黄曲霉毒素广谱特异性抗体的制备及总量检测免疫试纸方法的建立

发布时间：2018-02-25 16:23

本文关键词： 黄曲霉毒素总量检测人工免疫原单克隆抗体免疫试纸检测方法　出处：《河南科技学院》2017年硕士论文　论文类型：学位论文

【摘要】：黄曲霉毒素(aflatoxin,AF)是由曲霉菌属产生的具有毒性作用的次生代谢产物。AF对人体健康具有急性、慢性、致癌性、免疫抑制等多种毒性危害作用,自然条件下产生的AF主要包括B1、B2、G1、G2 4种,且这4种毒素残留对人体健康的毒性往往具有协同和加性效应,实施黄曲霉毒素总量(total aflatoxins,TAFs)(B1+B2+G1+G2)检测已成为当前食品质量安全检测领域发展的必然趋势。比较各种TAFs检测方法,GICA技术具有快速、简便、特异性强、稳定性好、可现场检测、筛检样品量大等优势,是实现TAFs检测的主要技术手段。本研究的目是建立TAFs免疫试纸检测方法,为食品TAFs快速检测提供可靠的技术支撑。本研究工作的主要内容与结论如下:1.根据AF的分子结构和活性位点,设计出4种B族AF和3种G族AF抗原制备方法,通过UV、SDS-PAGE和动物免疫对抗原进行鉴定。结果表明,OAE法是B族AF抗原合成的最佳方法,AFB1与BSA的分子结合比为8.46∶1,动物免疫所产生AFB1pAb具有高效价(免疫动物易产生抗体)、敏感(IC50为17.03μg/kg)、特异(100%识别AFB1)、广谱(同时100%识别AFB2)等特性;SA法是G族AF抗原合成的最佳方法,AFG1与BSA的分子结合比为4.32∶1,动物免疫所产生AFG1 pAb同样具有高效价(免疫动物易产生抗体)、敏感(IC50为13.6μg/kg)、特异(100%识别AFG1)、广谱(与AFG2的交叉反应率为82.19%)等特性。3.分别用AFB1-BSA(OAE)和AFG1-BSA(SA)免疫Balb/C小鼠,细胞融合技术筛选AFB1 mAb和AFG1 mAb细胞株,体内诱生腹水法制备AFB1 mAb和AFG1 mAb,并对其免疫学特性进行分析。结果表明,筛选出AFB1 mAb细胞株4株,其中2A11最好,经9次传代分泌抗体稳定,细胞培养上清抗体效价1∶(1.28×103),腹水抗体效价为1∶(5.12×105),Ka为1.05×109 L/moL,IC50为6.22μg/kg,可100%识别AFB1,与AFB2、AFG1和AFG2交叉反应率(cross-reactivity,CR)分别为82.31%、50.11%和12.48%,制备出了效价高、敏感性好、特异性强AFB1 mAb;筛选出AFG1 mAb细胞株2株,其中3F10最好,经6次传代分泌抗体稳定,细胞培养上清抗体效价1∶(1.28×104),腹水抗体效价为1∶(5.12×105),Ka为1.36×1010L/moL,IC50为5.04μg/kg,可100%识别AFG1,与AFG2的CR%为85.81%,与AFB1、AFB2无CR,制备出了效价高、敏感性好、特异性强AFG1 mAb,研究结果为AFs免疫试纸检测方法的建立奠定了抗体基础。4.根据胶体金免疫层析技术原理,建立了TAFs免疫试纸(TAFs-Strip)检测方法,TAFs-Strip具有快速(10 min)、灵敏(4μg/L)、特异(只与AF反应,与其他化合物无CR)、广谱(可同时检测AFB1、B2、G1、G2)、简便(不需要任何仪器与附加试剂)的优点,符合国内外TAFs检测限量标准,具有广阔的市场前景和应用价值。
[Abstract]:Aflatoxin AFB is a toxic secondary metabolite produced by Aspergillus. AF has acute, chronic, carcinogenic, immunosuppressive and other toxic effects on human health. Moreover, the toxicity of these four toxins to human health often has synergistic and additive effects. The application of total aflatoxins B1B2 G2) detection has become an inevitable trend in the field of food quality and safety detection. Compared with various TAFs detection methods, gica technique is quick, simple, specific, stable and can be detected in the field. The advantages of sieving large amount of samples are the main technical means to realize the detection of TAFs. The purpose of this study is to establish the test method of TAFs immune test paper. The main contents and conclusions of this study are as follows: 1. According to the molecular structure and active sites of AF, 4 kinds of group B AF and 3 kinds of G group AF antigens were designed. The antigens were identified by SDS-PAGE and animal immunity. The results showed that the best method for the synthesis of AF antigen in group B was the molecular binding ratio of AFB1 to BSA was 8.46: 1. The AFB1pAb produced by animal immunity had high titer (immunized animals were prone to produce antibodies). The sensitive IC50 is 17.03 渭 g / kg ~ (-1), the specificity is 100%, and the wide spectrum (100%) is the best method for the synthesis of G group AF antigen. The molecular binding ratio of AFG1 to BSA is 4.32: 1. The AFG1 pAb produced by animal immunity also has high titer (immune animals). The sensitive IC50 was 13.6 渭 g 路kg ~ (-1) 路kg ~ (-1), the specificity was 100%, and the broad spectrum (the cross reaction rate with AFG2 was 82.19). 3. The Balb/C mice were immunized with AFB1-BSA-OAE and AFG1-BSA-SAA, respectively. AFB1 mAb and AFG1 mAb cell lines were screened by cell fusion technique, and AFB1 mAb and AFG1 mAbwere prepared by inducing ascites in vivo, and their immunological characteristics were analyzed. The results showed that 4 AFB1 mAb cell lines were screened, of which 2A11 was the best. After 9 passages, the antibody titer of supernatant of cell culture was 1.28 脳 10 ~ 3g ~ (-1), the titer of ascites antibody was 1.05 脳 10 ~ (5) 脳 10 ~ (5) L ~ (-1) L ~ (-1) I = 1.05 脳 10 ~ (9) L ~ (-1) L ~ (-1) I = 6.22 渭 g 路kg ~ (-1), AFB _ (1) was recognized by 100%, and the cross reaction rate with AFB _ (2) AFG1 and AFG2 was 82.31% and 12.48%, respectively. Two AFG1 mAb cell lines were screened, of which 3F10 was the best, and the antibody secreted after 6 passages was stable. The antibody titer of supernatant of cell culture was 1: 1. 28 脳 10 ~ (4g), the antibody titer of ascites was 1: 1 / 10 ~ (5) 脳 10 ~ (5) C ~ (-1) KA = 1.36 脳 10 ~ (10) L / mol / L IC50 was 5.04 渭 g / kg, the recognition rate of AFG _ (1) was 100% 渭 g / kg, the CR% with AFG2 was 85.81%, and the antibody titer was higher and the sensitivity was better than that of AFB _ (1) and AFB _ (2). According to the principle of colloidal gold immunochromatographic technique, a method for the detection of TAFs-Strip in TAFs immunized test paper was established. The Tafs-Strip has the advantages of rapid detection of 10 mins, sensitivity of 4 渭 g / L 路L ~ (-1), and specificity (only with AF). Compared with other compounds, it is not CR-free, broad-spectrum (can simultaneously detect AFB _ 1B _ 2H _ 2H _ 1G _ (1) G _ (2)), simple (without any instruments and additional reagents). It conforms to the domestic and foreign TAFs detection limit standard, and has broad market prospect and application value.
【学位授予单位】：河南科技学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S859.8

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