黄曲霉毒素杂交瘤细胞株的选育及免疫层析检测技术研究
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摘要
黄曲霉毒素是一类由黄曲霉菌和寄生曲霉菌产生的剧毒代谢产物,产毒真菌分布于各级食物链,即使是在发达国家拥有先进农业生产技术和食品加工工艺的条件下,仍然无法完全避免和防止黄曲霉毒素的危害。黄曲霉毒素是迄今发现的最强致癌物质之一,多年来,世界各国由黄曲霉毒素引发的人畜等中毒死亡恶性事件时有发生,不但严重威胁人畜健康和生命安全,造成严重的经济损失,还极易引发社会恐慌,严重影响了和谐社会的构建,因此黄曲霉毒素污染已成为农产品质量与食品安全的重要热点难点问题,受到世界各国极大关注,并成为发达国家的技术贸易壁垒。近年来欧盟等通过制定苛刻的黄曲霉毒素限量标准对我国出口农产品设置贸易壁垒,致使我国出口的农产品尤其是粮油食品不断因黄曲霉毒素超标而遭拒绝、扣留、退货甚至销毁,给我国造成了巨大经济损失。
灵敏、准确、快速的检测技术是黄曲霉毒素污染监管和保障农产品安全消费必不可少的重要抓手。目前所采用的黄曲霉毒素分析方法专业性要求高、时间长、成本昂贵,严重制约了农产品中黄曲霉毒素污染监测工作的广泛开展：而缺乏核心试剂—高质量的黄曲霉毒素单克隆抗体又成为制约快速检测技术建立的瓶颈问题。为保护人畜健康和生命安全,减小贸易壁垒带来的不利影响,本研究针对以上迫切需要解决的问题,研制出了针对黄曲霉毒素B1、M1和总量的系列单克隆抗体,以此为基础,建立了系统配套的粮油食品及乳制品中黄曲霉毒素污染快速检测技术,研制了配套的黄曲霉毒素试纸条产品,并首次研制了一种黄曲霉毒素数码试纸条,为粮油等种植业大宗农产品及乳制品中黄曲霉毒素污染的监测提供了灵敏、准确、简单、快速、成本低廉的新方法。本论文的主要研究内容和创新点如下：
1.研制出一种超灵敏黄曲霉毒素通用单克隆抗体以及分别抗黄曲霉毒素B1和M1的特异性单克隆抗体。高质量的抗体是成功建立任何一种免疫学检测方法的关键因素之,为了制各高质量的黄曲霉毒素抗体,采用了常规半固体培养基法、干细胞培养基法以及液体培养基法三种方法选育杂交瘤细胞株,在前人研究基础上建立了新型两步筛选法,并通过此筛选法的应用选育出了一株分泌超高灵敏黄曲霉毒素通用抗体的细胞株1C11,1c11抗体对黄曲霉毒素B1、B2、G1和G2的灵敏度(用1C50表示)依次为1.2、1.3、2.2和18.0pg/mL,与国内外报道的抗体相比较,1C11是迄今灵敏度最高的黄曲霉毒素通用抗体。同时,采用干细胞培养基法先后选育出了另外两株高质量的杂交瘤细胞株3G1和2C9,其分泌的抗体分别对黄曲霉毒素B1和M1具有高特异性。其中3G1抗体对黄曲霉毒素B1、B2、G1和G2的交叉反应率依次为100%、6.4%、<0.02%、<0.02%,2C9抗体对以卜四种黄曲霉毒素均没有交叉反应。综合考虑灵敏度和特异性,与国内外报道相应抗体比较后发现,3G1和2C9均是迄今最好的黄曲霉毒素分量抗体。
2.研制出黄曲霉毒素B,高特异性免疫层析试纸条,与当前同类技术产品相比特异性最高。试纸条对黄曲霉毒素B1的检测限为1ng/mL,对黄曲霉毒素B2、G1和G2没有交叉反应。将该试纸条应用于7类62份农产品样品中黄曲霉毒素B1污染水平的检测并与高效液相色谱法进行比较,两种方法检测结果的符合率为98.39%(61/62),表明所研制试纸条能够有效满足农产品及食品中黄曲霉毒素B1的特异性检测。
3.研制出高特异性、高灵敏度黄曲霉毒素M1免疫层析试纸条。试纸条对牛奶检测液中黄曲霉毒素M1的检测限为0.3ng/mL,与黄曲霉毒素B1、B2、G1和G2没有交叉反应。将该试纸条应用于牛奶和奶粉中黄曲霉毒素M1的检测,与定量间接竞争ELISA检测结果的符合率为100%,充分表明了其在牛奶及奶制品中黄曲霉毒素M1污染监测中的巨大应用潜力。率先系统报道了黄曲霉毒素M。特异性免疫层析试纸条的研制及应用。
4.首次研制出黄曲霉毒素总量超灵敏免疫层析试纸条。试纸条对黄曲霉毒素B1、B2、G1和G2的检测限依次为0.03、0.06、0.12和0.25ng/mL。与国内外报道的分量黄曲霉毒素试纸条相比较,所研制的总量试纸条对每一种分量黄曲霉毒素的灵敏度均是目前最高的。将该试纸条应用于7类64份农产品中黄曲霉毒素污染水平的检测,与高效液相色谱法结果的符合率为95.31%(61/64),该试纸条在实际样品中对黄曲霉毒素B1、B2、G1和G2的检测浓度依次为0.23、0.45、0.90和1.88μg/kg,远远低于欧盟制定的限量标准值。因此,所研制的试纸条能够广泛应用于农产品及食品中总量黄曲霉毒素污染的监测。
5.率先研制出一种黄曲霉毒素数码免疫层析试纸条。首次构建了数码免疫层析分析模型,并以黄曲霉毒素为目标分析物对该模型进行了应用,建立了黄曲霉毒素数码免疫层析方法,研制出黄曲霉毒素数码试纸条。以黄曲霉毒素B1在实际样品中的检测为例,该数码试纸条能够满足<0.6ng/mL、0.6～1.25ng/mL、1.25～5.0ng/mL、5.0～20ng/mL和>20ng/mL五个污染水平的检测,15min内即能用肉眼读出黄曲霉毒素的含量范围。将该数码试纸条应用于花生和饲料中黄曲霉毒素B1污染水平的检测,与高效液相色谱法检测结果的符合率为100%,表明所设计、构建的数码免疫层析模型能够解决现有免疫层析技术无法满足可视化定量速测的技术难题,开辟了免疫层析定量分析新途径,为进一步研制其他目标分析物的数码试纸条提供了有力的技术支撑。
Aflatoxins are a group of extremely toxic metabolites produced by some Aspergillus species namely A. flavus and A. parasiticus. Toxigenic funguses distribute on each level of food chain, and the harm of aflatoxins can not be absolutely avoided or prevented even in developed countries with advanced technologies of agricultural production and food processing. Aflatoxin is one of the strongest carcinogens known up to date. In the past years, malignant events of poisoning death caused by aflatoxin constantly emerged, which not only seriously threatened the health and life safety of human and animals, caused a great loss of economics, but also easily resulted in panic of public and seriously affected the construction of harmonious society. Therefore, aflatoxin contamination has become one of the difficult problems of general interest in agro-product quality and food safety field and been paying great attention by countries all over the world. Besides, recently European Union, etc, set up rigorous technical trade barriers to exportation agro-products of China by establishing rather strict regulation of allowable limit standards for aflatoxins. Exported agro-products especially cereal and oilseeds products of China are continuously rejected, detained, returned or even destructed due to the exceeding aflatoxin content, which bring enormous economic loss to China.
Therefore, sensitive, accurate and rapid test technologies become indispensable for the aflatoxin contamination supervision and ensuring of agro-product secure consumption. Aflatoxin analytical methods adopted presently are skilled technician requirement, time-consuming and costly, which greatly restrict the wide implementation of aflatoxin contamination monitoring. Besides, the lack of key reagent of high-quality monoclonal antibody (MAb) also becomes a vital problem for the establishment of rapid test technologies. To protect the health and life safety of human and animals, decrease adverse influences of trade barriers, a series of MAbs against aflatoxin B1,M1 and total aflatoxins, respectively, were prepared for the urgent problems. Based on these MAbs, associated rapid test technologies for aflatoxin contamination in foodstuff, oilseeds, milk products and so on were established and, correspondingly, matching aflatoxin strips were also prepared. What's more, an aflatoxin digital strip was firstly developed. The study provided sensitive, accurate, simple, rapid and low cost methods for monitoring of aflatoxin contamination in cropping staple agro-products (e.g., cereal and oilseeds) and milk products. Main research contents and novelties are as follows:
1. An ultra-sensitive anti-aflatoxin generic MAb and MAbs with highly specific to aflatoxin B1 or M1 were prepared. High-quality antibody is one of the key factors for successful establishment of any immunological method. In order to prepare high-quality anti-aflatoxin antibodies, regular semisolid medium, stem cell medium and liquid medium culturing methods were successively adopted to select and breed excellent monoclonal hybridoma cell lines. A novel two-step screening method was established based on the previous research. Finally, an ultra-sensitive anti-aflatoxin generic MAb (1C11) was screened out using the novel two-step screening method. The sensitivities (expressed by IC50) of MAb 1C11 to aflatoxinB1, B2, G1 and G2 were 1.2,1.3,2.2 and 18.0 pg/mL, respectively. Compared with those corresponding anbodies reported home and abroad,1C11 was the most sensitive generic MAb for aflatoxins up to now. Meanwhile, another two high-quality hybridoma cell lines 3G1 and 2C9 were selected out using stem cell medium culturing method. MAbs secreted from 3G1 and 2C9 showed high specificity to aflatoxin B1 and aflatoxin M,, respectively. MAb 3G1 showed 100%,6.4%,<0.02% and <0.02% cross-reactivities toward aflatoxin B1, B2, G1 and G2, respectively, MAb 2C9 showed no cross-reactivity to the above four aflatoxins. Comparing comprehensively specificity and sensitivity with corresponding anbodies reported home and abroad, it was found that 3G1 and 2C9 were both the best MAbs against single aflatoxin so far.
2. A high specific immunochromatographic strip for aflatoxin B1 was developed and showed the highest specific compared with the present aflatoxin B1 strips. Visual detection limit (VDL) of the immunochromatographic strip test (IST) to aflatoxin B1 was 1 ng/mL, and the IST showed no cross-reactivity to aflatoxins B2, G1 and G2, The IST was applied and validated by high performance liquid chromatography (HPLC) in the detection of aflatoxin B1 contamination level in 62 samples which belonged to 7 types of agro-products, the results showed that the coincidence rate of two methods was up to 98.39%(61/62) indicating that the developed strip could well meet the requirement of sepcific detection for aflatoxin B1 in agro-products and food samples.
3. A high specific and sensitive immunochromatographic strip for aflatoxin M1 was developed. VDL of the IST to AFM1 in milk detection solution was 0.3 ng/mL. The strip was applied for milk and milk power, and the results were compared with an indirect competitive ELISA. The results showed that the coincidence rate of IST and the ELISA was up to 100%, indicating great application potential of the developed strip for aflatoxin M1 contamination monitoring in milk and milk products.
4. An immunochromatographic strip for total aflatoxins was firstly developed. VDLs of the IST for aflatoxin B1, B2, G1, and G2 were 0.03,0.06,0.12 and 0.25 ng/mL, respectively. Compared with single aflatoxin ISTs reported home and abroad, the sensitivity of developed IST to each aflatoxin was the highest up to now. With HPLC for reference, the IST was applied in the detection of aflatoxin contamination in 64 samples which belonged to 7 types of agro-products, the coincidence rate of the results between IST and HPLC was 95.31%(61/64). The detectable concentrations of IST for aflatoxin B1, B2, G1, and G2 in real samples were 0.23,0.45,0.90 and 1.88μg/kg, respectively, which were far lower than the limit standard levels set by European Union. Therefore, the developed strip could be well applied in the contamination monitoring of total aflatoxins in real samples.
5. Digital immunochromatographic strip for aflatoxins was firstly developed. A model of the digital immunochromatographic assay was firstly constructed, taking aflatoxin as the target analyte, the model was applied, and an aflatoxin digital strip was prepared. Taking aflatoxin B1 detection in real samples as an exemple, the digital strip could provide five contamination levels' detection of <0.6 ng/mL,0.6～1.25 ng/mL,1.25～5.0 ng/mL,5.0～20 ng/mL and >20 ng/mL, and the concentration range of aflatoxins could be red out by naked eyes within 15 min. With HPLC for validation, the digital strip was applied in the detection of aflatoxin B, contamination level in peanut and feedstuff samples, the coincidence rate of detection results between two methods was up to 100%(31/31). the results showed that the designed and constructed digital immunochromatographic assay could solve the technical difficulties that current immunochromatographic assay can not meet visual quantitative rapid detection. The study provided a novel way for quantitative immunochromatographic assay and a powerful technical support for developing digital strip of other target analytes.
灵敏、准确、快速的检测技术是黄曲霉毒素污染监管和保障农产品安全消费必不可少的重要抓手。目前所采用的黄曲霉毒素分析方法专业性要求高、时间长、成本昂贵,严重制约了农产品中黄曲霉毒素污染监测工作的广泛开展：而缺乏核心试剂—高质量的黄曲霉毒素单克隆抗体又成为制约快速检测技术建立的瓶颈问题。为保护人畜健康和生命安全,减小贸易壁垒带来的不利影响,本研究针对以上迫切需要解决的问题,研制出了针对黄曲霉毒素B1、M1和总量的系列单克隆抗体,以此为基础,建立了系统配套的粮油食品及乳制品中黄曲霉毒素污染快速检测技术,研制了配套的黄曲霉毒素试纸条产品,并首次研制了一种黄曲霉毒素数码试纸条,为粮油等种植业大宗农产品及乳制品中黄曲霉毒素污染的监测提供了灵敏、准确、简单、快速、成本低廉的新方法。本论文的主要研究内容和创新点如下：
1.研制出一种超灵敏黄曲霉毒素通用单克隆抗体以及分别抗黄曲霉毒素B1和M1的特异性单克隆抗体。高质量的抗体是成功建立任何一种免疫学检测方法的关键因素之,为了制各高质量的黄曲霉毒素抗体,采用了常规半固体培养基法、干细胞培养基法以及液体培养基法三种方法选育杂交瘤细胞株,在前人研究基础上建立了新型两步筛选法,并通过此筛选法的应用选育出了一株分泌超高灵敏黄曲霉毒素通用抗体的细胞株1C11,1c11抗体对黄曲霉毒素B1、B2、G1和G2的灵敏度(用1C50表示)依次为1.2、1.3、2.2和18.0pg/mL,与国内外报道的抗体相比较,1C11是迄今灵敏度最高的黄曲霉毒素通用抗体。同时,采用干细胞培养基法先后选育出了另外两株高质量的杂交瘤细胞株3G1和2C9,其分泌的抗体分别对黄曲霉毒素B1和M1具有高特异性。其中3G1抗体对黄曲霉毒素B1、B2、G1和G2的交叉反应率依次为100%、6.4%、<0.02%、<0.02%,2C9抗体对以卜四种黄曲霉毒素均没有交叉反应。综合考虑灵敏度和特异性,与国内外报道相应抗体比较后发现,3G1和2C9均是迄今最好的黄曲霉毒素分量抗体。
2.研制出黄曲霉毒素B,高特异性免疫层析试纸条,与当前同类技术产品相比特异性最高。试纸条对黄曲霉毒素B1的检测限为1ng/mL,对黄曲霉毒素B2、G1和G2没有交叉反应。将该试纸条应用于7类62份农产品样品中黄曲霉毒素B1污染水平的检测并与高效液相色谱法进行比较,两种方法检测结果的符合率为98.39%(61/62),表明所研制试纸条能够有效满足农产品及食品中黄曲霉毒素B1的特异性检测。
3.研制出高特异性、高灵敏度黄曲霉毒素M1免疫层析试纸条。试纸条对牛奶检测液中黄曲霉毒素M1的检测限为0.3ng/mL,与黄曲霉毒素B1、B2、G1和G2没有交叉反应。将该试纸条应用于牛奶和奶粉中黄曲霉毒素M1的检测,与定量间接竞争ELISA检测结果的符合率为100%,充分表明了其在牛奶及奶制品中黄曲霉毒素M1污染监测中的巨大应用潜力。率先系统报道了黄曲霉毒素M。特异性免疫层析试纸条的研制及应用。
4.首次研制出黄曲霉毒素总量超灵敏免疫层析试纸条。试纸条对黄曲霉毒素B1、B2、G1和G2的检测限依次为0.03、0.06、0.12和0.25ng/mL。与国内外报道的分量黄曲霉毒素试纸条相比较,所研制的总量试纸条对每一种分量黄曲霉毒素的灵敏度均是目前最高的。将该试纸条应用于7类64份农产品中黄曲霉毒素污染水平的检测,与高效液相色谱法结果的符合率为95.31%(61/64),该试纸条在实际样品中对黄曲霉毒素B1、B2、G1和G2的检测浓度依次为0.23、0.45、0.90和1.88μg/kg,远远低于欧盟制定的限量标准值。因此,所研制的试纸条能够广泛应用于农产品及食品中总量黄曲霉毒素污染的监测。
5.率先研制出一种黄曲霉毒素数码免疫层析试纸条。首次构建了数码免疫层析分析模型,并以黄曲霉毒素为目标分析物对该模型进行了应用,建立了黄曲霉毒素数码免疫层析方法,研制出黄曲霉毒素数码试纸条。以黄曲霉毒素B1在实际样品中的检测为例,该数码试纸条能够满足<0.6ng/mL、0.6～1.25ng/mL、1.25～5.0ng/mL、5.0～20ng/mL和>20ng/mL五个污染水平的检测,15min内即能用肉眼读出黄曲霉毒素的含量范围。将该数码试纸条应用于花生和饲料中黄曲霉毒素B1污染水平的检测,与高效液相色谱法检测结果的符合率为100%,表明所设计、构建的数码免疫层析模型能够解决现有免疫层析技术无法满足可视化定量速测的技术难题,开辟了免疫层析定量分析新途径,为进一步研制其他目标分析物的数码试纸条提供了有力的技术支撑。
Aflatoxins are a group of extremely toxic metabolites produced by some Aspergillus species namely A. flavus and A. parasiticus. Toxigenic funguses distribute on each level of food chain, and the harm of aflatoxins can not be absolutely avoided or prevented even in developed countries with advanced technologies of agricultural production and food processing. Aflatoxin is one of the strongest carcinogens known up to date. In the past years, malignant events of poisoning death caused by aflatoxin constantly emerged, which not only seriously threatened the health and life safety of human and animals, caused a great loss of economics, but also easily resulted in panic of public and seriously affected the construction of harmonious society. Therefore, aflatoxin contamination has become one of the difficult problems of general interest in agro-product quality and food safety field and been paying great attention by countries all over the world. Besides, recently European Union, etc, set up rigorous technical trade barriers to exportation agro-products of China by establishing rather strict regulation of allowable limit standards for aflatoxins. Exported agro-products especially cereal and oilseeds products of China are continuously rejected, detained, returned or even destructed due to the exceeding aflatoxin content, which bring enormous economic loss to China.
Therefore, sensitive, accurate and rapid test technologies become indispensable for the aflatoxin contamination supervision and ensuring of agro-product secure consumption. Aflatoxin analytical methods adopted presently are skilled technician requirement, time-consuming and costly, which greatly restrict the wide implementation of aflatoxin contamination monitoring. Besides, the lack of key reagent of high-quality monoclonal antibody (MAb) also becomes a vital problem for the establishment of rapid test technologies. To protect the health and life safety of human and animals, decrease adverse influences of trade barriers, a series of MAbs against aflatoxin B1,M1 and total aflatoxins, respectively, were prepared for the urgent problems. Based on these MAbs, associated rapid test technologies for aflatoxin contamination in foodstuff, oilseeds, milk products and so on were established and, correspondingly, matching aflatoxin strips were also prepared. What's more, an aflatoxin digital strip was firstly developed. The study provided sensitive, accurate, simple, rapid and low cost methods for monitoring of aflatoxin contamination in cropping staple agro-products (e.g., cereal and oilseeds) and milk products. Main research contents and novelties are as follows:
1. An ultra-sensitive anti-aflatoxin generic MAb and MAbs with highly specific to aflatoxin B1 or M1 were prepared. High-quality antibody is one of the key factors for successful establishment of any immunological method. In order to prepare high-quality anti-aflatoxin antibodies, regular semisolid medium, stem cell medium and liquid medium culturing methods were successively adopted to select and breed excellent monoclonal hybridoma cell lines. A novel two-step screening method was established based on the previous research. Finally, an ultra-sensitive anti-aflatoxin generic MAb (1C11) was screened out using the novel two-step screening method. The sensitivities (expressed by IC50) of MAb 1C11 to aflatoxinB1, B2, G1 and G2 were 1.2,1.3,2.2 and 18.0 pg/mL, respectively. Compared with those corresponding anbodies reported home and abroad,1C11 was the most sensitive generic MAb for aflatoxins up to now. Meanwhile, another two high-quality hybridoma cell lines 3G1 and 2C9 were selected out using stem cell medium culturing method. MAbs secreted from 3G1 and 2C9 showed high specificity to aflatoxin B1 and aflatoxin M,, respectively. MAb 3G1 showed 100%,6.4%,<0.02% and <0.02% cross-reactivities toward aflatoxin B1, B2, G1 and G2, respectively, MAb 2C9 showed no cross-reactivity to the above four aflatoxins. Comparing comprehensively specificity and sensitivity with corresponding anbodies reported home and abroad, it was found that 3G1 and 2C9 were both the best MAbs against single aflatoxin so far.
2. A high specific immunochromatographic strip for aflatoxin B1 was developed and showed the highest specific compared with the present aflatoxin B1 strips. Visual detection limit (VDL) of the immunochromatographic strip test (IST) to aflatoxin B1 was 1 ng/mL, and the IST showed no cross-reactivity to aflatoxins B2, G1 and G2, The IST was applied and validated by high performance liquid chromatography (HPLC) in the detection of aflatoxin B1 contamination level in 62 samples which belonged to 7 types of agro-products, the results showed that the coincidence rate of two methods was up to 98.39%(61/62) indicating that the developed strip could well meet the requirement of sepcific detection for aflatoxin B1 in agro-products and food samples.
3. A high specific and sensitive immunochromatographic strip for aflatoxin M1 was developed. VDL of the IST to AFM1 in milk detection solution was 0.3 ng/mL. The strip was applied for milk and milk power, and the results were compared with an indirect competitive ELISA. The results showed that the coincidence rate of IST and the ELISA was up to 100%, indicating great application potential of the developed strip for aflatoxin M1 contamination monitoring in milk and milk products.
4. An immunochromatographic strip for total aflatoxins was firstly developed. VDLs of the IST for aflatoxin B1, B2, G1, and G2 were 0.03,0.06,0.12 and 0.25 ng/mL, respectively. Compared with single aflatoxin ISTs reported home and abroad, the sensitivity of developed IST to each aflatoxin was the highest up to now. With HPLC for reference, the IST was applied in the detection of aflatoxin contamination in 64 samples which belonged to 7 types of agro-products, the coincidence rate of the results between IST and HPLC was 95.31%(61/64). The detectable concentrations of IST for aflatoxin B1, B2, G1, and G2 in real samples were 0.23,0.45,0.90 and 1.88μg/kg, respectively, which were far lower than the limit standard levels set by European Union. Therefore, the developed strip could be well applied in the contamination monitoring of total aflatoxins in real samples.
5. Digital immunochromatographic strip for aflatoxins was firstly developed. A model of the digital immunochromatographic assay was firstly constructed, taking aflatoxin as the target analyte, the model was applied, and an aflatoxin digital strip was prepared. Taking aflatoxin B1 detection in real samples as an exemple, the digital strip could provide five contamination levels' detection of <0.6 ng/mL,0.6～1.25 ng/mL,1.25～5.0 ng/mL,5.0～20 ng/mL and >20 ng/mL, and the concentration range of aflatoxins could be red out by naked eyes within 15 min. With HPLC for validation, the digital strip was applied in the detection of aflatoxin B, contamination level in peanut and feedstuff samples, the coincidence rate of detection results between two methods was up to 100%(31/31). the results showed that the designed and constructed digital immunochromatographic assay could solve the technical difficulties that current immunochromatographic assay can not meet visual quantitative rapid detection. The study provided a novel way for quantitative immunochromatographic assay and a powerful technical support for developing digital strip of other target analytes.
引文
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