Radioimmunoassays for prostaglandins. I. Technical validation of prostaglandin F2alpha measurements in human plasma using sephadex G-25 gelfiltration

Abstract

Human plasma samples of 1 ml were processed according to three different procedures prior to Radioimmunoassay (RIA) of Prostaglandin F2alpha (PGF2alpha). Serial dilutions of ethyl acetate extracts as such, or combined with either silicic acid or Sephadex G-25 chromatography were assessed for linearity, homogeneity and parallelism with the corresponding standard dose response line. For plasma extracts used as such, non-parallelism is observed. Subsequent chromatography on silicic acid of such extracts gave only a limited linear and parallel portion upon serial dilution. However, purification of the extracts by gelfiltration on Sephadex G-25 results in linear and parallel lines over the full range of the standard dose response line (B/BO 0.9-0.2). Upon comparison of separation by Polyethylene glycol (PEG) and Dextran coated charcoal (DCC) in these systems, PEG proved to give the best results. It was found that in the Sephadex G-25 procedure, separation by PEG is essential. The method of gelfiltration on Sephadex G-25 is simple and reliable. Intra- and inter-assay coefficients of variation are 6% and 12%, respectively. Accuracy, as measured by recovery of added known amounts of PGF2alpha is 97.6%.