食品科学 ›› 2019, Vol. 40 ›› Issue (14): 186-191.doi: 10.7506/spkx1002-6630-20180724-290

• 生物工程 • 上一篇    下一篇

糖皮质激素广谱特异性单克隆抗体的制备及其ic-ELISA方法的建立

姚添淇,劳翠瑜,王士峰,胡桂平,张世伟   

  1. 广东省深圳市计量质量检测研究院,国家营养食品质量监督检验中心,广东 深圳 518060
  • 出版日期:2019-07-25 发布日期:2019-07-23
  • 基金资助:
    深圳市科技计划项目(JCYJ20170817162119873)

Establishment of Indirect Competitive-Enzyme Linked Immunosorbent Assay to Detect Glucocorticoids: Preparation of Broad-Spectrum Monoclonal Antibody

YAO Tianqi, LAO Cuiyu, WANG Shifeng, HU Guiping, ZHANG Shiwei   

  1. National Nutrition Food Testing Center, Shenzhen Academy of Metrology & Quality Inspection, Shenzhen 518060, China
  • Online:2019-07-25 Published:2019-07-23

摘要: 将氢化可的松半抗原HYD通过活化脂法与钥孔戚血蓝蛋白(keyhole limpet hemocyanin,KLH)偶联获得氢化可的松完整抗原KLH-HYD,并对小鼠肌肉注射此抗原,使小鼠免疫;通过杂交瘤技术获得1 株能够稳定分泌氢化可的松的单克隆抗体细胞株HYD-C1,并通过腹水制备大量抗体。将氢化可的松半抗原HYD和地塞米松半抗原DEX通过活化脂法分别与卵清蛋白(ovalbumin,OVA)偶联作为包被原,建立糖皮质激素的间接竞争酶联免疫吸附检测(indirect competitive-enzyme linked immunosorbent assay,ic-ELISA)方法。结果:在同源包被情况下氢化可的松灵敏度为1.63 ng/mL,异源包被情况下灵敏度为0.24 ng/mL;在交叉抑制实验中,异源策略中的各糖皮质激素灵敏度均高于同源策略中的结果,异源策略条件下很好地覆盖了本研究中所检测的糖皮质激素,使该抗体具备良好的广谱特异性。在异源策略添加回收实验中平均添加回收率在77.5%~111.3%之间,落在合理范围内,满足检测需求。结论:成功制备出氢化可的松单克隆抗体,建立了同源和异源策略糖皮质激素检测方法,结果发现异源策略中糖皮质激素的灵敏度高于同源策略,并使该方法具有更好的广谱特异性。

关键词: 糖皮质激素, 同源策略, 异源策略, 广谱特异性, 间接竞争酶联免疫吸附(ic-ELISA)法

Abstract: A synthetic derivative of hydrocortisone (HYD) as a hapten was coupled to keyhole limpet hemocyanin (KLH) using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The KLHHYD conjugate as an immunogen was used to immunize mice. A stable hybridoma cell line HYD-C1 secreting monoclonal antibody against HYD was generated to induce large amounts of antibodies in the ascites of mice. Moreover, the HYD hapten and another hapten synthesized from dexamethasone (DEX) were conjugated separately to ovalbumin (OVA) to obtain coating antigens. The final aim of this study was to develop an indirect competitive-enzyme linked immunosorbent assay (ic-ELISA). The results showed that the IC50 of homologous ic-ELISA was 1.63 ng/mL while that of heterologous ic- ELISA was 0.24 ng/mL. Cross inhibition tests revealed that the sensitivity of heterologous ic-ELISA for all glucocorticoids tested was higher than homologous ic-ELISA, which shows the broad-spectrum specificity of heterologous ic-ELISA. The average recoveries of glucocorticoids from spiked liquid milk by heterologous ic-ELISA were in a reasonable range of 77.5% to 111.3%, meeting the requirements for detection. In conclusions, a monoclonal antibody specific for HYD was successfully prepared and used for homologous and heterologous ic-ELISA. Heterologous ic-ELISA was found to be more sensitive and have a broader-spectrum specificity.

Key words: glucocorticoids, homologous strategy, heterologous strategy, broad-spectrum specificity, indirect competitiveenzyme linked immunosorbent assay (ic-ELISA)

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