Radioimmunoassay of 5-hydroxyindole acetic acid using an iodinated derivative.
A radioimmunoassay for the main catabolite of serotonin, 5-hydroxyindole acetic acid (5-HIAA), was developed by using specific antibodies and iodinated derivative. The synthesis of a 125I-iodinated analog was performed by coupling 5-HIAA to [125I-]glycyl-tyrosine without any contact between 5-HIAA and iodine or chloramine T. It was purified on a G25 Sephadex column and diluted in citrate buffer up to 2.5 X 10(5) cpm/ml. Antibodies were obtained by coupling 5-HIAA to human serum albumin with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and tested by equilibrium dialysis. After the third immunogen injection, the four rabbits gave antisera capable of binding 50% of iodinated 5-HIIA-glycyl-tyrosine at 1/2000 final dilution. A chemical conversion of the biological samples gives to the antigen molecules a better resemblance to the immunogen, thus conferring a 100-fold gain in specificity and sensitivity. This assay allows 5-HIAA to be determined in small amounts of tissue, blood, cerebrospinal fluid or perfusate without purification with a sensitivity threshold below 0.1 ng. Some applications in cat and rat are presented.
D000818:Animals; D000918:Antibody Specificity; D001923:Brain Chemistry; D002415:Cats; D004151:Dipeptides / Q000138:chemical synthesis; D006897:Hydroxyindoleacetic Acid / Q000031:analogs & derivatives* / Q000032:analysis* / Q000097:blood / Q000138:chemical synthesis; D007457:Iodine Radioisotopes; D011863:Radioimmunoassay / Q000379:methods; D051381:Rats
D004151:Dipeptides; D007457:Iodine Radioisotopes; D006897:Hydroxyindoleacetic Acid; C030156:iodoglycyltyrosine 5-hydroxyindole acetic acid