本文選題：定向偶聯(lián) + 抗體　； 參考：《華中科技大學》2011年碩士論文

【摘要】：蛋白質(zhì)偶聯(lián)技術是采用一定的技術手段將具有生物活性的蛋白質(zhì)與其它載體分子或標記物結合在一起的一種方法,在生命科學和醫(yī)學領域有著廣泛的應用。在標記免疫分析方法中,抗體與酶等標記物的偶聯(lián)效果會直接影響到免疫方法的靈敏度和可靠性。 常用的偶聯(lián)方法如戊二醛法、碳二亞胺法、過碘酸鈉氧化法法等不可避免地會產(chǎn)生酶或抗體的自身交聯(lián)產(chǎn)物或多聚物,致使偶聯(lián)效率降低、結合物活性減弱。在此基礎上發(fā)展起來的異性雙功能偶聯(lián)劑雖然可以克服這一不足,但酶在抗體上的連接位點具有隨機性,導致抗體和酶的活性會受到影響。因此,在實際應用中,需建立一種能定向偶聯(lián)的抗體偶聯(lián)技術。 本研究采用SMCC法,研究優(yōu)化了免疫球蛋白IgG與辣根過氧化物酶HRP的偶聯(lián)條件,主要研究內(nèi)容包括: 1)采用體內(nèi)誘生腹水法制備氯霉素單克隆抗體,并對其活性進行測定,建立競爭曲線。 2)從投料比、反應溫度、反應時間三個方面對抗體的DTT還原反應過程進行了研究,探討了還原條件對免疫球蛋白的結構、活性等方面的影響。 3)采用SMCC法將抗體與HRP偶聯(lián),并對其活性進行鑒定,并將其用于酶聯(lián)免疫吸附實驗。 研究結果表明,DTT還原反應的溫度和時間對抗體結構和活性影響不大,而DTT的加入量則有明顯影響:在封閉巰基的條件下,投料比小于為600時,還原產(chǎn)物以大分子片段居多,當投料比大于600后,產(chǎn)物主要為小分子片段,且抗體活性開始下降,到6000時活性幾乎完全喪失;在未封閉巰基條件下,由于游離巰基會重新聚合,當投料比在600～12000之間,抗體活性與產(chǎn)物的組成差異均不明顯。在將其應用于羊抗小鼠二抗與HRP的偶聯(lián)中,確定優(yōu)化的SMCC法偶聯(lián)條件為DTT與抗體投料的比為8000,反應溫度為25℃,反應時間在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，

本文編號：2008724