Tritium labeling of insulin and its application to the double isotope derivative dilution analysis.

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A method of tritiation and application to chemical determination of insulin are presented. Tritium labeling of insulin is performed by a catalytic exchange method with tritiated phosphoric acid-boron trifluoride complex, which is prepared by the use of tritiated water (100 mCi/ml). Bovine crystalline insulin was tritiated with this reagent for 12 hr at room temperature, and the product was confirmed to be identical with unlabeled intact insulin by several chromatographic techniques. Specific radioactivity of 3H-insulin was 6.2-6.5 mCi/mmol. The most effective coupling condition for 3H-insulin with phenyl isothiocyanate [35S] was at 40°and pH 9.0 in N, N-dimethylallylamine-TFA buffer for 4 hr, which gave a single radioactive peak of 35S-bis (phenylthiocarbamyl)-3H-insulin on radio-paper scannogram. On the double isotope derivative dilution analysis, the radioactive portion was eluted from the paper and the radioactivities of 3H and 35S were measured by a liquid scintillation spectrometer. The loss of insulin through the analytical procedures can be corrected by the use of 3H-insulin added as an internal indicator. The recovery of authentic insulin was 101.5±1.4 to 104.1±3.4%. When 3H-insulin of the present specific radioactivity is used, the determination limit of the method for insulin is 100 μg (0.017 μmol). This isotope derivative method will be available for corrections of the overestimated values obtained by the routine radioimmunoassay as total immunoreactive insulin levels.

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