DEVELOPMENT AND APPLICATION OF 3 RADIOIMMUNOASSAYS OF ENDOTHELIN OF VARYING SPECIFICITIES

We have developed three radioimmunoassays (RIAs) of varying specificities toward the endothelin (ET) isoforms. The assays are called the endothelin-1,2 [I-125] assay system (RPA535), the endothelin 1-21 Specific [I-125] assay system (RPA555), and the endothelin-1,2 high-sensitivity [I-125] assay system (RPA545). We have fully characterized their cross-reactivities: RPA535-ED50 approximately 12 fmol/tube, ET-1 100%, ET-2 204%, ET-3 0.0024%, hBig ET 37.9%, pBig ET 32.9%; RPA545-ED50 approximately 4.8 fmol/tube, ET-1 100%, ET-2 1,300%, ET-3 < 0.001%, hBig ET 189%, pBig ET 63%; RPA555-ED50 approximately 3.6 fmol/tube, ET-1 100%, ET-2 144%, ET-3 52%, hBig ET 0.4%, pBig ET 0.26%. These assays have also been applied to the measurement of ET immunoreactivity (ir) in normal human plasma following Amprep extraction. Further validation of the use of these assays has included the measurement of endothelin-1 (ET-1) derived from big ET by in vitro enzymatic conversion by cathepsin E, which has demonstrated that cathepsin E possesses the appropriate specificity of cleavage to be considered the endothelin-converting enzyme (ECE).