Corticotrophin‐releasing Factor‐41 in the human and rat: utility of a highly sensitive enzyme amplified immunometric assay

We have developed a highly sensitive and specific immunoassay for human/rat corticotrophin‐releasing factor‐41 (CRF‐41) to enable determination of immunoreactive CRF‐41 levels in biological samples. To achieve high specificity, sensitivity and speed we have used two antisera in a sandwich enzyme immunoassay combined with enzyme amplification. The assay has a sensitivity of 0.08 fmol/well compared with radioimmunoassay sensitivities of 0.5 fmol/tube and is highly specific for the intact CRF‐41 molecule. Measurement of samples is complete within 24 h compared with the 5 days required to obtain sensitive radioimmunoassay measurement. The assay has been used to measure both rat hypothalamic CRF‐41 tissue content and release in vitro with good correlation when compared to radioimmunoassay measurement using antisera rC70 (0.983) or R1 (0.953). The assay only measures immunoreactive CRF‐41 coeluting with human/rat CRF‐41 and its oxidized form Met [O^21,38]CRF‐41 in human and rat tissue extracts separated by high‐performance liquid chromatography. The ability to measure immunoreactive CRF‐41 in unextracted plasma allows rapid measurement and eliminates multiple extraction steps.