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  • Title: Marked improvement of a substance P radioimmunoassay by reduction of 125I-labelled [Tyr8]-substance P prepared by the chloramine-T method with mercaptoethanol and subsequent purification by reversed-phase liquid chromatography.
    Author: Rissler K, Cramer H.
    Journal: J Chromatogr; 1991 Mar 08; 564(1):67-79. PubMed ID: 1713598.
    Abstract:
    [Tyr8]-substance P was radiolabelled with 125I by the application of the chloramine-T method. Due to the high oxidative potential of the 125I-chloramine-T system the purified reaction product was converted into a derivative, which presumably had been oxidized to the corresponding sulphoxide at Met11. This conversion was shown by reversed-phase high-performance liquid chromatography after consecutive reduction-oxidation experiments of the freshly prepared radiopeptide. The oxidized derivative exhibited only negligible binding to substance P receptors isolated from rat brain homogenates. However, in contrast, it showed marked cross-reaction to the antibody raised in rabbits against synthetic SP(1-11). The variance in the quantification of identical samples was marked, and the measurement of concentrations in the lower pg/ml range was not sensitive enough to determine levels of substance P-like immunoreactivity in human cerebrospinal fluid. Assay sensitivity could be substantially improved and variance significantly decreased by the use of a radiopeptide, which had been labelled by the chloramine-T method and which had been subsequently reduced with mercaptoethanol and purified by liquid chromatography.
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