Development of a highly specific enzyme immunoassay for oxytocin and its use in plasma samples
Development of a highly specific enzyme immunoassay for oxytocin and its use in plasma samples
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DOI：
10.1177/0004563216645122
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发表时间：
2017-01-01
影响因子：
2.2
通讯作者：
Arakawa, Hidetoshi
中科院分区：
文献类型：
--
作者：
Haraya, Shiomi;Karasawa, Koji;Arakawa, Hidetoshi
背景：肽激素催产素作用于中枢神经系统，在各种复杂的社会行为中起重要作用。我们报道了一种高亲和力和特异性的催产素抗体的产生及其在高灵敏度酶免疫测定中的应用。生物素与含有0到6个赖氨酸的催产素衍生物化学结合，作为桥梁被标记抗原。7种标记抗原用于开发高灵敏度的酶免疫测定。方法：兔免疫后叶催产素-牛甲状腺球蛋白偶联物获得抗后叶催产素抗血清。将催产素样品加入第二抗体包被的微滴板，在4℃下反应过夜，然后在4℃下加入生物素化的催产素1小时，加入辣根过氧化物酶标记的亲和素，在室温下孵育1小时。然后盘子被洗了。用邻苯二胺（490 nm）比色法测定辣根过氧化物酶活性。结果：随着赖氨酸残基数的增加，酶免疫分析法的灵敏度提高；因此，生物素化的催产素与5赖氨酸桥接被使用。催产素的标准曲线范围为1.0至1000 pg/次。该方法的检出限为2.36 pg，重复性为3.6% （n = 6）。与后叶加压素、后叶催产素的交叉反应性均小于0.01%。结论：增加生物素标记抗原上赖氨酸残基的数量可以提高酶免疫分析法的灵敏度。该方法比传统的催产素免疫测定法更敏感、更特异，可用于测定血浆催产素浓度。
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 degrees C, then biotinylated oxytocin was added 1 h at 4 degrees C, 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.