMA。EIA和IRMA的检出限分别为0.01fmol/管和0.03fmol/管。我们还可以开发一种对脑利钠肽(BNP)高度敏感的IRMA。由于这些检测系统可以在不提取的情况下测量血浆ANP和BNP水平，这些将是实用的工具在临床上使用。

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.