Shiomi Haraya, Koji Karasawa, Yoshihiro Sano, Kimiko Ozawa, Nobumasa Kato, Hidetoshi Arakawa

The peptide hormone oxytocin acts in the central nervous system and plays an important role in various complex social behaviors. We report the production of a high affinity and specificity antibody for oxytocin and its use in a highly sensitive enzyme immunoassay (EIA). Biotin that was chemically bound to oxytocin derivative containing zero to six lysines as bridge was the labeled antigen. Seven labeled antigens were used to develop a highly sensitive EIA. Anti-oxytocin antiserum was obtained by immunization of oxytocin-bovine thyrogloblin conjugate to rabbit.Oxytocin sample was added to the second antibody coated microtiter plate and allowed to react overnight at 4°C, then biotinylated oxytocin was added 1 hour at 4°C, and HRP labeled avidin was added and incubated for 1 hours at room temperature. The plate was then washed. HRP activity was measured by a colorimetric method using o-phenylenediamine (490nm). The sensitivity of the EIA improved as the number of lysine residues increased; consequently, biotinylated oxytocin bridged with five lysines was used. A standard curve for oxytocin ranged from 1.0 to 1000 pg/assay. The detection limit of the assay was 2.36 pg, and the reproducibility was 3.18% as CV% (n=5). Cross-reactivity with vasopressin and vasotocin was less than 0.01%. The sensitivity of the EIA could be improved by increasing the number of lysine residues on the biotin-labeled antigen. The proposed method is sensitive and more specific than conventional immunoassays for oxytocin and can be used to determine plasma oxytocin levels.