title: Development of a highly specific enzyme immunoassay for oxytocin and its use in plasma samples.
abstract: Background The peptide hormone oxytocin acts in the central nervous system and plays an important role in various complex social behaviours. We report the production of a high affinity and specificity antibody for oxytocin and its use in a highly sensitive enzyme immunoassay. Biotin that was chemically bound to oxytocin derivative containing zero to six lysines as bridge was the labelled antigen. Seven labelled antigens were used to develop a highly sensitive enzyme immunoassay. Methods Antioxytocin antiserum was obtained by immunization of oxytocin-bovine thyrogloblin conjugate to rabbit. Oxytocin sample was added to the second antibody-coated microtitre plate and allowed to react overnight at 4℃, then biotinylated oxytocin was added 1 h at 4℃, and horseradish peroxidase-labelled avidin was added and incubated for 1 h at room temperature. The plate was then washed. Horseradish peroxidase activity was measured by a colorimetric method using o-phenylenediamine (490 nm). Results The sensitivity of the enzyme immunoassay 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 3.6% as CV% ( n = 6). Cross-reactivity with vasopressin and vasotocin was less than 0.01%. Conclusion The sensitivity of the enzyme immunoassay could be improved by increasing the number of lysine residues on the biotin-labelled antigen. The proposed method is sensitive and more specific than conventional immunoassays for oxytocin and can be used to determine plasma oxytocin concentrations.
authors.affiliation: 1 Department of Analytical Biochemistry, School of Pharmacy, Showa University, Shinagawa, Tokyo, Japan.
authors.forename: Shiomi
authors.initials: S
authors.lastname: Haraya
authors.affiliation: 1 Department of Analytical Biochemistry, School of Pharmacy, Showa University, Shinagawa, Tokyo, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Karasawa
authors.affiliation: 1 Department of Analytical Biochemistry, School of Pharmacy, Showa University, Shinagawa, Tokyo, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Sano
authors.affiliation: 1 Department of Analytical Biochemistry, School of Pharmacy, Showa University, Shinagawa, Tokyo, Japan.
authors.forename: Kimiko
authors.initials: K
authors.lastname: Ozawa
authors.affiliation: 2 Karasuyama Hospital, Showa University School of Medicine, Tokyo, Japan.
authors.forename: Nobumasa
authors.initials: N
authors.lastname: Kato
authors.affiliation: 1 Department of Analytical Biochemistry, School of Pharmacy, Showa University, Shinagawa, Tokyo, Japan.
authors.forename: Hidetoshi
authors.initials: H
authors.lastname: Arakawa
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000302:isolation & purification; D001321:Autistic Disorder / Q000097:blood* / Q000175:diagnosis; D001360:Avidin / Q000737:chemistry; D001710:Biotin / Q000737:chemistry*; D019921:Biotinylation; D002417:Cattle; D003124:Colorimetry / Q000379:methods; D006735:Horseradish Peroxidase / Q000737:chemistry; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry; D007124:Immunoenzyme Techniques / Q000592:standards*; D010121:Oxytocin / Q000097:blood*; D011107:Polylysine / Q000737:chemistry*; D011817:Rabbits; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D000906:Antibodies; D018796:Immunoconjugates; D001360:Avidin; D011107:Polylysine; D010121:Oxytocin; D001710:Biotin; D006735:Horseradish Peroxidase
keywords: Oxytocin; autistic spectrum disorder; enzyme immunoassay; peptide hormone; the biotin-labelled antigen