Radioimmunoassay of 5-hydroxy-3-indole acetic acid.

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 5hydroxytryptamine 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 5hydroxytryptamine 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 I-labeled Bolton-Hunter reagent5-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 s diluents, contributing to the fiii l dilution of l : 1100. In the analysis of serum, a deproteination step (ethanol precipitation) prior to methylation was necessary to obtain reproducible results. The methylated 5hydroxy-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 μιηοΐ/l (0.21 mg/1 5-hydroxy-3-indole acetic acid) urine or 100 fmol/tube. The intra-assay precision (CV) for urine samples was 6.4% (n = 20) at a level of 22 jimol/l, and 9.6% (n = 20) at a level of 230 μπιοΐ/ΐ. The interassay CV was 1 1 % at a level of 230 μιηοΐ/ΐ. The only substance crpss-reacting with the antibody was Nacetylserotonin which was not detectable in urine when the esterification step was omitted. To validate the clinical usefulness of this assay, a comparison with the commercially available BioRad® column assay was performed. Both radioimmunoassay and fluorescence determination accurately identified two patients with known carcinoid syndrome. A correlation of r = 0.817 was demonstrated between the two assays in a comparison of normal and pathological urines. A simultaneous determination of serotonin and its metabolite 5-hydroxy-3-indole acetic acid in normal and pathological sera showed that both parameters were raised in carcinoid syndrome.