本文選題：赭曲霉毒素A + 單克隆抗體　； 參考：《山東大學(xué)》2010年碩士論文

【摘要】： 目的制備抗赭曲霉毒素A(ochratoxin A, OA)的單克隆抗體(monoclonal antibody, McAb)并對其進(jìn)行初步鑒定,在此基礎(chǔ)上建立競爭抑制酶聯(lián)免疫吸附試驗(competitive inhibition enzyme linked immunosorbent assay, ci-ELISA)用于OA的檢測。優(yōu)化小米和玉米中OA的提取方法,應(yīng)用建立的ci-ELISA檢測濟南市五個區(qū)的集市和超市出售的小米和玉米樣品中OA的含量,初步了解污染情況。 方法采用小劑量長周期的免疫方案,OA-牛血清白蛋白(bovine serum albumin, BSA)偶聯(lián)物免疫雌性BALB/c小鼠,分別以O(shè)A-BSA. BSA為包被抗原,用酶聯(lián)免疫吸附試驗(enzyme linked immunosorbent assay, ELISA)檢測小鼠血清中抗體的效價,觀察該McAb與BSA的交叉反應(yīng)情況。采用細(xì)胞融合的方法,以O(shè)A-BSA作為包被抗原,同時以BSA作對照,用ELISA法檢測雜交瘤細(xì)胞培養(yǎng)上清中的McAb,獲得分泌抗OA的McAb的雜交瘤細(xì)胞株,研究該McAb與BSA的交叉反應(yīng)情況,以評價McAb的特異性。以有限稀釋法對特異性分泌抗OA McAb的雜交瘤細(xì)胞進(jìn)行克隆化,以ci-ELISA法進(jìn)一步確定McAb的特異性,腹水誘生法大量制備McAb,采用辛酸-飽和硫酸銨法純化腹水中的McAb,以ELISA檢測純化前和純化后腹水效價,同時以BSA作包被抗原對照,研究交叉反應(yīng)情況,以O(shè)A為競爭抗原,建立ci-ELISA方法用于OA的檢測。選用不同濃度甲醇和氯化鈉的溶液作提取溶劑提取樣品中的OA,比較不同提取溶劑的加標(biāo)回收率,觀察提取溶劑中甲醇和氯化鈉的濃度對小米、玉米樣品中OA提取效果的影響,優(yōu)化OA提取方法。采集濟南市市中、歷下、槐蔭、天橋、歷城五個區(qū)的集市出售的小米和玉米、濟南市區(qū)超市出售的小米和玉米各50份,用所建立的ci-ELISA,采用標(biāo)準(zhǔn)加入法,對這些小米、玉米樣品中OA含量進(jìn)行了檢測,計算檢出率,并依照《中國國家標(biāo)準(zhǔn)GB2715—2005(糧食衛(wèi)生標(biāo)準(zhǔn))》中對谷類、豆類的OA的限量要求,計算超標(biāo)率。所有數(shù)據(jù)均經(jīng)SPSS軟件包處理。 結(jié)果OA-BSA免疫的BALB/c小鼠血清抗體效價為1：512000,與BSA有強烈的交叉反應(yīng)。細(xì)胞融合后,ELISA篩選抗體分泌陽性的雜交瘤細(xì)胞株,抗OA-BSA的McAb與BSA的交叉反應(yīng)率僅為3.50%,對分泌抗OA-BAS特異的McAb的細(xì)胞株經(jīng)3輪克隆化,抗體分泌陽性率達(dá)到100%,建立了1株能穩(wěn)定分泌抗OA-BSAMcAb的雜交瘤細(xì)胞株,ci-ELISA進(jìn)一步證明了該抗體是特異針對OA的。腹水誘生法制備了大量的McAb,純化后腹水抗體效價為1：512000,而且與BSA無交叉反應(yīng)。ci-ELISA線性范圍為0.04ng／ml-1250.OOng／ml,標(biāo)準(zhǔn)曲線的線性方程y=-0.1368x+0.6796,相關(guān)系數(shù)r=0.9965,檢測下限為0.04ng／ml。當(dāng)提取溶劑中甲醇含量為60%且含有5%氯化鈉時對OA的提取效果較為理想,OA加標(biāo)量為50.OOng／g、25.OOng／g、12.50ng／g時,以該提取溶劑進(jìn)行確證實驗,小米各加標(biāo)量的平均回收率(%)分別為93.52%、96.86%、95.66%；變異系數(shù)(CV%)分別為4.59%、8.42%、8.94%,平均7.32%；玉米各加標(biāo)量的平均回收率分別為96.30%、98.67%、98.13%,變異系數(shù)(CV%)分別為5.74%、5.59%、9.56%,平均6.96%。用于檢測小米中OA的ci-ELISA工作曲線線性方程為y=-0.3249x+1.079,相關(guān)系數(shù)r=0.9959,用于檢測玉米中OA的ci-ELISA工作曲線線性方程為y=-0.2554x+0.9561,相關(guān)系數(shù)r=0.9971。小米、玉米OA檢測下限均為0.04ng／ml,OA最低限出量均為0.2ng／g。采集的所有樣品中,小米OA檢出率為41%(41／100),玉米OA的檢出率為38%(38／100),二者無顯著性差異(P0.05),其中,集市小米OA的檢出率為26%(13／50),集市玉米OA檢出率為32%(16／50),二者無顯著性差異(P0.05)；超市小米OA檢出率為56%(28／50),超市玉米OA檢出率為44%(22／50),二者無顯著性差異(P0.05)；檢測的所有小米樣品中,超市小米OA檢出率(56%)明顯高于集市小米(26%)(P0.05)；檢測的所有玉米樣品中,超市玉米(44%)和集市玉米(32%)OA檢出率無顯著性差異(P0.05)。采集的所有樣品中,小米(0.40-4.45ug/kg,中位數(shù)為1.73 ug/kg)和玉米(0.48-4.02ug/kg,中位數(shù)為1.61 ug/kg) OA含量無顯著性差異(P0.05)。其中,集市小米(0.41-3.94ug／kg,中位數(shù)為1.63 ug／kg)和集市玉米(0.53-3.99ug/kg,中位數(shù)為1.52 ug/kg)、超市小米(0.40-4.45ug/kg,中位數(shù)為1.78ug/kg)和超市玉米(0.48-4.02ug/kg,中位數(shù)為1.74 ug/kg) OA含量均無顯著性差異(P0.05)。檢測的所有小米樣品中,超市小米(中位數(shù)為1.78ug/kg)與集市小米(中位數(shù)為1.63ug/kg)OA含量無顯著性差異(P0.05),檢測的所有玉米樣品中超市玉米(中位數(shù)為1.74ug/kg)和集市玉米(中位數(shù)為1.52ug/kg) OA含量均無顯著性差異(P0.05)。本研究檢測的所有樣品的OA含量在0.40-4.45ug/kg之間,中位數(shù)為1.67ug/kg,根據(jù)《中國國家標(biāo)準(zhǔn)GB2715—2005(糧食衛(wèi)生標(biāo)準(zhǔn))》中對谷類、豆類的OA的限量≤5ug／kg的要求,所有樣品中的OA含量均未超標(biāo)。 結(jié)論獲得了分泌抗OA的McAb的雜交瘤細(xì)胞株。半抗原單抗的制備過程中,免疫抗原和檢測抗原可以用同一種載體,從而使完全抗原的制備大大簡化。在此抗OA-McAb基礎(chǔ)上,建立了ci-ELISA方法,用于OA檢測。該檢測方法靈敏度較高,滿足了《中國國家標(biāo)準(zhǔn)GB2715—2005(糧食衛(wèi)生標(biāo)準(zhǔn))》中對谷類、豆類的OA的限量≤5ug/kg的要求。以含有5%氯化鈉的60%甲醇水溶液對小米、玉米中的OA的提取效果較為理想,方法重復(fù)性良好。濟南市集市和超市出售的小米和玉米中均不同程度地存在OA污染情況；其中,超市小米的OA檢出率高于集市小米OA的檢出率。依照《中國國家標(biāo)準(zhǔn)GB2715—2005(糧食衛(wèi)生標(biāo)準(zhǔn))》中對谷類、豆類的OA的限量≤5ug／kg要求,所檢測的樣品中OA含量均未超標(biāo),中位數(shù)為1.67ug/kg。
[Abstract]:Objective to prepare a monoclonal antibody (monoclonal antibody, McAb) against ochratoxin A (ochratoxin A, OA) and to identify it preliminarily. On this basis, a competitive inhibition enzyme linked immunosorbent assay (competitive inhibition enzyme linked immunosorbent) was established to optimize the detection of millet and corn. Methods ci-ELISA was used to detect the content of OA in millet and corn samples sold in bazaar and supermarkets in five districts of Ji'nan, and preliminarily understand the pollution situation.
Methods OA- bovine serum albumin (bovine serum albumin, BSA) conjugate was used to immunize female BALB/c mice with OA-BSA. BSA as the antigen, and the antibody titer of the mice was detected by the enzyme linked immunosorbent assay (enzyme linked immunosorbent assay), and the intersections of the antibody were observed by the enzyme linked immunosorbent assay (enzyme linked immunosorbent assay). Reaction conditions. Using the method of cell fusion, using OA-BSA as the envelope antigen and BSA as the control, the McAb in the hybridoma cell culture supernatant was detected by ELISA method, and the hybridoma cell line secreting McAb against OA was obtained. The cross reaction between the McAb and BSA was studied to evaluate the specificity of McAb. The specific secretion of anti O by the finite dilution method was used. The hybridoma cells of A McAb were cloned, and the specificity of McAb was further determined by ci-ELISA method. McAb was prepared in a large number of ascites. The McAb in ascites was purified by octanoic acid saturated ammonium sulfate method. The titer of ascites before and after purification was detected by ELISA. At the same time, the cross reaction was studied with BSA as the anti original control. OA was the competitive antigen. The ci-ELISA method was established for the detection of OA. The solution of different concentration of methanol and sodium chloride was selected as the extraction solvent to extract the OA from the sample, and the recovery rate of the different extraction solvents was compared. The effects of methanol and sodium chloride concentration on the extraction effect of OA in millet and corn samples were observed, and the extraction method of OA was optimized. The Ji'nan city was collected. Millet and maize, millet and corn sold in five districts of almanac, millet and corn sold in five districts of the city, 50 millet and corn each sold in Ji'nan city supermarket. Using the standard addition method, the content of OA in the millet and corn samples was tested, the rate of detection was calculated, and according to the China national standard GB2715 - 2005 (food hygienic standard) The limit of OA for cereals and legumes is calculated, and the exceeding standard rate is calculated. All data are processed by SPSS software package.
Results the serum antibody titer of BALB/c mice immunized with OA-BSA was 1:512000, and there was a strong cross reaction with BSA. After the fusion, ELISA screened the hybridoma cell lines with positive antibody secretion, and the cross reaction rate of McAb and BSA against OA-BSA was only 3.50%. The cell lines secreting anti OA-BAS specific McAb were cloned in 3 rounds, and the positive rate of antibody secretion was reached. To 100%, 1 hybridoma cell lines that could stabilize the secretion of anti OA-BSAMcAb were established. Ci-ELISA further demonstrated that the antibody was specific to OA. A large number of McAb were prepared by the ascites induced method. The antibody titer of the purified ascites was 1:512000, and the.Ci-ELISA linear range of.Ci-ELISA was 0.04ng / ml-1250.OOng / ml, and the standard curve of BSA was no cross reaction to BSA. The linear equation y=-0.1368x+0.6796, the correlation coefficient r=0.9965, the detection limit is 0.04ng / ml. when the content of methanol in the extraction solvent is 60% and the extraction effect of 5% sodium chloride is ideal. OA plus the scalar is 50.OOng / g, 25.OOng / g, 12.50ng / g, the extraction solvent is confirmed by the extraction solvent, the average recovery rate of the millet is added to the scalar (%). The coefficient of variation (CV%) was 4.59%, 8.42%, 8.94%, and 7.32%, respectively, and the average recovery of each additive was 96.30%, 98.67% and 98.13%, respectively, and the coefficient of variation (CV%) was 5.74%, 5.59%, 9.56%, respectively, and the average 6.96%. used to detect the ci-ELISA working curve of OA in millet was y=-0.3249x+1.079, related lines, respectively. The linear equation of ci-ELISA working curve used to detect OA in maize was y=-0.2554x+0.9561, the correlation coefficient r=0.9971. millet was r=0.9971., the detection limit of maize OA was 0.04ng / ml, and the minimum output limit of OA was all 0.2ng / g. in all samples. The detection rate of millet OA was 41% (41 / 100), and the detection rate of maize was 38% (38 / 100) and two were not obvious. P0.05, among them, the detection rate of millet OA was 26% (13 / 50), the OA detection rate was 32% (16 / 50) and two (P0.05), the detection rate of OA in supermarket millet was 56% (28 / 50), the detection rate of OA in supermarket corn was 44% (22 / 50), and there was no significant difference (P0.05) in all millet samples, and the millet OA in all the samples of the millet was detected. The detection rate (56%) was significantly higher than that of Millet (26%) (P0.05), and there was no significant difference in the detection rate of corn (44%) and market corn (32%) OA in all corn samples (P0.05). Among all the samples, millet (0.40-4.45ug/kg, median of 1.73 ug/kg) and corn (0.48-4.02ug/kg, median of 1.61 ug/kg) had no significant OA content. Difference (P0.05). Among them, millet (0.41-3.94ug / kg, median 1.63 UG / kg) and market corn (0.53-3.99ug/kg, median of 1.52 ug/kg), supermarket millet (0.40-4.45ug/kg, median is 1.78ug/kg) and supermarket corn (0.48-4.02ug/kg, median of 1.74 ug/kg) no significant difference in OA content. All millet samples detected. There was no significant difference in the content of Millet (median 1.78ug/kg) and millet (median 1.63ug/kg) of market millet (median of 1.63ug/kg) in supermarkets (P0.05). There was no significant difference in OA content between supermarket corn (median is 1.74ug/kg) and market corn (median of 1.52ug/kg) in all corn samples (P0.05). The OA content of all the samples tested in this study was found to be no significant difference (P0.05). The median of 0.40-4.45ug/kg is 1.67ug/kg. According to the Chinese national standard GB2715 - 2005 (grain health standard), the limit of the OA of the beans is less than 5ug / kg, and the OA content in all the samples is not exceeding the standard.
Conclusion the hybridoma cell line that secretes anti OA McAb is obtained. During the preparation of the antigen monoclonal antibody, the immune antigen and detection antigen can be used the same carrier so that the preparation of the complete antigen is greatly simplified. On the basis of the anti OA-McAb, the ci-ELISA method is established and used for the test of OA. The sensitivity of this method is high and satisfies China. The standard GB2715 - 2005 (grain health standard) > the requirement for the limit of OA in cereals and legumes is less than 5ug/kg. The extraction effect of OA in millet and maize with 60% methanol water containing 5% sodium chloride is ideal, and the method repeatability is good. There are OA pollution in the millet and corn sold in Ji'nan market and supermarkets in different degrees. Among them, the OA detection rate of millet in supermarket is higher than that of millet OA in market market. According to China national standard GB2715 - 2005 (grain health standard), the limit of OA of the beans is less than 5ug / kg, and the content of OA in the samples is not exceeding the standard, the median is 1.67ug/kg.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392

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