| Abstract: | A direct radioimmunoassay of the methyl ester of urinary and serum 5-hydroxy-3-indole acetic acid is described. The antiserum, raised in a rabbit against a conjugate of bovine serum albumin with 5-hydroxytryptamine hemisuccinamide, contained two antigenic fractions, one binding N-acyl 5-hydroxytryptamine, and the other binding methyl ester of 5-hydroxy-3-indole acetic acid, and N-acyl 5-hydroxytryptamine. The N-acyl 5-hydroxytryptamine binding fraction was removed by affinity chromatography on a N-acyl 5-hydroxytryptamine agarose gel in the presence of excess methyl ester of 5-hydroxy-3-indole acetic acid. The antibody methyl ester of 5-hydroxy-3-indole acetic acid complexes were dissociated and this affinity-purified antiserum was used in all experiments. Polyethylene glycol in combination with goat anti-rabbit IgG was used to separate bound and unbound 125I-labeled Bolton-Hunter reagent- 5-hydroxytryptamine conjugate. Sample preparation (esterification of 5-hydroxy-3-indole acetic acid to its methyl ester) was performed with trimethylsilyldiazomethane in dioxane. In the analysis of urine, the reagents used in the methylation served as diluents, contributing to the final dilution of 1:1100. In the analysis of serum, a deproteination step (ethanol precipitation) prior to methylation was necessary to obtain reproducible results. The methylated 5-hydroxy-3-indole acetic acid was then extracted with ethyl acetate and the extract redissolved in assay buffer. The minimal detectable concentration of methyl ester of 5-hydroxy-3-indole acetic acid was 1.1 mumol/l (0.21 mg/l 5-hydroxy-3-indole acetic acid) urine or 100 fmol/tube.(ABSTRACT TRUNCATED AT 250 WORDS) |
