SMCC法抗体定向偶联技术的优化与应用研究

发布时间：2018-06-12 06:32

  本文选题：定向偶联 + 抗体　； 参考：《华中科技大学》2011年硕士论文

【摘要】：蛋白质偶联技术是采用一定的技术手段将具有生物活性的蛋白质与其它载体分子或标记物结合在一起的一种方法,在生命科学和医学领域有着广泛的应用。在标记免疫分析方法中,抗体与酶等标记物的偶联效果会直接影响到免疫方法的灵敏度和可靠性。 常用的偶联方法如戊二醛法、碳二亚胺法、过碘酸钠氧化法法等不可避免地会产生酶或抗体的自身交联产物或多聚物,致使偶联效率降低、结合物活性减弱。在此基础上发展起来的异性双功能偶联剂虽然可以克服这一不足,但酶在抗体上的连接位点具有随机性,导致抗体和酶的活性会受到影响。因此,在实际应用中,需建立一种能定向偶联的抗体偶联技术。 本研究采用SMCC法,研究优化了免疫球蛋白IgG与辣根过氧化物酶HRP的偶联条件,主要研究内容包括: 1)采用体内诱生腹水法制备氯霉素单克隆抗体,并对其活性进行测定,建立竞争曲线。 2)从投料比、反应温度、反应时间三个方面对抗体的DTT还原反应过程进行了研究,探讨了还原条件对免疫球蛋白的结构、活性等方面的影响。 3)采用SMCC法将抗体与HRP偶联,并对其活性进行鉴定,并将其用于酶联免疫吸附实验。 研究结果表明,DTT还原反应的温度和时间对抗体结构和活性影响不大,而DTT的加入量则有明显影响:在封闭巯基的条件下,投料比小于为600时,还原产物以大分子片段居多,当投料比大于600后,产物主要为小分子片段,且抗体活性开始下降,到6000时活性几乎完全丧失;在未封闭巯基条件下,由于游离巯基会重新聚合,当投料比在600～12000之间,抗体活性与产物的组成差异均不明显。在将其应用于羊抗小鼠二抗与HRP的偶联中,确定优化的SMCC法偶联条件为DTT与抗体投料的比为8000,反应温度为25℃,反应时间在20～30min。
[Abstract]:Protein coupling is a method of combining bioactive proteins with other carrier molecules or markers by certain means. It has been widely used in life sciences and medicine. In labeling immunoassay, the coupling effect of antibody and enzyme will directly affect the sensitivity and reliability of the immunological method, such as glutaraldehyde method, carbodiimide method, and so on. Sodium periodate oxidation process will inevitably produce autocrosslinking products or polymers of enzymes or antibodies, resulting in lower coupling efficiency and weaker conjugate activity. Although the heterosexual bifunctional coupling agent developed on this basis can overcome this deficiency, the binding site of enzyme on antibody is random, which results in the influence of antibody and enzyme activity. Therefore, in practical application, it is necessary to establish an antibody coupling technique that can be directional coupled. In this study, the coupling conditions of immunoglobulin IgG and horseradish peroxidase HRP were optimized by SMCC method. The main research contents are as follows: 1) Monoclonal antibody of chloramphenicol was prepared by in vivo induced ascites method, and its activity was determined to establish competition curve. 2) from feed ratio, reaction temperature, The DTT reduction process of antibody was studied from three aspects of reaction time. The effects of reduction conditions on the structure and activity of immunoglobulin were discussed. 3) the antibody was coupled with HRP by SMCC method and its activity was identified. The results showed that the temperature and time of DTT reduction reaction had little effect on the structure and activity of antibody, but the addition amount of DTT had obvious effect: when the feed ratio was less than 600 under the condition of closed sulfhydryl group, When the feed ratio was greater than 600, the reduction products were mainly small molecular fragments, and the antibody activity began to decrease, and the activity was almost completely lost at 6000, and the free sulfhydryl group would be repolymerized under the condition of unenclosed sulfhydryl group. When the feed ratio was between 600 and 12000, there was no significant difference in antibody activity and product composition. In the coupling of sheep anti-mouse second antibody and HRP, the optimized coupling conditions of SMCC method were determined as follows: the ratio of DTT to antibody was 8,000, the reaction temperature was 25 鈩，

本文编号：2008724