本文關(guān)鍵詞： 膝溝藻毒素 單克隆抗體 酶免疫檢測法　出處：《暨南大學(xué)》2006年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：制備膝溝藻毒素2，3(GTX2，3)的單克隆抗體(McAbs)，并對其進(jìn)行鑒定，初步建立起GTX2，3的間接及直接競爭酶免疫學(xué)檢測方法(ELISA)，為該毒素酶免疫學(xué)快速檢測試劑盒的研制奠定基礎(chǔ)。 方法：利用醛化法將GTX2，3與載體蛋白血藍(lán)蛋白(KLH)偶聯(lián)，制備完全抗原。以GTX2，3-KLH作為免疫原免疫Balb／C小鼠，同時(shí)用高碘酸鹽氧化法將該毒素與葡萄糖氧化酶(GOX)偶聯(lián)，得到的GTX2，3-GOX偶聯(lián)物作為檢測抗原。應(yīng)用雜交瘤技術(shù)制備抗GTX2，3的單克隆抗體。常規(guī)方法鑒定該單克隆抗體的亞類及親和力，間接ELISA檢測腹水中抗體效價(jià)和抗體的特異性，Protein A親和層析純化腹水中單克隆抗體。初步建立檢測該毒素的間接競爭、直接競爭酶免疫學(xué)檢測方法，比較兩種方法的靈敏度、檢測限、工作范圍。 結(jié)果：分別用醛化法和高碘酸鹽氧化法成功制備出GTX2，3-KLH和GTX2，3-GOX偶聯(lián)物；獲得一株穩(wěn)定分泌抗GTX2，3單克隆抗體的雜交瘤細(xì)胞株CD1，該細(xì)胞株分泌的抗體屬于IgG1，腹水中抗體效價(jià)為2.5×10~5，親和力為1.2×10~(-8)mol／1，該抗體與載體蛋白BSA、KLH和GOX均無交叉反應(yīng)，特異性好。初步建立了檢測麻痹性貝類毒素的間接、直接競爭酶免疫學(xué)檢測方法，兩種方法的靈敏度分別為15μg／ml和6μg／ml，檢測限分別為6μg／ml和0.6μg／ml，工作范圍分別為6-50μg／ml和0.6-50μg／ml。 結(jié)論：運(yùn)用醛化法或高碘酸鹽氧化法能夠?qū)⒍舅谿TX2，，3與載體蛋白偶聯(lián)。利用小鼠雜交瘤技術(shù)獲得了一株分泌抗GTX2，3單克隆抗體的雜交瘤細(xì)胞株，該單克隆抗體親和力高，特異性好。利用該單克隆抗體初步建立了GTX2，3的間接和直接競爭酶免疫學(xué)檢測方法，但兩種檢測方法的靈敏度尚未達(dá)到海產(chǎn)品中毒素殘留最低限量(80μg／100g)的要求，還需進(jìn)一步優(yōu)化。
[Abstract]:Objective: to prepare and identify the monoclonal antibody McAbsN of geniculatoxin 2, 3, GTX2, and to establish a preliminary GTX2. 3. The indirect and direct competitive enzyme immunological detection method (ELISAA) lays a foundation for the development of the rapid detection kit for the toxin enzyme immunology. Methods: the complete antigen was prepared by coupling GTX2H3 with carrier protein hemocyanin KLH. GTX2O3-KLH was used as immunogen to immunize Balb/C mice. At the same time, the toxin was coupled with glucose oxidase (GOX) by periodate oxidation method, and the GTX2O3-GOX conjugate was used as the detection antigen. The hybridoma technique was used to prepare anti-GTX2O3-GOX. Routine method was used to identify the subclass and affinity of the monoclonal antibody, and indirect ELISA was used to detect the titer and specificity of the antibody in ascites. Protein A affinity chromatography was used to purify monoclonal antibody in ascites. Indirect competition and direct competition enzyme immunoassay were established to detect the toxin. The sensitivity and detection limit of the two methods were compared. Scope of work Results: GTX2O3-KLH and GTX2O3-GOX coupling compounds were successfully prepared by aldehyde method and periodate oxidation method, respectively. A hybridoma cell line CD1 was obtained, which secreted monoclonal antibody against GTX2H3 stably. The antibody secreted by the cell line belonged to IgG1, and the titer of antibody in ascites was 2.5 脳 10 ~ (5). The affinity was 1.2 脳 10 ~ (-8) mol / 1, and the antibody had no cross reaction with the carrier protein BSA-KLH and GOX, and had good specificity. The indirect detection of paralytic shellfish toxin was established. The sensitivity of the two methods were 15 渭 g / ml and 6 渭 g / ml, respectively, and the detection limits were 6 渭 g / ml and 0.6 渭 g / ml, respectively. The working ranges were 6-50 渭 g / ml and 0.6-50 渭 g / ml, respectively. Conclusion: the toxin GTX2O3 can be conjugated to the carrier protein by the method of formaldehyde or periodate oxidation. A strain secreting anti-#en0# was obtained by using mouse hybridoma technique. (3) the hybridoma cell line of monoclonal antibody has high affinity and good specificity. The indirect and direct competitive enzyme immunoassay method of GTX2H3 was established by using this monoclonal antibody. However, the sensitivity of the two methods is not up to the minimum limit of 80 渭 g / 100 g of residual toxin in seafood, which needs further optimization.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 卞正和 ,郭皓;海洋赤潮——大海向人類發(fā)出的警告[J];大自然;2004年06期

2 許敏,趙以軍,程凱;水華和赤潮的毒素及其檢測與分析[J];湖泊科學(xué);2001年04期

3 胡顥琰,唐靜亮,王益鳴,黃備,陳逸華,毛宏躍;浙江近岸海域有害赤潮發(fā)生區(qū)麻痹性貝毒素研究[J];環(huán)境污染與防治;2005年06期

4 林燕棠,楊美蘭,陳瑞雯,胡石金,金桂英;廣東沿海麻痹性貝類毒素的研究[J];海洋與湖沼;1994年02期

5 周名江,李鈞,于仁誠,顏天,傅萌;赤潮藻毒素研究進(jìn)展[J];中國海洋藥物;1999年03期

6 朱小兵,向軍儉;赤潮藻毒素檢測研究進(jìn)展(綜述)[J];暨南大學(xué)學(xué)報(bào)(自然科學(xué)與醫(yī)學(xué)版);2002年05期

7 向軍儉,朱小兵,呂頌輝,楊紅宇,凌欽婕,沈曉濤;五種赤潮藻單克隆抗體的制備[J];暨南大學(xué)學(xué)報(bào)(自然科學(xué)與醫(yī)學(xué)版);2003年03期

8 秦志高;海洋中的赤潮現(xiàn)象及其防治對策[J];南通航運(yùn)職業(yè)技術(shù)學(xué)院學(xué)報(bào);2005年03期

9 劉智勇;計(jì)融;;麻痹性貝類毒素研究進(jìn)展(綜述)[J];中國熱帶醫(yī)學(xué);2006年02期

10 黃長江;赤潮研究及其展望[J];生命科學(xué);1999年S1期

本文編號：1446350