本文選題：氯霉素 + 獸藥殘留��； 參考：《河南科技大學(xué)》2017年碩士論文

【摘要】：氯霉素(chloramphenicol,ACP)作為動(dòng)物源性食品中殘留問題較為突出的藥物,建立嚴(yán)格的檢測(cè)方法已勢(shì)在必行。酶聯(lián)免疫分析法用于檢測(cè)獸藥殘留具有簡便快速,高效穩(wěn)定等優(yōu)點(diǎn),適合動(dòng)物性食品殘留的現(xiàn)場(chǎng)檢測(cè)。本課題以合成抗原的為基礎(chǔ),經(jīng)過細(xì)胞融合后,制備單克隆抗體,并建立間接競(jìng)爭ELISA的檢測(cè)方法。利用混合酸酐法使氯霉素半抗原(CAP-HS)與牛血清白蛋白(BSA)偶聯(lián),獲得免疫原CAP-HS-BSA;與雞卵清蛋白(OVA)偶聯(lián),制備檢測(cè)抗原CAP-OVA,利用紫外掃描(UV)和SDS一聚丙烯酞胺凝膠電泳(SDS-PAGE)鑒定CAP-HS-BSA:CAP-HS-BSA紫外掃描圖的最大吸收峰發(fā)生偏移,CAPBSA的泳動(dòng)率小于BSA,判定CAP-BSA成功偶聯(lián),并推算出CAP-HS與BSA分子結(jié)合比為12.1∶1。CAP-HS-BSA免疫3只BALB/c小鼠,4次免疫后用間接ELISA檢測(cè)CAP鼠源多克隆抗體(CAP pAb),其效價(jià)均大于1∶6.4×103,用間接競(jìng)爭ELISA(ci-ELISA)檢測(cè)CAP pAb的半數(shù)抑制濃度(IC50),其中測(cè)定結(jié)果最好的3號(hào)小鼠的IC50為16.4μg/L,CAP pAb與CAP的結(jié)構(gòu)類似物及常用抗生素沒有交叉反應(yīng),獲得高效價(jià),特異性強(qiáng)的抗CAP多克隆抗體。ELISA篩選細(xì)胞融合鼠,融合小鼠脾細(xì)胞與SPNS0瘤細(xì)胞,得到雜交瘤細(xì)胞株,體內(nèi)誘生腹水法生產(chǎn)CAP單克隆抗體(CAP mAb),選取4株敏感度和選擇性均好的抗CAP雜交瘤細(xì)胞:1B8 CAP mAb、2C4 CAP mAb、4A1 CAP mAb,其細(xì)胞上清效價(jià):1∶2.4×102、1∶6.4×102、1∶1.2×102,腹水效價(jià):1∶2.0×105、1∶4.8×105、1∶1.2×105。ci-ELISA測(cè)定親和力最高的2C4株的IC50為0.53μg/L,與其類似物沒有交叉反應(yīng)。以CAP mAb為基礎(chǔ),優(yōu)化了ci-ELISA最佳實(shí)驗(yàn)條件:CAP-HS-OVA的包被濃度:0.4μg/mL,CAP mAb的工作濃度:1:6.4×104;羊抗鼠酶標(biāo)二抗(GaMIgG-HRP)的稀釋濃度為1:1000;包被條件:包被原CAP-OVA的包被濃度0.4μg/mL,37℃,孵育2h,5%的豬血清封閉,37℃,孵育1h;室溫條件下,顯色液作用時(shí)間為9min。繪制出的CAP殘留的ci-ELISA的標(biāo)準(zhǔn)曲線顯示為典型的S型,與4參數(shù)logit擬合曲線相吻合,IC50為0.53 ng/m L,檢測(cè)限為0.6 ng/mL;添加回收實(shí)驗(yàn),陰性的魚肉、牛奶的平均回收率分別為97.3%和97.5%,平均變異系數(shù)為4.9%、5.5%,CV均小于10%。HPLC對(duì)比實(shí)驗(yàn):CiELISA測(cè)定結(jié)果與樣品值的差異是3.30%~4.40%,Ci-ELISA與HPLC的測(cè)定值差異是1.13%~4.85%;實(shí)際樣品的測(cè)定:河蝦樣品中,Ci-ELISA對(duì)HPLC的差異為4.43%。
[Abstract]:Chloramphenicolium (ACP) is an important drug in animal food. It is imperative to establish a strict method for detection of chloramphenicolium. Enzyme-linked immunosorbent assay (Elisa) has the advantages of simplicity, rapidity, high efficiency and stability, and is suitable for field detection of animal food residues. Based on synthetic antigen, monoclonal antibody was prepared after cell fusion, and indirect competitive Elisa method was established. Chloramphenicol hapten (CAP-HS) was coupled with bovine serum albumin (BSA) by mixed anhydride method to obtain the immunogen CAP-HS-BSAand chicken ovalbumin (OVA). The detection antigen CAP-OVA was prepared. The UV scanning and SDS-PAGE were used to identify the maximum absorption peak of CAP-HS-BSA-HS-BSA. The swimming mobility of CAP-OVA was less than that of BSA-BSA, and the successful coupling of CAP-BSA was determined. The binding ratio of CAP-HS to BSA was calculated to be 12.1: 1. 1. CAP-HS-BSA was used to immunize three BALB / c mice. After 4 times immunization, the titers of CAP pAb), were detected by indirect Elisa. The titers of CAP pAb), were all greater than 1: 6.4 脳 103, and the half inhibitory concentration of CAP was detected by indirect competitive Elisa (ci-ELISA). (IC50), in which the IC50 of mice with the best results was 16.4 渭 g / L, and there was no cross-reaction between the structural analogues of CAP and CAP and common antibiotics. High titer and strong specificity of anti-CAP polyclonal antibody. Elisa was used to screen the fusion cells of mouse spleen cells and SPNS0 tumor cells, and the hybridoma cell lines were obtained. In vivo induced ascites production of CAP monoclonal antibody (CAP mAb), selection of four anti-CAP hybridoma cell lines with good sensitivity and selectivity: 1B8 CAP mAbmAb2CAP mAb2CAP 4A1 CAP mAb. the supernatant titer of the cell supernatant was 12.4 脳 102: 1: 1. 4 脳 10 2: 1. 2 脳 10 2% 1.2 脳 10 2). Ascites titer was 1: 2.0 脳 10 5% 1: 4.8 脳 10 5% 1: 1.2 脳 105.ci-ELISA. The IC50 of 2C4 strain with the highest affinity was 0.53 渭 g / L, and there was no cross reaction with its analogues. On the basis of CAP mAb, the optimal conditions for ci-ELISA were optimized. The optimal conditions for ci-ELISA were as follows: the encapsulation concentration of: CAP-HS-OVA was: 1: 0.4 渭 g / mL / min, the working concentration of CAP mAb was 1: 1: 6.4 脳 10 ~ (4), the dilution concentration of goat anti-mouse enzyme-labeled second antibody (GaMIgG-HRP) was 11000: the encapsulation conditions were as follows: the encapsulation concentration of the original CAP-OVA was 0.4 渭 g / mL ~ (-1) at 37 鈩，

本文編號(hào)：2107865