黄曲霉毒素B_1单克隆抗体的制备及基于该抗体的黄曲霉毒素B_1免疫学检测方法的建立
|
摘要
为了快速准确检测粮食与饲料中黄曲霉毒素B_1(AFB_1)含量,本试验制备了具有高灵敏度的AFB_1单克隆抗体,并将其应用于AFB_1间接竞争酶联免疫吸附测定(ELISA)方法的建立。采用碳二亚胺(EDC)法制备AFB_1完全抗原[AFB_1-牛血清白蛋白(BSA)和AFB_1-鸡卵白蛋白(OVA)],并免疫B_ALB/c小鼠。经细胞融合,筛选出能够分泌高灵敏度AFB_1单克隆抗体的杂交瘤细胞株。采用体内诱生腹水法,大量制备AFB_1单克隆抗体,并对其免疫学特性进行鉴定。基于制备的AFB_1单克隆抗体,建立AFB_1间接竞争ELISA检测方法。结果显示:通过筛选,获得稳定产生抗体的杂交瘤细胞株9H1F5,经间接ELISA方法测定,获得的AFB_1单克隆抗体的效价高达5.12×105,亲和力常数(Ka)=6.72×107L/mol,抗体亚型为免疫球蛋白G_2(IgG_2)。采用间接竞争ELISA方法测得AFB_1的半数抑制浓度(IC_(50))为0. 232 ng/mL,检测范围为0.014～1.920 ng/mL,最低检测限为0.014 ng/mL。同时,该AFB_1单克隆抗体与黄曲霉毒素B_2(AFB_2)、黄曲霉毒素G_1(AFG_1)、黄曲霉毒素G_2(AFG_2)、黄曲霉毒素M1(AFM1)的交叉反应率分别为40.21%、33.19%、31.96%、4.40%,与其他霉菌毒素无交叉反应。由上述结果可知,本试验成功制备了灵敏度高、特异性强的AFB_1单克隆抗体,并基于该抗体建立了检测AFB_1的间接竞争ELISA方法,为粮食与饲料中AFB_1快速免疫学检测奠定了基础。
In order to detect aflatoxin B_1 (AFB_1) content in cereals and feeds rapidly and correctly,a highly sensitive AFB_1 monoclonal antibody was prepared and an indirect competitive enzyme-linked immunosorbent assay (ELISA) for AFB_1 was established. AFB_1 complete antigens [AFB_1-bovine serum albumin (BSA) and AFB_1-ovalbumin (OVA) ]were prepared via carbodiimide (EDC) method and used to immunize the BALB/c mice. After the cell fusion,hybridoma cell line which could secret highly sensitive AFB_1 monoclonal antibody was selected. Then a lot of AFB_1 monoclonal antibodies were prepared using inducing ascites in vivo,and the AFB_1 monoclonal antibody properties were identified. Finally,an indirect competitive ELISA method for detecting AFB_1 based on AFB_1 monoclonal antibody was established. A hybridoma cell line 9 H1 F5 was selected,and the titer of AFB_1 monoclonal antibody was up to 5.12×105,the affinity constant (Ka) = 6.72×107 L/mol,and the subtype was immunoglobulin G_2 (IgG_2) measured by indirect ELISA method. According to the indirect competitive ELISA,the median inhibitory concentration (IC_(50)) of AFB_1 was 0.232 ng/mL,the detection range was 0.014 to 1. 920 ng/mL,and the limit of detection (LOD) of AFB_1 was 0. 014 ng/mL. Besides,cross reaction rates of AFB_1 monoclonal antibody with aflatoxin B_2 (AFB_2),aflatoxin G_1 (AFG_1),aflatoxin G_2 (AFG_2) and aflatoxin M1 (AFM1) were 40.21%,33.19%,31.96% and 4.40%,respectively,but there were no cross reactions with other mycotoxins. According to the above results,the highly sensitive and specific AFB_1 monoclonal antibody is successfully prepared,and the indirect competitive ELISA method for AFB_1 detection is established on the base of AFB_1 monoclonal antibody,which can provide the foundation for AFB_1 rapid immunological detection in cereals and feeds.
In order to detect aflatoxin B_1 (AFB_1) content in cereals and feeds rapidly and correctly,a highly sensitive AFB_1 monoclonal antibody was prepared and an indirect competitive enzyme-linked immunosorbent assay (ELISA) for AFB_1 was established. AFB_1 complete antigens [AFB_1-bovine serum albumin (BSA) and AFB_1-ovalbumin (OVA) ]were prepared via carbodiimide (EDC) method and used to immunize the BALB/c mice. After the cell fusion,hybridoma cell line which could secret highly sensitive AFB_1 monoclonal antibody was selected. Then a lot of AFB_1 monoclonal antibodies were prepared using inducing ascites in vivo,and the AFB_1 monoclonal antibody properties were identified. Finally,an indirect competitive ELISA method for detecting AFB_1 based on AFB_1 monoclonal antibody was established. A hybridoma cell line 9 H1 F5 was selected,and the titer of AFB_1 monoclonal antibody was up to 5.12×105,the affinity constant (Ka) = 6.72×107 L/mol,and the subtype was immunoglobulin G_2 (IgG_2) measured by indirect ELISA method. According to the indirect competitive ELISA,the median inhibitory concentration (IC_(50)) of AFB_1 was 0.232 ng/mL,the detection range was 0.014 to 1. 920 ng/mL,and the limit of detection (LOD) of AFB_1 was 0. 014 ng/mL. Besides,cross reaction rates of AFB_1 monoclonal antibody with aflatoxin B_2 (AFB_2),aflatoxin G_1 (AFG_1),aflatoxin G_2 (AFG_2) and aflatoxin M1 (AFM1) were 40.21%,33.19%,31.96% and 4.40%,respectively,but there were no cross reactions with other mycotoxins. According to the above results,the highly sensitive and specific AFB_1 monoclonal antibody is successfully prepared,and the indirect competitive ELISA method for AFB_1 detection is established on the base of AFB_1 monoclonal antibody,which can provide the foundation for AFB_1 rapid immunological detection in cereals and feeds.
引文
[1]BENNETT J W,KLICH M.Mycotoxins[J].Clinical M icrobiology Review s,2003,16(3):497-516.
[2]MACHIDA M,ASAI K,SANO M,et al.Genome sequencing and analysis of Aspergillus oryzae[J].Nature,2005,438(7071):1157-1161.
[3]WU F,GROOPMAN J D,PESTKA J J.Public health impacts of foodborne mycotoxins[J].Annual Review of Food Science and Technology,2014,5:351-372.
[4]陈兴祥,黄克和.黄曲霉毒素对畜禽免疫机能的影响[J].中国兽医杂志,2002,38(10):33-35.
[5]OSTRY V,MALIR F,TOMAN J,et al.Mycotoxins as human carcinogens-the IARC monographs classification[J].M ycotoxin Research,2017,33(1):65-73.
[6]IARC.International Agency for Research on Cancer,WHO IARC M onographs on the evaluation of carcinogenic risks to humans Some traditional herbal medicines,some mycotoxins naphthalene and styrene aflatoxins[C].Lyon:IARC,2002.
[7]CHOI S,JUN H,BANG J,et al.Behaviour of Aspergillus flavus and Fusarium graminearum on rice as affected by degree of milling,temperature,and relative humidity during storage[J].Food M icrobiology,2015,46:307-313.
[8]HU X F,HU R,ZHANG Z W,et al.Development of a multiple immunoaffinity column for simultaneous determination of multiple mycotoxins in feeds using UP-LC-M S/M S[J].Analytical and Bioanalytical Chemistry,2016,408(22):6027-6036.
[9]GARCA-MORALEJA A,FONT G,MAES J,et al.Development of a new method for the simultaneous determination of 21 mycotoxins in coffee beverages by liquid chromatography tandem mass spectrometry[J].Food Research International,2015,72:247-255.
[10]ZHENG W L,TENG J,CHENG L,et al.Hetero-enzyme-based tw o-round signal amplification strategy for trace detection of aflatoxin B1using an electrochemical aptasensor[J].Biosensors and Bioelectronics,2016,80:574-581.
[11]GARCA-FONSECA S,BALLESTEROS-GMEZA,RUBIO S.Restricted access supramolecular solvents for sample treatment in enzyme-linked immuno-sorbent assay of mycotoxins in food[J].Analytica Chimica Acta,2016,935:129-135.
[12]LEE N A,WANG S,ALLAN R D,et al.A rapid aflatoxin B1ELISA:development and validation w ith reduced matrix effects for peanuts,corn,pistachio,and soybeans[J].Journal of Agricultural and Food Chemistry,2004,52(10):2746-2755.
[13]HU X,YAO J,WANG F,et al.Eu3+-labeled IgG-based time-resolved fluoroimmunoassay for highly sensitive detection of aflatoxin B1in feed[J].Journal of the Science of Food and Agriculture,2018,98(2):674-680.
[14]WANG D,ZHANG Z W,LI P W,et al.Europium nanospheres-based time-resolved fluorescence for rapid and ultrasensitive determination of total aflatoxin in feed[J].Journal of Agricultural and Food Chemistry,2015,63(47):10313-10318.
[15]HANG D H,LI P W,ZHANG Q,et al.Ultrasensitive nanogold probe-based immunochromatographic assay for simultaneous detection of total aflatoxins in peanuts[J].Biosensors and Bioelectronics,2010,26(6):2877-2882.
[16]LI P W,ZHANG Z W,ZHANG Q,et al.Current development of microfluidic immunosensing approaches for mycotoxin detection via capillary electromigration and lateral flow technology[J].Electrophoresis,2012,33(15):2253-2265.
[17]CHEN Y Q,CHEN Q,HAN M M,et al.Development and optimization of a multiplex lateral flow immunoassay for the simultaneous determination of three mycotoxins in corn,rice and peanut[J].Food Chemistry,2016,213:478-484.
[18]KOLOSOVA A Y,SHIM W B,YANG Z Y,et al.Direct competitive ELISA based on a monoclonal antibody for detection of aflatoxin B1.Stabilization of ELISA kit components and application to grain samples[J].Analytical and Bioanalytical Chemistry,2006,384(1):286-294.
[19]王磊,胡骁飞,滕蔓,等.抗黄曲霉毒素B1单抗制备及免疫学定量方法建立[J].中国公共卫生,2012,28(1):58-60.
[20]谢珲,章先,王歆,等.黄曲霉毒素B1单克隆抗体的制备及间接竞争ELISA检测技术研究[J].微生物学通报,2015,42(10):2033-2040.
[21]KONG D Z,XIE Z J,LIU L Q,et al.Development of ic-ELISA and lateral-flow immunochromatographic assay strip for the detection of vancomycin in raw milk and animal feed[J].Food and Agricultural Immunology,2017,28(3):414-426.
[22]宋青龙,李成洪,傅巍,等.黄曲霉毒素B1胶体金快速定量检测试剂盒的研发[J].动物营养学报,2017,29(10):3703-3709.
[23]KEMP H A,MORGAN M R A.Studies on the detrimental effects of bivalent binding in a microtitration plate ELISA and possible remedies[J].Journal of Immunological M ethods,1986,94(1/2):65-72.
[24]王磊,胡骁飞,职爱民,等.黄曲霉毒素B1人工抗原及鼠源多克隆抗血清的制备[J].食品科技,2011,36(3):277-281.
[25]LANE R D,CRISSMAN R S,GINN S.High efficiency fusion procedure for producing monoclonal antibodies against w eak immunogens[J].M ethods in Enzymology,1986,121:183-192.
[26]刘晓波,蔡美英,王霞,等.一种简单实用纯化腹水M cAb方法---辛酸/硫酸铵法[J].华西医科大学学报,1999,30(4):455-456.
[27]BEATTY J D,BEATTY B G,VLAHOS W G.Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay[J].Journal of Immunological M ethods,1987,100(1/2):173-179.
[28]万文徽.单克隆抗体亲和常数的测定[J].单克隆抗体通讯,1993,9(2):72-75.
[29]YAO J J,WANG F Y,HAN J L,et al.Novel fluoroimmunoassays for detecting ochratoxin A using CdTe quantum dots[J].Journal of Biophotonics,2017,10:657-663.
[2]MACHIDA M,ASAI K,SANO M,et al.Genome sequencing and analysis of Aspergillus oryzae[J].Nature,2005,438(7071):1157-1161.
[3]WU F,GROOPMAN J D,PESTKA J J.Public health impacts of foodborne mycotoxins[J].Annual Review of Food Science and Technology,2014,5:351-372.
[4]陈兴祥,黄克和.黄曲霉毒素对畜禽免疫机能的影响[J].中国兽医杂志,2002,38(10):33-35.
[5]OSTRY V,MALIR F,TOMAN J,et al.Mycotoxins as human carcinogens-the IARC monographs classification[J].M ycotoxin Research,2017,33(1):65-73.
[6]IARC.International Agency for Research on Cancer,WHO IARC M onographs on the evaluation of carcinogenic risks to humans Some traditional herbal medicines,some mycotoxins naphthalene and styrene aflatoxins[C].Lyon:IARC,2002.
[7]CHOI S,JUN H,BANG J,et al.Behaviour of Aspergillus flavus and Fusarium graminearum on rice as affected by degree of milling,temperature,and relative humidity during storage[J].Food M icrobiology,2015,46:307-313.
[8]HU X F,HU R,ZHANG Z W,et al.Development of a multiple immunoaffinity column for simultaneous determination of multiple mycotoxins in feeds using UP-LC-M S/M S[J].Analytical and Bioanalytical Chemistry,2016,408(22):6027-6036.
[9]GARCA-MORALEJA A,FONT G,MAES J,et al.Development of a new method for the simultaneous determination of 21 mycotoxins in coffee beverages by liquid chromatography tandem mass spectrometry[J].Food Research International,2015,72:247-255.
[10]ZHENG W L,TENG J,CHENG L,et al.Hetero-enzyme-based tw o-round signal amplification strategy for trace detection of aflatoxin B1using an electrochemical aptasensor[J].Biosensors and Bioelectronics,2016,80:574-581.
[11]GARCA-FONSECA S,BALLESTEROS-GMEZA,RUBIO S.Restricted access supramolecular solvents for sample treatment in enzyme-linked immuno-sorbent assay of mycotoxins in food[J].Analytica Chimica Acta,2016,935:129-135.
[12]LEE N A,WANG S,ALLAN R D,et al.A rapid aflatoxin B1ELISA:development and validation w ith reduced matrix effects for peanuts,corn,pistachio,and soybeans[J].Journal of Agricultural and Food Chemistry,2004,52(10):2746-2755.
[13]HU X,YAO J,WANG F,et al.Eu3+-labeled IgG-based time-resolved fluoroimmunoassay for highly sensitive detection of aflatoxin B1in feed[J].Journal of the Science of Food and Agriculture,2018,98(2):674-680.
[14]WANG D,ZHANG Z W,LI P W,et al.Europium nanospheres-based time-resolved fluorescence for rapid and ultrasensitive determination of total aflatoxin in feed[J].Journal of Agricultural and Food Chemistry,2015,63(47):10313-10318.
[15]HANG D H,LI P W,ZHANG Q,et al.Ultrasensitive nanogold probe-based immunochromatographic assay for simultaneous detection of total aflatoxins in peanuts[J].Biosensors and Bioelectronics,2010,26(6):2877-2882.
[16]LI P W,ZHANG Z W,ZHANG Q,et al.Current development of microfluidic immunosensing approaches for mycotoxin detection via capillary electromigration and lateral flow technology[J].Electrophoresis,2012,33(15):2253-2265.
[17]CHEN Y Q,CHEN Q,HAN M M,et al.Development and optimization of a multiplex lateral flow immunoassay for the simultaneous determination of three mycotoxins in corn,rice and peanut[J].Food Chemistry,2016,213:478-484.
[18]KOLOSOVA A Y,SHIM W B,YANG Z Y,et al.Direct competitive ELISA based on a monoclonal antibody for detection of aflatoxin B1.Stabilization of ELISA kit components and application to grain samples[J].Analytical and Bioanalytical Chemistry,2006,384(1):286-294.
[19]王磊,胡骁飞,滕蔓,等.抗黄曲霉毒素B1单抗制备及免疫学定量方法建立[J].中国公共卫生,2012,28(1):58-60.
[20]谢珲,章先,王歆,等.黄曲霉毒素B1单克隆抗体的制备及间接竞争ELISA检测技术研究[J].微生物学通报,2015,42(10):2033-2040.
[21]KONG D Z,XIE Z J,LIU L Q,et al.Development of ic-ELISA and lateral-flow immunochromatographic assay strip for the detection of vancomycin in raw milk and animal feed[J].Food and Agricultural Immunology,2017,28(3):414-426.
[22]宋青龙,李成洪,傅巍,等.黄曲霉毒素B1胶体金快速定量检测试剂盒的研发[J].动物营养学报,2017,29(10):3703-3709.
[23]KEMP H A,MORGAN M R A.Studies on the detrimental effects of bivalent binding in a microtitration plate ELISA and possible remedies[J].Journal of Immunological M ethods,1986,94(1/2):65-72.
[24]王磊,胡骁飞,职爱民,等.黄曲霉毒素B1人工抗原及鼠源多克隆抗血清的制备[J].食品科技,2011,36(3):277-281.
[25]LANE R D,CRISSMAN R S,GINN S.High efficiency fusion procedure for producing monoclonal antibodies against w eak immunogens[J].M ethods in Enzymology,1986,121:183-192.
[26]刘晓波,蔡美英,王霞,等.一种简单实用纯化腹水M cAb方法---辛酸/硫酸铵法[J].华西医科大学学报,1999,30(4):455-456.
[27]BEATTY J D,BEATTY B G,VLAHOS W G.Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay[J].Journal of Immunological M ethods,1987,100(1/2):173-179.
[28]万文徽.单克隆抗体亲和常数的测定[J].单克隆抗体通讯,1993,9(2):72-75.
[29]YAO J J,WANG F Y,HAN J L,et al.Novel fluoroimmunoassays for detecting ochratoxin A using CdTe quantum dots[J].Journal of Biophotonics,2017,10:657-663.