Development and validation of an improved radioimmunoassay for serotonin in platelet-rich plasma.

Abstract

A radioimmunoassay (RIA) using a 125I-tracer is described for measurement of serotonin (5-hydroxytryptamine, 5-HT) in human platelet-rich plasma (PRP). Antisera were raised against 5-HT-succinamate conjugated to bovine albumin and, to improve assay sensitivity, the analyte was made chemically similar to the immunogen by conversion to N-acetylserotonin prior to assay, using the specific amino reagent N-acetoxysuccinimide. The assay shows good correlation with a high-pressure liquid chromatography (HPLC) reference method (5-HT RIA = 1.007 X 5-HT HPLC + 29.3, r = 0.936, p less than 0.001, n = 40), indicating that no significant cross-reactions were detected. Samples of PRP are diluted 1/20 to fall within the working range (80-15% B/B0) of the assay, which is 4.75-325 nmol/l, (0.95-65.0 pmol/tube), corresponding to 95-6500 nmol/l in PRP. Intra- and interassay coefficients of variation were 5.0-10.5% and 12.0-21.2% respectively for serotonin concentrations of 250-2,500 nmol/l added to platelet-poor plasma. With this improved assay, it is possible to analyse up to 100 samples/day, compared with 10-20 samples/day by HPLC.