雌二醇单克隆抗体的制备与酶联免疫检测试剂盒的初步研究
摘要
为建立雌二醇(estradiol, E2)在畜产品中残留的快速检测方法,本研究从完全抗原的制备着手,采用碳化二亚胺法制备E2免疫原,免疫BALB/c 小鼠,用间接酶联免疫吸附法(ELISA)对抗体效价进行检测,利用杂交瘤技术筛选稳定分泌E2的单克隆抗体,用过碘酸钠氧化法制备酶标雌二醇抗原(E2-BSA-HRP)和酶标雌二醇抗体(1E3-HRP),用方阵滴定法确定包被原最适工作浓度及单抗最适稀释倍数。最终对用于检测雌二醇残留的三种酶联免疫检测方法(酶标抗原直接竞争法、酶标抗体直接抑制法、双抗夹心法)进行了筛选。实验结果为:E2抗血清效价为1∶3.2×104。利用杂交瘤技术获得了两株稳定分泌E2单克隆抗体的杂交瘤细胞株1E3和1B4,经鉴定1E3细胞株分泌的单克隆抗体为IgG2b 亚型,1B4 细胞株分泌的单克隆抗体为IgG1 亚型;杂交瘤细胞染色体数目为92~102 条;间接ELISA 测定腹水效价为1∶1×105,其亲和常数为2.9×108L/mol。1E3单克隆抗体的分子量为179KD,其中轻链分子量为28KD,重链分子量为61.2KD。该单抗与雌三醇、雌酮和孕酮交叉反应率均小于0.5%。该细胞株体外传代和冻存复苏后抗体分泌稳定。用方阵滴定法确定包被原最适工作浓度为0.625 μg/ml,单抗最适稀释倍数为1∶1000,辣根过氧化物酶标记羊抗鼠IgG(酶标二抗)最适稀释倍数为l∶5000。采用间接竞争ELISA 方法建立检测E2的标准曲线,在0.01 ng/ml~1μg/ml 之间,呈良好的线性相关,线性回归方程为:y = -8.201x + 47.814,R2 = 0.9965。以抑制率为50%时对应的浓度计算,该方法对E2的检测限为0.54 ng/ml。通过比较三种ELISA 方法,从中选出了试剂盒的检测方法为酶标抗原直接竞争法。
To esabolish the quick test for the residues of Estradiol(E2) in the animal products, E2 was coupled to carrier protein of bovine serum albumin(BSA) and ovalbumin(OVA) using carbodiimide method. The E2-BSA and the E2-OVA were used as immunogen and coating antigen, respectively, then BALB/c mice were immunised with E2-BSA. The titer of anti-E2 serum was tested with indirectly competitive ELISA. The constantly secreting anti-E2 monoclonal antibody was screened with hybridomas techinque. HRP-labelled estradiol antigen and antibody (E2-BSA-HRP and 1E3-HRP) were prepared by coupling the E2-BSA and 1E3 with HRP by the improved sodium periodate method. The optimised working concentrations of coated antigen and antibody were performed with phalanx titration. Consequently, the enzyme-labelled antigen directly competitive, enzyme-labelled antibody directly inhibition and double antibody sandwich enzyme linked immunosorbent assay (ELISA) methods for E2 residues testing were compared to select the optimised one. Results were that the titer of anti-E2 serum was 1:3.2×104. Two hybridomas producing anti-E2 monoclonal antibodies (McAb) were obtained by limited dilution, named 1E3 and 1B4. Aditionally, the characteristic of McAb was performed by ELISA, the subclass of the McAb was IgG2b and IgG1, the titer of ascites was 1:1×105, the affinity constant of E2-McAb for coated complete antigen was 2.9 ×108L/mol, the molecular weight was 179KD. Chromosomal numbers of the hybridoma 92-102. The cross-reaction rate of McAb to estriol, estrone and progesterone was all below 0.5%. The antibody secretion of 1E3 was stable after in vitro passage and the anabiosis from the frozened store. The optimised working concentration and dilution ratio of coated antigen and McAb were 0.625μg/ml, 1:1000, respectively. The optimised dilution ratio of HRP linked goat anti-mouse IgG antibody was 1:5000. The linear detecting range of standard curves was between 0.01ng -1μg/ml. The linear regression equation was y = -8.201x + 47.814 with R2 = 0.9965. When regarding the 50% of inhibition concentration (IC50) as the detecting limitation it was 0.54ng/ml. It is concluded that the enzyme labelled antigen direct competitive ELISA could be used in the kit-test for the E2 residues.
To esabolish the quick test for the residues of Estradiol(E2) in the animal products, E2 was coupled to carrier protein of bovine serum albumin(BSA) and ovalbumin(OVA) using carbodiimide method. The E2-BSA and the E2-OVA were used as immunogen and coating antigen, respectively, then BALB/c mice were immunised with E2-BSA. The titer of anti-E2 serum was tested with indirectly competitive ELISA. The constantly secreting anti-E2 monoclonal antibody was screened with hybridomas techinque. HRP-labelled estradiol antigen and antibody (E2-BSA-HRP and 1E3-HRP) were prepared by coupling the E2-BSA and 1E3 with HRP by the improved sodium periodate method. The optimised working concentrations of coated antigen and antibody were performed with phalanx titration. Consequently, the enzyme-labelled antigen directly competitive, enzyme-labelled antibody directly inhibition and double antibody sandwich enzyme linked immunosorbent assay (ELISA) methods for E2 residues testing were compared to select the optimised one. Results were that the titer of anti-E2 serum was 1:3.2×104. Two hybridomas producing anti-E2 monoclonal antibodies (McAb) were obtained by limited dilution, named 1E3 and 1B4. Aditionally, the characteristic of McAb was performed by ELISA, the subclass of the McAb was IgG2b and IgG1, the titer of ascites was 1:1×105, the affinity constant of E2-McAb for coated complete antigen was 2.9 ×108L/mol, the molecular weight was 179KD. Chromosomal numbers of the hybridoma 92-102. The cross-reaction rate of McAb to estriol, estrone and progesterone was all below 0.5%. The antibody secretion of 1E3 was stable after in vitro passage and the anabiosis from the frozened store. The optimised working concentration and dilution ratio of coated antigen and McAb were 0.625μg/ml, 1:1000, respectively. The optimised dilution ratio of HRP linked goat anti-mouse IgG antibody was 1:5000. The linear detecting range of standard curves was between 0.01ng -1μg/ml. The linear regression equation was y = -8.201x + 47.814 with R2 = 0.9965. When regarding the 50% of inhibition concentration (IC50) as the detecting limitation it was 0.54ng/ml. It is concluded that the enzyme labelled antigen direct competitive ELISA could be used in the kit-test for the E2 residues.
引文
[1] 阮芳赋编著.性激素的发现.北京:科学出版社, 1979, 43
[2] 王明运主编.激素生物化学.北京:人民卫生出版社, 1987, 313~316
[3] Fortune J. E. Sirois J., Quirk S. M. Theriogenology [J], 1988, 29:95~109
[4] Lin La, Tomlinson J A, Satzger R D. J Chromatogr A, 1997, 762(1/2):275
[5] Sangiorgi E, Cruatlo M. J Chromatogr, B: Biomed Sci Appl, 1997, 693(2):468
[6] Plsyniak A. Polish J. Environ Studies, 1995, 5(5):33
[7]Simpson J L,Elias S,Morgan C D,et al.2nd Trimester Maternal Serum Human Chorionie-gonadotropin and Anconju-gated Estriol Levels in Blacks and Whites. Lancet 1990, 335 :1 459~1 460
[8] Leporrier N, Herrou M, Herlicoviez M, et al. The Usefulness of hCG and Unconjugated Oestriol in Prenatal Diagnosis of Trisomy 18. Br J Obst Gynaecol, 1996, 103:335~338
[9] Forest J C, 1Ylasse J, Rousseau F, et al. Screening for Dowrrsyndrome during the First and 2nd Trimesters-impact of Risk estimation Parameters. (3in Biochem, 1995, 28:443~449
[10] Keren D F, Canick J A, Johnson M J, et al. Low Maternal Serum Unconjugated Estriol during Prenatal Screening as an Indiction of Placental Sulfatase Deficiency and xlinked Ichthyosis. Am J Clin Pathol, 1995, 103 :400~403
[11] Petruzzelli E, Ius A, Berta S, et al. Food Agric Immunol, 1996, 8(1):3
[12] 冯海,徐光明,刘迪霞,等. 近红外光谱法同时测定多种雌、孕激素. 分析化学,2001,29(2):175~177
[13] 胡伟,黄荣斌,陈小君,等. 雌二醇的气相色谱分析衍生化方法研究. 宁波高等专科学校学报,2001,13:45~47
[14] 林奕芝,张世英,梁伟,等. 高效液相色谱法测定肉中激素残留量. 现代预防医学,2002,29(6):766~767
[15] 杨利国等. 酶免疫测定技术. 南京大学出版社,1998
[16] Frank Z, et al. Use of 125I-histamine-labeled steroid derivatives as radioligands for radioimmunoassay of natural and synthetic steroids. J Steroid Biochem. 1981, 14:53~62
[17] 汪现,翁玲玲,张祖兵. 17-α雌二醇3-醚化合物的合成及初步生理活性研究. 华西药学杂志,2003,18(1):30~31
[18] 李晓莉,郑虎,翁玲玲. 转位法制备α-雌二醇的工艺. 华西药学杂志,2003,18(3):191~192
[19] Mares A, Boever J De, Osher J, et al. Adirect non-com petitive idiom etricenzyme immunassay for serum Estradiol. Immun Methods, 1995, 181:83
[20] Mares A, Boever J De, Stans G, et al. Synthesis of a novel biotin-estradiol conjugate its use for the development of a direct broad range enzyme immunassay for plasma estradiol. J Immun Methods, 1995, 183:211
[21] 金声,周浣芳,常文保,等. 酶联免疫吸附法直接测定血清雌二醇. 分析化学,1994,22(2):115~120
[22] 顾鸣,韩伟,孙文清. 应用自动酶联免疫荧光仪(VIDAS)检测禽类组织中雌二醇水平. 检验检疫科学,2003,13(4):19~21
[23] 王永成,张新祥,李元宗,等. 基于温度敏感水凝胶的雌二醇荧光免疫分析. 高等学校化学学报,2002,23(5):792~795
[24] L?rgren T. J. Steroid Biochem. 1987, 27(1~3):47-51
[25]Altamirano-Bustamante A, Barnard G, Kohen F. J. Immunlogical Methods, 1991, 138:95~101
[26] Mikola H, Sundell A C, Hanninen E. Steroids, 1993,58:330~334
[27] 康棣,王永成,常文保,等. 时间分辨荧光免疫分析法直接测定雌二醇. 分析化学,1999,27(8):899~903
[28] 李振甲,等激素的放射免疫分析北京:科学技术文献出版社,1984,393~399
[29] 郭大智,等应用同位素技术测定山羊血浆和乳中孕酮、雌二醇浓度及甲状腺功能原子能农业应用1986,2,61~64
[30] 赵丹慧,李成文,薛延,等. 高价特异半抗原雌二醇抗体及酶标雌二醇复合物的研制. 卫生研究, 1998, 27:191~193
[31] 孙家秀. 直接法雌二醇(E2)放射免疫分析试剂盒的研制. 中国原子能科学研究院年报, 1997, 124
[32] 张丁联, 潘家荣, 王斌. 血清雌二醇放射免疫试剂盒的研制. 核农学报, 2003, 17(4):304~307
[33] K?hler G, Milstein C. Continuous Cultures of Fused Cells Secreting Antibody of Redefined Specificity. Nature, 1975, 256:495~497
[34] 李建科, 张海彬, 畜产食品中有害物质残留分析及安全性评价, 畜牧与兽医, 2003, 35(2):35~372.
[35]张雨梅, 动物源性食品的安全性及药物残留监控, 江苏农业科学, 2001(6):60~63
[36] Ishida J., Kai M., Ohkura Y. J. Chromatogr. [J], 1988, 431:249~257
[37] Jamila E. L., Jabri J.. Steroid Biochem. Molec. Biol. [J], 1991, 38:339~343
[38]Chan K. C., Muschik G. M., Issaq H. J. et al. J. Chromtogr. A [J], 1995, 690:149~154
[39]杨杏芬.环境雌激素污染与毒效应研究的现状与展望[J]. 广东卫生防疫, 2001, 27(1):20~24.
[40] Boyd D, O’Keeffe M, Smyth R. Analyst, 1994, 119:1467
[41] Van Ginkel L A. J Chromatogr, 1991, 564:363
[42] Nausch T. Archiv Fur Lebensmittelhygiene, 1997, 48(3):56
[43] Dhar T K,Samanta A K,Ali E. Steroids,1988,51(5~6):519~526
[44] 徐志凯.实用单克隆抗体技术.陕西科学技术出版社,1992
[45] 吴冠芳主编.生物化学与分子生物学实验常用数据手册.科学出版社,1999
[46] Heddy Zola. 单克隆技术手册.周宗安译.南京大学出版社,1991
[47] 张龙翔等. 生化实验方法和技术.教育出版社,1997
[48][美]F·奥斯伯等著,颜子颖等译.精编分子生物学指南.科学出版社,1998
[49] 周顺伍主编.生物化学实验技术.北京农业大学出版社,1991
[50] 鄂征.组织培养技术(第二版).人民卫生出版,1987
[51] 郭晓君编著.蛋白质电泳实验技术.科学出版社,1999
[52] John GR, Monoclonal hybridoma antibodies: Techniques and applications CRC Press Boca Raton. Florida 1982:36
[53] Beatty J D, Barbara B G, William G V. Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay. J. Immunol Methods, 1978, 100:173~179
[54] Erlanger B F, Borek F, Beiser S M, et al. J Bio Chem, 1957, 228:713
[55] Erlanger B F, Borek F, Beiser S M, et al. J Bio Chem, 1959, 234:1090
[56] C. W.帕克.生物活性化合物的放射免疫测定法.北京:科学出版社, 1981
[57] Samant A K, AliE. Enzyme immunoassay of testosterone using nitrocellose discs as the solid phase. JClin Chem Clin Biochem, 1990, 28(12):943
[58] AliE, Sengupta J, Dhar T K. Sandwich immunoassay of small molecules. ImmunlMethods, 1992, 147:173
[59] 张国龙.大豆胰蛋白酶抑制因子酶联免疫吸附测定方法的研究:[硕士学位论文].北京:中国农业大学,1995
[60] 郭春祥, 郭锡琼. 介绍一种简单、快速、高效的辣根过氧化物酶标记抗体的过碘酸钠法. 上海免疫学杂志, 1983, 3(2):97~100
[2] 王明运主编.激素生物化学.北京:人民卫生出版社, 1987, 313~316
[3] Fortune J. E. Sirois J., Quirk S. M. Theriogenology [J], 1988, 29:95~109
[4] Lin La, Tomlinson J A, Satzger R D. J Chromatogr A, 1997, 762(1/2):275
[5] Sangiorgi E, Cruatlo M. J Chromatogr, B: Biomed Sci Appl, 1997, 693(2):468
[6] Plsyniak A. Polish J. Environ Studies, 1995, 5(5):33
[7]Simpson J L,Elias S,Morgan C D,et al.2nd Trimester Maternal Serum Human Chorionie-gonadotropin and Anconju-gated Estriol Levels in Blacks and Whites. Lancet 1990, 335 :1 459~1 460
[8] Leporrier N, Herrou M, Herlicoviez M, et al. The Usefulness of hCG and Unconjugated Oestriol in Prenatal Diagnosis of Trisomy 18. Br J Obst Gynaecol, 1996, 103:335~338
[9] Forest J C, 1Ylasse J, Rousseau F, et al. Screening for Dowrrsyndrome during the First and 2nd Trimesters-impact of Risk estimation Parameters. (3in Biochem, 1995, 28:443~449
[10] Keren D F, Canick J A, Johnson M J, et al. Low Maternal Serum Unconjugated Estriol during Prenatal Screening as an Indiction of Placental Sulfatase Deficiency and xlinked Ichthyosis. Am J Clin Pathol, 1995, 103 :400~403
[11] Petruzzelli E, Ius A, Berta S, et al. Food Agric Immunol, 1996, 8(1):3
[12] 冯海,徐光明,刘迪霞,等. 近红外光谱法同时测定多种雌、孕激素. 分析化学,2001,29(2):175~177
[13] 胡伟,黄荣斌,陈小君,等. 雌二醇的气相色谱分析衍生化方法研究. 宁波高等专科学校学报,2001,13:45~47
[14] 林奕芝,张世英,梁伟,等. 高效液相色谱法测定肉中激素残留量. 现代预防医学,2002,29(6):766~767
[15] 杨利国等. 酶免疫测定技术. 南京大学出版社,1998
[16] Frank Z, et al. Use of 125I-histamine-labeled steroid derivatives as radioligands for radioimmunoassay of natural and synthetic steroids. J Steroid Biochem. 1981, 14:53~62
[17] 汪现,翁玲玲,张祖兵. 17-α雌二醇3-醚化合物的合成及初步生理活性研究. 华西药学杂志,2003,18(1):30~31
[18] 李晓莉,郑虎,翁玲玲. 转位法制备α-雌二醇的工艺. 华西药学杂志,2003,18(3):191~192
[19] Mares A, Boever J De, Osher J, et al. Adirect non-com petitive idiom etricenzyme immunassay for serum Estradiol. Immun Methods, 1995, 181:83
[20] Mares A, Boever J De, Stans G, et al. Synthesis of a novel biotin-estradiol conjugate its use for the development of a direct broad range enzyme immunassay for plasma estradiol. J Immun Methods, 1995, 183:211
[21] 金声,周浣芳,常文保,等. 酶联免疫吸附法直接测定血清雌二醇. 分析化学,1994,22(2):115~120
[22] 顾鸣,韩伟,孙文清. 应用自动酶联免疫荧光仪(VIDAS)检测禽类组织中雌二醇水平. 检验检疫科学,2003,13(4):19~21
[23] 王永成,张新祥,李元宗,等. 基于温度敏感水凝胶的雌二醇荧光免疫分析. 高等学校化学学报,2002,23(5):792~795
[24] L?rgren T. J. Steroid Biochem. 1987, 27(1~3):47-51
[25]Altamirano-Bustamante A, Barnard G, Kohen F. J. Immunlogical Methods, 1991, 138:95~101
[26] Mikola H, Sundell A C, Hanninen E. Steroids, 1993,58:330~334
[27] 康棣,王永成,常文保,等. 时间分辨荧光免疫分析法直接测定雌二醇. 分析化学,1999,27(8):899~903
[28] 李振甲,等激素的放射免疫分析北京:科学技术文献出版社,1984,393~399
[29] 郭大智,等应用同位素技术测定山羊血浆和乳中孕酮、雌二醇浓度及甲状腺功能原子能农业应用1986,2,61~64
[30] 赵丹慧,李成文,薛延,等. 高价特异半抗原雌二醇抗体及酶标雌二醇复合物的研制. 卫生研究, 1998, 27:191~193
[31] 孙家秀. 直接法雌二醇(E2)放射免疫分析试剂盒的研制. 中国原子能科学研究院年报, 1997, 124
[32] 张丁联, 潘家荣, 王斌. 血清雌二醇放射免疫试剂盒的研制. 核农学报, 2003, 17(4):304~307
[33] K?hler G, Milstein C. Continuous Cultures of Fused Cells Secreting Antibody of Redefined Specificity. Nature, 1975, 256:495~497
[34] 李建科, 张海彬, 畜产食品中有害物质残留分析及安全性评价, 畜牧与兽医, 2003, 35(2):35~372.
[35]张雨梅, 动物源性食品的安全性及药物残留监控, 江苏农业科学, 2001(6):60~63
[36] Ishida J., Kai M., Ohkura Y. J. Chromatogr. [J], 1988, 431:249~257
[37] Jamila E. L., Jabri J.. Steroid Biochem. Molec. Biol. [J], 1991, 38:339~343
[38]Chan K. C., Muschik G. M., Issaq H. J. et al. J. Chromtogr. A [J], 1995, 690:149~154
[39]杨杏芬.环境雌激素污染与毒效应研究的现状与展望[J]. 广东卫生防疫, 2001, 27(1):20~24.
[40] Boyd D, O’Keeffe M, Smyth R. Analyst, 1994, 119:1467
[41] Van Ginkel L A. J Chromatogr, 1991, 564:363
[42] Nausch T. Archiv Fur Lebensmittelhygiene, 1997, 48(3):56
[43] Dhar T K,Samanta A K,Ali E. Steroids,1988,51(5~6):519~526
[44] 徐志凯.实用单克隆抗体技术.陕西科学技术出版社,1992
[45] 吴冠芳主编.生物化学与分子生物学实验常用数据手册.科学出版社,1999
[46] Heddy Zola. 单克隆技术手册.周宗安译.南京大学出版社,1991
[47] 张龙翔等. 生化实验方法和技术.教育出版社,1997
[48][美]F·奥斯伯等著,颜子颖等译.精编分子生物学指南.科学出版社,1998
[49] 周顺伍主编.生物化学实验技术.北京农业大学出版社,1991
[50] 鄂征.组织培养技术(第二版).人民卫生出版,1987
[51] 郭晓君编著.蛋白质电泳实验技术.科学出版社,1999
[52] John GR, Monoclonal hybridoma antibodies: Techniques and applications CRC Press Boca Raton. Florida 1982:36
[53] Beatty J D, Barbara B G, William G V. Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay. J. Immunol Methods, 1978, 100:173~179
[54] Erlanger B F, Borek F, Beiser S M, et al. J Bio Chem, 1957, 228:713
[55] Erlanger B F, Borek F, Beiser S M, et al. J Bio Chem, 1959, 234:1090
[56] C. W.帕克.生物活性化合物的放射免疫测定法.北京:科学出版社, 1981
[57] Samant A K, AliE. Enzyme immunoassay of testosterone using nitrocellose discs as the solid phase. JClin Chem Clin Biochem, 1990, 28(12):943
[58] AliE, Sengupta J, Dhar T K. Sandwich immunoassay of small molecules. ImmunlMethods, 1992, 147:173
[59] 张国龙.大豆胰蛋白酶抑制因子酶联免疫吸附测定方法的研究:[硕士学位论文].北京:中国农业大学,1995
[60] 郭春祥, 郭锡琼. 介绍一种简单、快速、高效的辣根过氧化物酶标记抗体的过碘酸钠法. 上海免疫学杂志, 1983, 3(2):97~100