Haraya Shiomi, Karasawa Koji, Sano Yoshihiro, Ozawa Kimiko, Kato Nobumasa, Arakawa Hidetoshi
1 Department of Analytical Biochemistry, School of Pharmacy, Showa University, Shinagawa, Tokyo, Japan.
2 Karasuyama Hospital, Showa University School of Medicine, Tokyo, Japan.
Ann Clin Biochem. 2017 Jan;54(1):101-106. doi: 10.1177/0004563216645122. Epub 2016 Sep 28.
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.
背景 肽类激素催产素在中枢神经系统中发挥作用，在各种复杂的社会行为中起重要作用。我们报告了一种针对催产素的高亲和力和特异性抗体的产生及其在高灵敏度酶免疫测定中的应用。化学结合到含有零至六个赖氨酸作为桥连的催产素衍生物上的生物素是标记抗原。使用七种标记抗原来开发高灵敏度酶免疫测定。方法 通过用催产素-牛甲状腺球蛋白偶联物免疫兔子获得抗催产素抗血清。将催产素样品加入包被有第二抗体的微量滴定板中，于4℃过夜反应，然后在4℃加入生物素化的催产素1小时，再加入辣根过氧化物酶标记的抗生物素蛋白并在室温下孵育1小时。然后洗涤平板。使用邻苯二胺（490nm）通过比色法测量辣根过氧化物酶活性。结果 随着赖氨酸残基数量的增加，酶免疫测定的灵敏度提高；因此，使用与五个赖氨酸桥连的生物素化催产素。催产素的标准曲线范围为每次测定1.0至1000 pg。该测定的检测限为2.36 pg，重复性为3.6%（变异系数，n = 6）。与加压素和催产素的交叉反应性小于0.01%。结论 通过增加生物素标记抗原上的赖氨酸残基数量可以提高酶免疫测定的灵敏度。所提出的方法对催产素具有敏感性且比传统免疫测定更具特异性，可用于测定血浆催产素浓度。
