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.
[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.
Rissler
K
K
Dr. Gerhard Friedrich, Pharmbiodyn, Auftragsforschung, Biochemical Unit, Denzlingen, Germany.
Cramer
H
H
D002700:Chloramines; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D006801:Humans; D007457:Iodine Radioisotopes; D007553:Isotope Labeling / Q000379:methods*; D008623:Mercaptoethanol; D011863:Radioimmunoassay / Q000706:statistics & numerical data; D013373:Substance P / Q000031:analogs & derivatives* / Q000032:analysis* / Q000134:cerebrospinal fluid / Q000302:isolation & purification; D014105:Tosyl Compounds
D002700:Chloramines; D007457:Iodine Radioisotopes; D014105:Tosyl Compounds; C016300:chloramine-T; D013373:Substance P; C055640:substance P, Tyr(8)-; D008623:Mercaptoethanol