抗黄曲霉毒素B_1单克隆抗体的制备与特性鉴定
摘要
黄曲霉毒素(Aflatoxins, AFT)是黄曲霉(Aspergillus flavus)和寄生曲霉(A.parasiticus)等产生的一组结构类似的次生代谢产物。其中黄曲霉毒素B1(Aflatoxin B1, AFB_1)是迄今发现的毒性最大、致癌性最强的一种真菌毒素。调查表明,世界范围内AFT污染十分普遍。因此,AFT受到最广泛的重视,各国政府都制定了十分严格的食品中AFT的限量标准。自20世纪六十年代以来,已逐渐探索出了多种AFT的检测方法。这些方法都曾在各个历史时期为检测AFT作出了一定贡献,但也存在着操作复杂、污染大、对仪器设备要求高、假阳性率高、检测时间长等缺陷。随着时代发展,大部分方法已经不能适应当代社会要求的污染小、灵敏度高、检测时间短及操作简单的检测要求。以免疫分析为基础的准确、快速、无污染、智能化检测技术已成为AFB_1快速检测方法研究的热点。其中免疫亲和层析技术由于其对AFT的提取效率高、净化程度高,越来越受研究人员的推崇,该方法是目前公认的最有效的AFB_1净化方法。而高亲和力、高灵敏度、质量稳定的抗体是建立理想检测方法的前提,是应首先解决的问题。
本研究的主要内容包括:抗AFB_1单克隆抗体的制备和特性鉴定及其免疫亲和柱的初步研制。
目的:通过杂交瘤细胞技术制备抗AFB_1单克隆抗体,并对所制备的单克隆抗体进行特性鉴定和初步应用。
方法:(1)以AFB_1-BSA作为抗原免疫BALB/c小鼠;(2)取免疫效果最佳的小鼠脾细胞与骨髓瘤细胞SP2/0进行融合;(3)以AFB_1-OVA作为检测抗原,通过酶联免疫吸附实验(enzyme-linked immunosorbent assay, ELISA)检测杂交瘤细胞的上清进行筛选,保留阳性孔,用有限稀释法进行三次克隆化,得到能稳定分泌单克隆抗体的杂交瘤细胞株;(4)探索大量制备和纯化所得抗体的方法和条件;(5)鉴定对所得单克隆抗体的特性(6)初步应用该单克隆抗体,研制AFB_1免疫亲和柱。
结果:(1)得到一株能稳定分泌抗AFB_1单克隆抗体的杂交瘤细胞株5E10。此单克隆抗体的亚类为IgG_2b,亲和常数是4.79×10~8M~(-1),与AFB_2、AFG_1、AFG_2、AFM_1的交叉反应率分别为<26.06%、63.68%、26.06%、39.26%。(2)单克隆抗体的大量制备采用动物体内生产的方法,即以不完全弗氏佐剂(freund's incomplete adjuvant, FIA)为致敏剂预处理12周龄以上的BALB/c小鼠。一周内腹腔注射杂交瘤细胞1～5×10~6个/只,10天后便可采集腹水;辛酸硫酸铵法(CA-AS)适用于该单抗的大量纯化。(3)初步制备AFB_1单抗免疫亲和柱,采用荧光分光光度法确定AFB_1单抗免疫亲和柱的使用条件。使用条件为:5mL 0.01M PBS(pH7.4)平衡;上样缓冲液中甲醇浓度为10%;上样后2mL超纯水洗涤;1mL80%甲醇-水洗脱,20mL0.01M PBS(pH7.4)再生。制备的AFB_1单抗免疫亲和柱其抗体浓度与柱容量的关系为y=85.63x-0.043,其中R~2=0.9732。制备柱容量为40ng的免疫亲和柱,使用1次后,柱容量下降至原柱容量的43.6%。
结论:杂交瘤细胞株5E10能稳定的分泌目的单克隆抗体,利用该抗体初步研制的AFB_1免疫亲和柱的性能仍需进一步优化。
Aflatoxin (AFT) is a group of structurally similar secondary metabolites, produced by Aspergillus flavus and A.parasiticu, aflatoxin B1 (AFB_1) is the most serious occurring chemical carcinogens and is one of the most toxic known to a fungal toxin. Investigation shows that aflatoxin contamination is widespread in the world. Therefore, the most widely attention pays to aflatoxin, governments have enacted very stringent standards to limit amount of aflatoxin in food. Since the 1950s, it gradually explored many ways to detect AFB_1, these methods have made some contribution in various historical periods for detecting aflatoxin, however, it also has shortcomings, such as complicated operating, pollution, high equipment requirement, the high false positive rate, long detection time; with the development of the times, it has been unable to meet the requirements of contemporary society that small pollution, high sensitivity, short detection time, simple testing. The accurate, rapid, clean, intelligent detection technology on the basis of immunoassay has become the hot of the rapid test to AFB_1. Because of the high extraction efficiency, high degree of purification, immunoaffinity chromatography technology to aflatoxin is increasingly respected by the research staff, this method is recognized as the most effective purification methods. High affinity, high sensitivity, stable quality antibody is the premise which should be solved firstly to establish the ideal testing method.
In this study, monoclonal antibodies (McAb) against aflatoxin B1 was prepared and its characterizations were identified, then we preliminary apply the monoclonal antibodies to prepare the immunoaffinity column.
Objective: Prepared the monoclonal antibodies against aflatoxin B1 by hybridoma technique, identify its characterizations and preliminary apply the monoclonal antibodies.
Method: (1) BALB/c mice were immunized with AFB_1-BSA; (2) Hybridomas were produced by fusing SP2/0 myeloma cells with spleen cells from the immunized BALB/c which was immune best; (3) Supernatant of the growing hybirdoma was used to screen for the presence of anti-AFB_1-BSA antibody with ELISA. The positive clone was subcloned for three cycles by limiting dilution to select stable secreting hybridoma; (4) Explored the method and conditions to prepare the large number of antibodies and antibodies purification; (5) Identified monoclonal antibodies’characterizations; (6) Preliminary apply the monoclonal antibodies to prepare the immunoaffinity column.
Result: (1) One hybirdoma secreting McAb against AFB_1 was produced and its subclass of the McAb was the IgG_2b, its affinity constant was 4.79×10~8M~(-1), and its cross-reaction rates with AFB_2, AFG_1, AFG_2, AFM_1 were <26.06%、63.68%、26.06%、39.26% ; (2) Freund’s Incomplete adjuvant retreated 10 to 12 weeks mice to prepare the mice ascites, then purified by the method of CA-AS; (3) AFB_1 immunoaffinity column’s procedures were 5mL 0.01M PBS (pH7.4) balanced the column, methanol concentration of the sample buffer was 10%, then 2mL ultrapure water wash, 1mL 80% methanol-water elute, 20mL0.01M PBS (pH7.4) regenerate; column capacity and antibodies’concentration relation formula is y=85.63x-0.043, R2=0.9732; the column that the column capacity was 40ng desend to 43.6% after one test.
Conclusion: The cell line 5E10 could secret the purpose McAb, AFB_1 immunoaffinity column prepared by the McAb needs to be further optimized.
本研究的主要内容包括:抗AFB_1单克隆抗体的制备和特性鉴定及其免疫亲和柱的初步研制。
目的:通过杂交瘤细胞技术制备抗AFB_1单克隆抗体,并对所制备的单克隆抗体进行特性鉴定和初步应用。
方法:(1)以AFB_1-BSA作为抗原免疫BALB/c小鼠;(2)取免疫效果最佳的小鼠脾细胞与骨髓瘤细胞SP2/0进行融合;(3)以AFB_1-OVA作为检测抗原,通过酶联免疫吸附实验(enzyme-linked immunosorbent assay, ELISA)检测杂交瘤细胞的上清进行筛选,保留阳性孔,用有限稀释法进行三次克隆化,得到能稳定分泌单克隆抗体的杂交瘤细胞株;(4)探索大量制备和纯化所得抗体的方法和条件;(5)鉴定对所得单克隆抗体的特性(6)初步应用该单克隆抗体,研制AFB_1免疫亲和柱。
结果:(1)得到一株能稳定分泌抗AFB_1单克隆抗体的杂交瘤细胞株5E10。此单克隆抗体的亚类为IgG_2b,亲和常数是4.79×10~8M~(-1),与AFB_2、AFG_1、AFG_2、AFM_1的交叉反应率分别为<26.06%、63.68%、26.06%、39.26%。(2)单克隆抗体的大量制备采用动物体内生产的方法,即以不完全弗氏佐剂(freund's incomplete adjuvant, FIA)为致敏剂预处理12周龄以上的BALB/c小鼠。一周内腹腔注射杂交瘤细胞1～5×10~6个/只,10天后便可采集腹水;辛酸硫酸铵法(CA-AS)适用于该单抗的大量纯化。(3)初步制备AFB_1单抗免疫亲和柱,采用荧光分光光度法确定AFB_1单抗免疫亲和柱的使用条件。使用条件为:5mL 0.01M PBS(pH7.4)平衡;上样缓冲液中甲醇浓度为10%;上样后2mL超纯水洗涤;1mL80%甲醇-水洗脱,20mL0.01M PBS(pH7.4)再生。制备的AFB_1单抗免疫亲和柱其抗体浓度与柱容量的关系为y=85.63x-0.043,其中R~2=0.9732。制备柱容量为40ng的免疫亲和柱,使用1次后,柱容量下降至原柱容量的43.6%。
结论:杂交瘤细胞株5E10能稳定的分泌目的单克隆抗体,利用该抗体初步研制的AFB_1免疫亲和柱的性能仍需进一步优化。
Aflatoxin (AFT) is a group of structurally similar secondary metabolites, produced by Aspergillus flavus and A.parasiticu, aflatoxin B1 (AFB_1) is the most serious occurring chemical carcinogens and is one of the most toxic known to a fungal toxin. Investigation shows that aflatoxin contamination is widespread in the world. Therefore, the most widely attention pays to aflatoxin, governments have enacted very stringent standards to limit amount of aflatoxin in food. Since the 1950s, it gradually explored many ways to detect AFB_1, these methods have made some contribution in various historical periods for detecting aflatoxin, however, it also has shortcomings, such as complicated operating, pollution, high equipment requirement, the high false positive rate, long detection time; with the development of the times, it has been unable to meet the requirements of contemporary society that small pollution, high sensitivity, short detection time, simple testing. The accurate, rapid, clean, intelligent detection technology on the basis of immunoassay has become the hot of the rapid test to AFB_1. Because of the high extraction efficiency, high degree of purification, immunoaffinity chromatography technology to aflatoxin is increasingly respected by the research staff, this method is recognized as the most effective purification methods. High affinity, high sensitivity, stable quality antibody is the premise which should be solved firstly to establish the ideal testing method.
In this study, monoclonal antibodies (McAb) against aflatoxin B1 was prepared and its characterizations were identified, then we preliminary apply the monoclonal antibodies to prepare the immunoaffinity column.
Objective: Prepared the monoclonal antibodies against aflatoxin B1 by hybridoma technique, identify its characterizations and preliminary apply the monoclonal antibodies.
Method: (1) BALB/c mice were immunized with AFB_1-BSA; (2) Hybridomas were produced by fusing SP2/0 myeloma cells with spleen cells from the immunized BALB/c which was immune best; (3) Supernatant of the growing hybirdoma was used to screen for the presence of anti-AFB_1-BSA antibody with ELISA. The positive clone was subcloned for three cycles by limiting dilution to select stable secreting hybridoma; (4) Explored the method and conditions to prepare the large number of antibodies and antibodies purification; (5) Identified monoclonal antibodies’characterizations; (6) Preliminary apply the monoclonal antibodies to prepare the immunoaffinity column.
Result: (1) One hybirdoma secreting McAb against AFB_1 was produced and its subclass of the McAb was the IgG_2b, its affinity constant was 4.79×10~8M~(-1), and its cross-reaction rates with AFB_2, AFG_1, AFG_2, AFM_1 were <26.06%、63.68%、26.06%、39.26% ; (2) Freund’s Incomplete adjuvant retreated 10 to 12 weeks mice to prepare the mice ascites, then purified by the method of CA-AS; (3) AFB_1 immunoaffinity column’s procedures were 5mL 0.01M PBS (pH7.4) balanced the column, methanol concentration of the sample buffer was 10%, then 2mL ultrapure water wash, 1mL 80% methanol-water elute, 20mL0.01M PBS (pH7.4) regenerate; column capacity and antibodies’concentration relation formula is y=85.63x-0.043, R2=0.9732; the column that the column capacity was 40ng desend to 43.6% after one test.
Conclusion: The cell line 5E10 could secret the purpose McAb, AFB_1 immunoaffinity column prepared by the McAb needs to be further optimized.
引文
[1] 居乃琥. 黄曲霉毒素. 北京轻工业出版社, 1980.
[2] 杨建伯. 真菌毒素与人类疾病. 中国地方病学杂志,2002,21(4):314-317.
[3] Machinski M J, Soares LMV, Sawazaki E, et al. Aflatoxins, ochratoxin A and zearalenone in Brazilian corn cultivars. J Science of food and agriculture. 2001, 81: 1001-1007.
[4] Arora DK, Bridge PD, Bhatnagar D, et al. Fungal biotechnology in agriculture, food and environmental applications. Mycotoxins. 2004,21: 325-342.
[5] Olivier M, Hussain SP, Hainaut P, et al. TP53 mutation spectra and load: a tool for generating hypotheses on the etiology of cancer. IARC Sci Publ. 2004, 157: 247-270.
[6] 徐进,罗雪云. 黄曲霉毒素合成的分子生物学. 卫生研究. 2003,32: 628-631.
[7] Luderitz O, Westphal O, Staub AM, et al. Microbial Toxins. New York Academic Press. 1971, 163-168.
[8] 食品中黄曲霉毒素的测定. 免疫亲和层析净化高效液相色谱法.中华人民共和国国家标准 GB/T 18979-2003.
[9] 叶雪珠,王小骊,等. 黄曲霉毒素 B1 检测方法的分析. 食品与发酵工业. 29: 90-92.
[10] Groopman JD, Trudel LJ, Donahue PR, et al. High-affinity monoclonal antibodies for aflatoxins and their application to solid-phase immunoassays. Proc Natl Acad Sci USA. 1984, 81: 7728-7731.
[11] Trucksess MW, Young KN, Donahue KF, et al. Comparation of two immunochemical methods with thin layer chromatographic methods for determination of aflatoxins. Journal of AOAC Int. 1990, 73: 425-428.
[12] 江湖,熊勇华,许扬. 黄曲霉毒素分析方法研究进展. 卫生研究. 2005, 34: 252-255.
[13] Maurice OM. Risk assessment for aflatoxins in food stuffs. International Biodeterioration and Biodegradation. 2002, 50, 137-142.
[14] Shan G, Stoutamire DW, Wengatz I, et al. Development of an immunoassay for the pyrethroid insecticide esfenvalerate. J Agric Food Chem. 1999, 47: 2145-2155.
[15] 朱立平,陈雪清. 免疫学实验方法. 北京人民军医出版社. 2000, 120-150.
[16] 王重庆. 分子免疫学基础. 北京大学出版社. 1999, 24-28.
[17] Engcall E., Jonsson K, Perlmann P. Enzyme-linked immunosorbent assay. II. Quantitative assay of protein antigen, immunoglobulin G, by means of enzyme-labelled antigen and antibody-coated tubes. Biochim Biophys Acta. 1971, 251: 427-434.
[18] 陈福生,李根久. 酱油中黄曲霉毒素的酶联免疫检测. 中国调味品,1998: 26-30.
[19] 刘冬儿. 酶联免疫吸附分析法测定食品中的黄曲霉毒B1. 包装与机械. 2002, 23: 79-89.
[20] Powell RD, Halsey CM, Hainfel R,et al. Combined fluoresecent and gold immuno-probes: reagents and methods for correlative light and electron microscopy. Microsc Res Tech. 1998, 42: 2-12.
[21] Putalun W. Tanaka H., Shoyama Y. Rapid detection of glycyrrhizin by immunochromatographic assay. Phytochem. Anal. 2005, 16: 370-374.
[22] Zhang GP, Wang XN, Yang JF, et al. Development of an immunochromatographic lateral flow test strip for detection of β-adrenergic agonist Clenbuterol residues. J Immunol. Methods. 2006, 312: 27-33.
[23] Sun XL, Zhao XL, Tang J, et al. Preparation of gold-labeled antibody probe and its use in immunochromatography assay for detection of aflatoxin B1. International Journal of Food Microbiology. 2005, 99: 185-194.
[24] 刘景梅,刘兆军. 金标试纸条对测定生化指标的影响. 现代检验医学杂志,2006, 21: 7.
[25] 羿国香,赵静,李刚,等. 氯嘧磺隆胶体金免疫检测试纸条的制备与应用.分析化学. 2006, 34: 1679-1682.
[26] Gao YT. Study on the preptation of colloidal gold probe. Journal of Yunnan Institute of the Nationalities. 1999,1: 41-43.
[27] 李晓华. 胶体金标技术及应用. 生物学通报. 2004, 39: 54-55.
[28] 李振甲,陈泮藻,高平,等. 时间分辨荧光分析技术与应用. 科学出版社. 1996.
[29] 黄飚,陶文沂,张莲芬,等. 黄曲霉毒素 B1 的高灵敏时间分辨荧光免疫分析. 核技术. 2006, 29: 295-300.
[30] Anthony PFT. Biosensors sense and sensitivity. Science. 2000, 290: 1315-1317.
[31] Rodriguez-Mozaz S, Alda MJL, Marco MP, et al. Biosensors for environmental monitoring: A global perspective. Talanta. 2005, 65:291-297.
[32] Jamett A, Aguayo J, Miquel A, et al. Characteristics of monoclonal antibodies against Piscirickettsia salmonis. Journal of Flash Diseases. 2001, 24: 205-215.
[33] Minunni M, Tombelli S, Mascini M, et al. An optical DNA-based biosensor for the analysis of bioactive constituents with application in drug and herbal drug screening. Talanta. 2005, 65: 578-585.
[34] Hajighasemi F, Gharagozlou S, Moheghi N, et al. Production and characterization of murine monoclonal antibodies recognizing conformational and linear epitopes localized on human IgA2 molecules. IJI. 2005, 2: 125-133.
[35] David SH. Survey of recent advances in analytical applications of immunoaffinity chromatography. Journal of Chromatography. 1998, 715: 3-28.
[36] Gurbay A, Aydin S, Girgin G., et al. Assessment of aflatoxin M1 levels in milk in Ankara, Turkey. Food Control. 2006, 17:1-4.
[37] 邵秀金,刘曙照,冯大和,等. 免疫亲合色谱及在农药残留分析中的应用. 农药学学报. 2003, 5: 9-13.
[38] Carvajal M, Mulholland F, Carner RC. Comparison of the EASI-EXTRACT immunoaffinity concentration procedure with the AOAC CB method for the extraction and quantitation of aflatoxin B1 in raw ground unskinned peanuts. J Chromatography. 1990, 511: 379-393.
[39] Stroka J, Anklam E, Jorissen U, et al. Immunoaffinity column cleanup with liquid chromatography using post-column bromination for determination of aflatoxins in peanut butter, pistachio paste, fig paste,and paprika powder: collaborative. J AOAC Int. 2000, 83: 320-340.
[40] Groopman JD, Donahue KF. Aflatoxin, a human carcinogen: determination in foods and biological samples by monoclonal antibody affinity chromatography. J Assoc Off Anal Chem. 1988, 71: 861-867.
[41] 李培武,马良等. 粮油产品中黄曲霉毒素 B1 检测技术研究进展. 中国油料作物学报. 2005, 27: 77-81.
[42] 金涌. 单克隆抗体技术及其在食品卫生检验中的应用. 农牧产品开发. 2001, 5: 9-12.
[43] Xia HC, Hu WG, Yan XX, et al. Preparation and primary application of monoclonal antibodies against a novel ribosome-inactivating protein moschatin from pumpkin seeds. Acta Biochimica et Biophysica Sinica. 2004, 36: 105-110.
[44] Lane RD, Crissman RS, Ginn S. High efficiency fusion procedure for producing monoclonal antibodies against weak immunogens. Methods Enzymol.1986, 121: 183-192.
[45] 江涛,王环宇,等. 抗赭曲霉素 A 单克隆杂交瘤细胞系的建立及特性. 中国食品卫生杂志. 2004, 16:14-17.
[46] Lin YL, Shen JH, Lee LH. A monoclonal antibody-based competitive enzyme-linked immunosorbent assay for detecting antibody production against avian reovirus protein σA. Journal of Virological Methods. 2006, 136: 71-77.
[47] Kim A, Li CR, Jin CF, et al. A sensitive and reliable quantification method for Bisphenol. Chemosphere. 2007, 68: 1204-1209.
[48] Tran TM, Ivanyi P, Hilgert I, et a1. The Epitope Recognized by Pan-HLA Class I-Reactive Monoclonal Antibody W6/32 and Its Relationship to Unusual Stability of the HLA-B27/beta2-Microglobulin Complex. Immunogenetics. 2001,53: 440-446.
[49] K?hler G., Milstein C. Continuous culture of fused cells secreting antibody of predefined specificity. Nature. 1975, 256: 495-497.
[50] 张文元,杨亚冬. 不同温度冻存对杂交瘤细胞及其单克隆抗体活力的影响.中国卫生检验杂志. 2006,16: 536-537.
[51] Harris ELV, Angal S. Protein purification methods, a practical approach. IRL Press. 1989.
[52] 周华蕾,吕茂民,王娜,等. 应用 A 蛋白亲和层析法纯化单克隆抗体. 生物技术通讯. 2005, 5: 72-74.
[53] Zhu QZ, Haupt K, Knopp D, et al Molecularly imprinted polymer for metsulfuron methyl and its binding charaterictics for sulfonylurea herbicides. Anal. Chim. Acta. 2002, 468: 217-227.
[54] Luzi E, Minunni M, Tombelli S, et al. New trends in affinity sensing: aptamers for ligand binding. Trends Anal Chem. 2003, 22: 810-818.
[55] 郭利军,王晶,申元英,等. 三种免疫血清制备方法的比较. 大理学院学报. 2004, 3: 7-8.
[56] Diano M, Le Bivic A, Hirn M. Raising polyclonal antibodies using nitrocellulose-bound antigen. Methods Mol Biol. 1998, 80: 5-13.
[57] 郑继平, 刘向昕, 王令春, 等. 产肠毒素大肠杆菌混合载体疫苗的免疫原性评价. 现代免疫学. 2004, 24: 482-485.
[58] 周宗安译. 单克隆抗体手册. 南京大学出版社. 1991, 48-63.
[59] 王文聪. 单克隆抗体制备中的几个关键问题 .西昌农业高等专科学校学报. 2002, 16: 78-80.
[60] 李义良, 宋海, 赵静, 等. LPTS 抗体的制备和活性检测. 中国生物工程杂志. 2004, 24: 44-47.
[61] 徐修礼,张建芳,于文彬,等. 杂交瘤腹水产量的影响因素. 第四军医大学学报. 2002, 23: 1632.
[62] Michella MM, Andrew P. A simple,non-chromatographic procedure to purify immunoglobulins from serum and ascites fluid. Journal of Immunological Methods. 1987, 96: 271-278.
[63] Huang YF, Yang SH, Li BT. Experimental study of Pristane,liquid paraffin,Freund's incomplete adjuvant induced mice producing ascite-McAb. Bulletin of McAb. 1988, 4: 78.
[64] James D, Gokin SE, Eastwell KC. Identification and differentiation prunus virus isolates that cross-react with plum pox virus and apple stem pitting virus antisers. Plant Disease. 1998, 81: 855-861.
[65] 杨婉娟,高凯,饶春明,等. 抗 DNA 单克隆抗体杂交瘤细胞株的建立及其抗体的鉴定.中国生物制品学杂志. 2006, 19: 182-186.
[66] Beatty JD, Beatty BG. Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay. J Immunol Methods. 1987, 100: 173-179.
[67] Hajighasemi F, Saboor-Yaraghi AA, Shokri F. Measurement of affinity constant of anti-human IgG monoclonal antibodies by an Elisa-based method. Iranian J Immunol. 2004, 1: 154-161.
[68] 邓舜洲,余宙,章英,等. 脱氧雪腐镰刀菌烯醇免疫亲和层析柱的研制. 食品工业科技. 2007, 2: 220-223.
[69] Brimfield AA, Lenz DE, Graham C, et al. Mouse monoclonal antibodies against paraoxon: potential reagents for immunoassay with constant immunochemical characteristics. 1985, 33: 1237-1242.
[70] Ejimaa D, Yumiokaa R, Tsumotob K, et al. Effective elution of antibodies by arginine and arginine derivatives in affinity column chromatography. Analytical Biochemistry. 2005, 345: 250-257.
[71] Hennion MC, Pichon V. Immuno-based sample preparation for trace analysis. J Chromatogr A. 2003, 1000: 29 -52.
[72] Pfeiffer NE, Wylie DE, Schuster SM, et al. Immunoaffinity chromatography utilizing monoclonal antibodies: factors which influence antigen-binding capacity. J Immunol Methods. 1987, 97: 1-9.
[73] Hage D, Nelson M. Chromatographic Immunoassays. Anal Chem. 2001, 73: 199-205.
[2] 杨建伯. 真菌毒素与人类疾病. 中国地方病学杂志,2002,21(4):314-317.
[3] Machinski M J, Soares LMV, Sawazaki E, et al. Aflatoxins, ochratoxin A and zearalenone in Brazilian corn cultivars. J Science of food and agriculture. 2001, 81: 1001-1007.
[4] Arora DK, Bridge PD, Bhatnagar D, et al. Fungal biotechnology in agriculture, food and environmental applications. Mycotoxins. 2004,21: 325-342.
[5] Olivier M, Hussain SP, Hainaut P, et al. TP53 mutation spectra and load: a tool for generating hypotheses on the etiology of cancer. IARC Sci Publ. 2004, 157: 247-270.
[6] 徐进,罗雪云. 黄曲霉毒素合成的分子生物学. 卫生研究. 2003,32: 628-631.
[7] Luderitz O, Westphal O, Staub AM, et al. Microbial Toxins. New York Academic Press. 1971, 163-168.
[8] 食品中黄曲霉毒素的测定. 免疫亲和层析净化高效液相色谱法.中华人民共和国国家标准 GB/T 18979-2003.
[9] 叶雪珠,王小骊,等. 黄曲霉毒素 B1 检测方法的分析. 食品与发酵工业. 29: 90-92.
[10] Groopman JD, Trudel LJ, Donahue PR, et al. High-affinity monoclonal antibodies for aflatoxins and their application to solid-phase immunoassays. Proc Natl Acad Sci USA. 1984, 81: 7728-7731.
[11] Trucksess MW, Young KN, Donahue KF, et al. Comparation of two immunochemical methods with thin layer chromatographic methods for determination of aflatoxins. Journal of AOAC Int. 1990, 73: 425-428.
[12] 江湖,熊勇华,许扬. 黄曲霉毒素分析方法研究进展. 卫生研究. 2005, 34: 252-255.
[13] Maurice OM. Risk assessment for aflatoxins in food stuffs. International Biodeterioration and Biodegradation. 2002, 50, 137-142.
[14] Shan G, Stoutamire DW, Wengatz I, et al. Development of an immunoassay for the pyrethroid insecticide esfenvalerate. J Agric Food Chem. 1999, 47: 2145-2155.
[15] 朱立平,陈雪清. 免疫学实验方法. 北京人民军医出版社. 2000, 120-150.
[16] 王重庆. 分子免疫学基础. 北京大学出版社. 1999, 24-28.
[17] Engcall E., Jonsson K, Perlmann P. Enzyme-linked immunosorbent assay. II. Quantitative assay of protein antigen, immunoglobulin G, by means of enzyme-labelled antigen and antibody-coated tubes. Biochim Biophys Acta. 1971, 251: 427-434.
[18] 陈福生,李根久. 酱油中黄曲霉毒素的酶联免疫检测. 中国调味品,1998: 26-30.
[19] 刘冬儿. 酶联免疫吸附分析法测定食品中的黄曲霉毒B1. 包装与机械. 2002, 23: 79-89.
[20] Powell RD, Halsey CM, Hainfel R,et al. Combined fluoresecent and gold immuno-probes: reagents and methods for correlative light and electron microscopy. Microsc Res Tech. 1998, 42: 2-12.
[21] Putalun W. Tanaka H., Shoyama Y. Rapid detection of glycyrrhizin by immunochromatographic assay. Phytochem. Anal. 2005, 16: 370-374.
[22] Zhang GP, Wang XN, Yang JF, et al. Development of an immunochromatographic lateral flow test strip for detection of β-adrenergic agonist Clenbuterol residues. J Immunol. Methods. 2006, 312: 27-33.
[23] Sun XL, Zhao XL, Tang J, et al. Preparation of gold-labeled antibody probe and its use in immunochromatography assay for detection of aflatoxin B1. International Journal of Food Microbiology. 2005, 99: 185-194.
[24] 刘景梅,刘兆军. 金标试纸条对测定生化指标的影响. 现代检验医学杂志,2006, 21: 7.
[25] 羿国香,赵静,李刚,等. 氯嘧磺隆胶体金免疫检测试纸条的制备与应用.分析化学. 2006, 34: 1679-1682.
[26] Gao YT. Study on the preptation of colloidal gold probe. Journal of Yunnan Institute of the Nationalities. 1999,1: 41-43.
[27] 李晓华. 胶体金标技术及应用. 生物学通报. 2004, 39: 54-55.
[28] 李振甲,陈泮藻,高平,等. 时间分辨荧光分析技术与应用. 科学出版社. 1996.
[29] 黄飚,陶文沂,张莲芬,等. 黄曲霉毒素 B1 的高灵敏时间分辨荧光免疫分析. 核技术. 2006, 29: 295-300.
[30] Anthony PFT. Biosensors sense and sensitivity. Science. 2000, 290: 1315-1317.
[31] Rodriguez-Mozaz S, Alda MJL, Marco MP, et al. Biosensors for environmental monitoring: A global perspective. Talanta. 2005, 65:291-297.
[32] Jamett A, Aguayo J, Miquel A, et al. Characteristics of monoclonal antibodies against Piscirickettsia salmonis. Journal of Flash Diseases. 2001, 24: 205-215.
[33] Minunni M, Tombelli S, Mascini M, et al. An optical DNA-based biosensor for the analysis of bioactive constituents with application in drug and herbal drug screening. Talanta. 2005, 65: 578-585.
[34] Hajighasemi F, Gharagozlou S, Moheghi N, et al. Production and characterization of murine monoclonal antibodies recognizing conformational and linear epitopes localized on human IgA2 molecules. IJI. 2005, 2: 125-133.
[35] David SH. Survey of recent advances in analytical applications of immunoaffinity chromatography. Journal of Chromatography. 1998, 715: 3-28.
[36] Gurbay A, Aydin S, Girgin G., et al. Assessment of aflatoxin M1 levels in milk in Ankara, Turkey. Food Control. 2006, 17:1-4.
[37] 邵秀金,刘曙照,冯大和,等. 免疫亲合色谱及在农药残留分析中的应用. 农药学学报. 2003, 5: 9-13.
[38] Carvajal M, Mulholland F, Carner RC. Comparison of the EASI-EXTRACT immunoaffinity concentration procedure with the AOAC CB method for the extraction and quantitation of aflatoxin B1 in raw ground unskinned peanuts. J Chromatography. 1990, 511: 379-393.
[39] Stroka J, Anklam E, Jorissen U, et al. Immunoaffinity column cleanup with liquid chromatography using post-column bromination for determination of aflatoxins in peanut butter, pistachio paste, fig paste,and paprika powder: collaborative. J AOAC Int. 2000, 83: 320-340.
[40] Groopman JD, Donahue KF. Aflatoxin, a human carcinogen: determination in foods and biological samples by monoclonal antibody affinity chromatography. J Assoc Off Anal Chem. 1988, 71: 861-867.
[41] 李培武,马良等. 粮油产品中黄曲霉毒素 B1 检测技术研究进展. 中国油料作物学报. 2005, 27: 77-81.
[42] 金涌. 单克隆抗体技术及其在食品卫生检验中的应用. 农牧产品开发. 2001, 5: 9-12.
[43] Xia HC, Hu WG, Yan XX, et al. Preparation and primary application of monoclonal antibodies against a novel ribosome-inactivating protein moschatin from pumpkin seeds. Acta Biochimica et Biophysica Sinica. 2004, 36: 105-110.
[44] Lane RD, Crissman RS, Ginn S. High efficiency fusion procedure for producing monoclonal antibodies against weak immunogens. Methods Enzymol.1986, 121: 183-192.
[45] 江涛,王环宇,等. 抗赭曲霉素 A 单克隆杂交瘤细胞系的建立及特性. 中国食品卫生杂志. 2004, 16:14-17.
[46] Lin YL, Shen JH, Lee LH. A monoclonal antibody-based competitive enzyme-linked immunosorbent assay for detecting antibody production against avian reovirus protein σA. Journal of Virological Methods. 2006, 136: 71-77.
[47] Kim A, Li CR, Jin CF, et al. A sensitive and reliable quantification method for Bisphenol. Chemosphere. 2007, 68: 1204-1209.
[48] Tran TM, Ivanyi P, Hilgert I, et a1. The Epitope Recognized by Pan-HLA Class I-Reactive Monoclonal Antibody W6/32 and Its Relationship to Unusual Stability of the HLA-B27/beta2-Microglobulin Complex. Immunogenetics. 2001,53: 440-446.
[49] K?hler G., Milstein C. Continuous culture of fused cells secreting antibody of predefined specificity. Nature. 1975, 256: 495-497.
[50] 张文元,杨亚冬. 不同温度冻存对杂交瘤细胞及其单克隆抗体活力的影响.中国卫生检验杂志. 2006,16: 536-537.
[51] Harris ELV, Angal S. Protein purification methods, a practical approach. IRL Press. 1989.
[52] 周华蕾,吕茂民,王娜,等. 应用 A 蛋白亲和层析法纯化单克隆抗体. 生物技术通讯. 2005, 5: 72-74.
[53] Zhu QZ, Haupt K, Knopp D, et al Molecularly imprinted polymer for metsulfuron methyl and its binding charaterictics for sulfonylurea herbicides. Anal. Chim. Acta. 2002, 468: 217-227.
[54] Luzi E, Minunni M, Tombelli S, et al. New trends in affinity sensing: aptamers for ligand binding. Trends Anal Chem. 2003, 22: 810-818.
[55] 郭利军,王晶,申元英,等. 三种免疫血清制备方法的比较. 大理学院学报. 2004, 3: 7-8.
[56] Diano M, Le Bivic A, Hirn M. Raising polyclonal antibodies using nitrocellulose-bound antigen. Methods Mol Biol. 1998, 80: 5-13.
[57] 郑继平, 刘向昕, 王令春, 等. 产肠毒素大肠杆菌混合载体疫苗的免疫原性评价. 现代免疫学. 2004, 24: 482-485.
[58] 周宗安译. 单克隆抗体手册. 南京大学出版社. 1991, 48-63.
[59] 王文聪. 单克隆抗体制备中的几个关键问题 .西昌农业高等专科学校学报. 2002, 16: 78-80.
[60] 李义良, 宋海, 赵静, 等. LPTS 抗体的制备和活性检测. 中国生物工程杂志. 2004, 24: 44-47.
[61] 徐修礼,张建芳,于文彬,等. 杂交瘤腹水产量的影响因素. 第四军医大学学报. 2002, 23: 1632.
[62] Michella MM, Andrew P. A simple,non-chromatographic procedure to purify immunoglobulins from serum and ascites fluid. Journal of Immunological Methods. 1987, 96: 271-278.
[63] Huang YF, Yang SH, Li BT. Experimental study of Pristane,liquid paraffin,Freund's incomplete adjuvant induced mice producing ascite-McAb. Bulletin of McAb. 1988, 4: 78.
[64] James D, Gokin SE, Eastwell KC. Identification and differentiation prunus virus isolates that cross-react with plum pox virus and apple stem pitting virus antisers. Plant Disease. 1998, 81: 855-861.
[65] 杨婉娟,高凯,饶春明,等. 抗 DNA 单克隆抗体杂交瘤细胞株的建立及其抗体的鉴定.中国生物制品学杂志. 2006, 19: 182-186.
[66] Beatty JD, Beatty BG. Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay. J Immunol Methods. 1987, 100: 173-179.
[67] Hajighasemi F, Saboor-Yaraghi AA, Shokri F. Measurement of affinity constant of anti-human IgG monoclonal antibodies by an Elisa-based method. Iranian J Immunol. 2004, 1: 154-161.
[68] 邓舜洲,余宙,章英,等. 脱氧雪腐镰刀菌烯醇免疫亲和层析柱的研制. 食品工业科技. 2007, 2: 220-223.
[69] Brimfield AA, Lenz DE, Graham C, et al. Mouse monoclonal antibodies against paraoxon: potential reagents for immunoassay with constant immunochemical characteristics. 1985, 33: 1237-1242.
[70] Ejimaa D, Yumiokaa R, Tsumotob K, et al. Effective elution of antibodies by arginine and arginine derivatives in affinity column chromatography. Analytical Biochemistry. 2005, 345: 250-257.
[71] Hennion MC, Pichon V. Immuno-based sample preparation for trace analysis. J Chromatogr A. 2003, 1000: 29 -52.
[72] Pfeiffer NE, Wylie DE, Schuster SM, et al. Immunoaffinity chromatography utilizing monoclonal antibodies: factors which influence antigen-binding capacity. J Immunol Methods. 1987, 97: 1-9.
[73] Hage D, Nelson M. Chromatographic Immunoassays. Anal Chem. 2001, 73: 199-205.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |