Radioimmunoassay for neurotensin, a hypothalamic peptide

Abstract

A highly sensitive and specific radioimmunoassay for neurotensin has been developed which utilizes 125I-labeled neurotensin and rabbit antisera raised toward synthetic neurotensin which has been coupled specifically through its lysine side chain to several proteins. The three antisera described have different specificities but are directed primarily towards the COOH-terminal region of neurotensin which is the biologically active portion of the molecule. Two of the antisera, poly(Glu60, Lys40) (from animal no. 4) and keyhole limpet hemocyanin (from animal no. 8), cross-react fully with COOH-terminal partial sequences of neurotensin while antiserum poly(Glu60, Lys40) (from animal no. 6) requires the entire molecule for full recognition. The assay can detect less than 3 fmol of neurotensin and the dose-response curves for synthetic and native neurotensin are superimposable, irrespective of the antiserum employed. Using these assay systems, the immunoactivity in acid/acetone extracts of 45 kg of bovine hypothalami was purified to homogeneity and shown to be attributable to intact neurotensin and not to fragments of neurotensin nor to related molecules. Radioimmunoassayable neurotensin (R-NT) obtained from bovine, rat, guinea pig, and rabbit hypothalami also gave dose-response curves which paralleled that of neurotensin and the neurotensin equivalents per g of wet tissue were in the range 45 to 70 pmol/g. Measurements with the three antisera were in agreement, especially after the extracts were chromatographed on Sephadex G-25; R-NT in these hypothalamic extracts has also been shown to be destroyed by treatment with various enzymes known to cleave neurotensin.