已烯雌酚人工抗原的研究
|
摘要
己烯雌酚（diethylstilbestrol，DES）属于人工合成的雌激素，在人和动物体内会产生雌激素样效应。在养殖业中，DES能够显著促进动物的生长，提高饲料转化率，但由于DES对人体的致癌和致畸作用，世界各国均规定食品动物养殖中不得以任何途径和方式使用DES，动物及动物性食品中不得检出DES。DES残留免疫分析是DES残留监控中的重要手段之一。本研究属于DES残留免疫检测试剂盒研制的前期工作内容，也是必不可少的研究。研究取得如下结果：
1.在室温条件下，将DES与丁二酸酐按一定比例溶解于无水吡啶中,搅拌反应生成棕黄色物质，将其分离纯化后对其进行高分辨质谱、红外光谱、核磁共振碳谱及氢谱等鉴定，试验结果表明，该棕黄色物质分子量为368，分子式为C22H24O5，含有酚羟基、羧基等关键基团，为所要制备的DES衍生物己烯雌酚半琥珀酸（DES-HS）。
2.利用混合酸酐法将DES-HS与载体蛋白（BSA，OVA）进行偶联得到DES人工抗原。 合成的人工抗原经紫外扫描、SDS-PAGE 凝胶电泳鉴定证明DES-HS与载体蛋白偶联成功；并用UVP Bioimaging system 中的labworks 4.0 Image Acquisition and Analysis software 分析估算BSA与DES-HS的偶联率为1：30～32。OVA与DES-HS的偶联率为1:25～28。
3.用合成的DES-HS-BSA结合物分别免疫家兔和Balb/c小鼠，采集免疫动物血清。应用琼脂双扩散试验和间接ELISA试验、阻断ELISA检测特异性DES抗体的产生，抗体效价及其与游离DES反应的特异性。结果表明：无论免疫兔还是免疫小鼠，其免疫血清均能够与DES-HS-OVA上的DES决定簇发生特异性结合反应；免疫兔血清效价不低于1：6000，免疫小鼠血清效价不低于1：12000；兔免疫血清能够与游离DES发生特异性反应。
通过上述研究和分析，可得出如下的结论：
建立了DES-HS制备方法；成功地制备出了DES的人工抗原；用DES-HS-BSA分别免疫兔和小鼠制备出了抗DES免疫血清；建立了DES抗体的间接ELISA检测方法。本研究为研制DES单克隆抗体及拥有我国自主知识产权的DES残留免疫检测试剂盒奠定了基础。
Diethylstilbestrol(DES) is a synthetic estrogen. It can produce the effects like natural estrogens in the bodies of human beings and animals.In feeding, it was used illegally to promote growth and feed conversion, but it can lead to cancer and deformity. So it was ruled not to use DES in feeding animal by any way all over the world. Animal and animal products could not be detected DES. The immunoassay of residual DES is one of the critical means in monitor of DES residues. The study is necessary and piror to develop DES residual immunoassay reagent box . The main results are as follows:
1.At the room temperature,the certain ratio of DES and succinate anhydride was stirred in anhydrous pyridine, and got the brown product. The analysis results of HRMS, IR , 13C-NMR and 1H-NMR showed that the separated and pured is product was the derivative of DES wanted, called as DES-HS. Its molecular weight is 368, Its molecular formula is C22H24O5, It includes the critical structure such as -OH ,–COOH and so on.
2.The artificial antigen of DES was synthesized by mixed anhydride method using DES-HS and bovine serum albumin(BSA) and ovabumin(OVA) respectively. The characterization of hapten-protein conjugates were examined ultraviolet spectrophotometry(UV) and sodium doclecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), the results showed that the binding is successful. The number of hapten molecules per molecules of protein estimated by UVP Bioimaging system of labworks 4.0 Image Acquisition and Analysis software was 30～32 and 25～28 respectively.
3.BSA-hapten conjugate synthesized was used as immunogen to immunized rabbits and Balb/c mice respectively and the polyclonal antisera were all emerged in rabbits and Balb/c mice. The specificity of antisera was tested by agar double-diffusion method and indirect enzyme-linked immunosorbent assay(ELISA), blocked ELISA detected the antisera titer at the same time. The results showed that the immune antisera titer of rabbits were not below 1:6000,the immune antisera titer of Balb/c mice were not below 1:12000. Rabbits antisera can react with free DES.
To sum up above all,the conclusions were drawn: the preparation method of DES-HS was built,the artificial antigen of DES were synthesized successfully,the polyclonal antisera of DES were prepared using the synthetic DES-HS-BSA conjugate; the indirect ELISA method was builded to detect DES antibody with prepared DES polyclonal
antibody. It established the base of preparing DES monoclonal and developing DES residual immunoassay reagent box which owned the independent knowledge authority of our country.
1.在室温条件下，将DES与丁二酸酐按一定比例溶解于无水吡啶中,搅拌反应生成棕黄色物质，将其分离纯化后对其进行高分辨质谱、红外光谱、核磁共振碳谱及氢谱等鉴定，试验结果表明，该棕黄色物质分子量为368，分子式为C22H24O5，含有酚羟基、羧基等关键基团，为所要制备的DES衍生物己烯雌酚半琥珀酸（DES-HS）。
2.利用混合酸酐法将DES-HS与载体蛋白（BSA，OVA）进行偶联得到DES人工抗原。 合成的人工抗原经紫外扫描、SDS-PAGE 凝胶电泳鉴定证明DES-HS与载体蛋白偶联成功；并用UVP Bioimaging system 中的labworks 4.0 Image Acquisition and Analysis software 分析估算BSA与DES-HS的偶联率为1：30～32。OVA与DES-HS的偶联率为1:25～28。
3.用合成的DES-HS-BSA结合物分别免疫家兔和Balb/c小鼠，采集免疫动物血清。应用琼脂双扩散试验和间接ELISA试验、阻断ELISA检测特异性DES抗体的产生，抗体效价及其与游离DES反应的特异性。结果表明：无论免疫兔还是免疫小鼠，其免疫血清均能够与DES-HS-OVA上的DES决定簇发生特异性结合反应；免疫兔血清效价不低于1：6000，免疫小鼠血清效价不低于1：12000；兔免疫血清能够与游离DES发生特异性反应。
通过上述研究和分析，可得出如下的结论：
建立了DES-HS制备方法；成功地制备出了DES的人工抗原；用DES-HS-BSA分别免疫兔和小鼠制备出了抗DES免疫血清；建立了DES抗体的间接ELISA检测方法。本研究为研制DES单克隆抗体及拥有我国自主知识产权的DES残留免疫检测试剂盒奠定了基础。
Diethylstilbestrol(DES) is a synthetic estrogen. It can produce the effects like natural estrogens in the bodies of human beings and animals.In feeding, it was used illegally to promote growth and feed conversion, but it can lead to cancer and deformity. So it was ruled not to use DES in feeding animal by any way all over the world. Animal and animal products could not be detected DES. The immunoassay of residual DES is one of the critical means in monitor of DES residues. The study is necessary and piror to develop DES residual immunoassay reagent box . The main results are as follows:
1.At the room temperature,the certain ratio of DES and succinate anhydride was stirred in anhydrous pyridine, and got the brown product. The analysis results of HRMS, IR , 13C-NMR and 1H-NMR showed that the separated and pured is product was the derivative of DES wanted, called as DES-HS. Its molecular weight is 368, Its molecular formula is C22H24O5, It includes the critical structure such as -OH ,–COOH and so on.
2.The artificial antigen of DES was synthesized by mixed anhydride method using DES-HS and bovine serum albumin(BSA) and ovabumin(OVA) respectively. The characterization of hapten-protein conjugates were examined ultraviolet spectrophotometry(UV) and sodium doclecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), the results showed that the binding is successful. The number of hapten molecules per molecules of protein estimated by UVP Bioimaging system of labworks 4.0 Image Acquisition and Analysis software was 30～32 and 25～28 respectively.
3.BSA-hapten conjugate synthesized was used as immunogen to immunized rabbits and Balb/c mice respectively and the polyclonal antisera were all emerged in rabbits and Balb/c mice. The specificity of antisera was tested by agar double-diffusion method and indirect enzyme-linked immunosorbent assay(ELISA), blocked ELISA detected the antisera titer at the same time. The results showed that the immune antisera titer of rabbits were not below 1:6000,the immune antisera titer of Balb/c mice were not below 1:12000. Rabbits antisera can react with free DES.
To sum up above all,the conclusions were drawn: the preparation method of DES-HS was built,the artificial antigen of DES were synthesized successfully,the polyclonal antisera of DES were prepared using the synthetic DES-HS-BSA conjugate; the indirect ELISA method was builded to detect DES antibody with prepared DES polyclonal
antibody. It established the base of preparing DES monoclonal and developing DES residual immunoassay reagent box which owned the independent knowledge authority of our country.
引文
[1]刘清，刘军，李志强，等.甘肃省猪肉、猪肝中激素类生长促进剂残留量检测[J].预防医学文献信息，2002，18, 6.
[2] 李俊锁，邱月明，王超. B-激动剂残留的高灵敏免疫测定法研究[J] .兽药残留分析，2002，2：106-107.
[3] 郭志峰，安秋荣，刘鹏岩.鸡蛋中DES的GC-MS法分析测定[J].质谱学报，1997，18(3)：36-40.
[4] 林奕芝，张世英，梁伟，等. HPLC法同时测定肉与肉制品中五种激素残留量[J]. 中国卫生检验杂志，2002，12(4)：411-412.
[5] 李薇，张娟. 高效液相色谱法测定儿童保健品中DES的方法[J].江苏预防医学，2001，12（1）：65.
[6] 陈永红，鲁长豪，陶锐，等.用HPLC法测定人尿中20种20种甾体激素[J] .中国公共卫生学报，1995，14(6)：372-375.
[7] 陈永红，鲁长豪，陶锐，等.反相高效液相色谱法同时测定保健口服液中20种甾体激素的研究 [J] .色谱，1996，14(5)：342-345.
[8] 陈杏琴，任永红，刘俊荣，等.石家庄市鸡肉中DES含量调查与分析[J] .中华预防医学杂志，2000，6(34)：332.
[9] 常碧影，张萍，袁汉成. LC/MC测定饲料中DES方法的研究[J] .现代科学仪器，2003，1：38－41.
[10] 林培英，武向锋，刘荔荔，等.毛细管区带电泳法测定DES片剂中DES的含量[J] .药学实践杂志，2002，20（5）：289－290.
[11] 李俊锁，邱月明，王超. 兽药残留分析[M] .上海：上海科学技术出版社，2002，106－107.
[12] 徐英宏，孙亚欣. 薄层色谱法同时鉴别复方DES栓的四种成分[J] .辽宁药物与临床，2002，5（增刊）：9.
[13] 潘祖亭，姚礼峰，马红燕. DES荧光性质的研究及其分析应用[J] . 武汉大学学报(自然科学版)，1997，43(6)：701－705.
[14] 尹伯元主编.发射免疫分析在医学中的应用[M] .北京： 原子能出版社，1991，1.
[15] 肖祥熊，邹颂海，沈德恩，等.实用放射免疫分析及其临床意义[M] .上海：同济大学出版社，1985，23－28.
[16] 胡鲲，杨先乐，张菊. 酶联免疫法检测中华鳖肌肉中DES[J] .上海水产大学学报，2002，11（3）：199-202.
[17] 何方洋，邱阳生，杨根海，等.克伦特罗单克隆抗体的制备与鉴定 [J] .中国兽医科技，2001，31(6)：30-31.
[18] 王永成，常文保，郭振泉. 雌三醇单克隆抗体的制备与表征[J] . 北京大学学报(自然科学版)，2002，38(4)：459-465.
[19] 覃雅丽，石德时，王桂枝，等.抗氯霉素单克隆抗体的制备及鉴定[J] . 中国兽医学报，2001，21（6）：556-558.
[20] 洪孝庄，孙曼荠.蛋白质连接技术[M].北京：中国医药科技出版社,1993，52-56.
[21] 姜玲，章文才，柯云，等. 抗槲皮素抗体的研制[J] .免疫学杂志，2000，16(5)：383-386.
[22] 黄世乐，陈祖义.混合酸酐法合成DES免疫原[J] .核农学通报, 1990，11(4)：183－184 .
[23] 黄世乐，陈祖义，成冰.RIA测定畜产品中的DES[J] .核农学报, 2001，27(1):20-24.
[24]吴颂如,万寅生. 植物小分子物质的免疫测定技术[J].植物生理通讯.1989,5:68-72.
[25] 荣康泰，龚雄麒.对氧磷-血蓝蛋白免疫家兔的抗毒试验[J] .中华预防医学杂志.1980，14(3)：301-304.
[26] 荣康泰，徐勤惠，李根令，等.半抗原与载体的最佳分子结合比[J] .生物化学杂志，1985，1(2)：27-31.
[27] 朱立平，陈学清.免疫学常用实验方法，人民军医出版社，2000.
[28] 李成文.免疫化学研究进展[M].北京：中国科学技术出版社，1992，1-5.
[29] 马宝骊，肖祥熊.医学免疫学[M].上海：同济大学出版社，1987，238-241.
[30] 朱力平，陈学清.免疫学常用实验方法[M].北京：人民军医出版社，2000,120-138.
[31] 龚非力.医学免疫学[M].北京：科学出版社，2000，187-193.
[32] 王振国，宋广义，王君双.食品农药残留免疫学检测技术[J].吉林农业大学学报，1997,19(3):113-120.
[33] 李银生, 曾振灵. 兽药残留的现状与危害[J].中国兽药杂志,2002,1：25-28.
[34] 焦彦朝.动物性食物中的抗生素残留对人体的影响[J].当代畜牧.1994,3:37.
[35] 刘忠，陈定中，吕植.用于放射免疫分析的胰岛素抗体的制备[J].王世真.竞争放射分析[M].北京：原子能出版社，1981，127.
[36]朱承漠，史爱兰，许叔祥，等.血清地高辛放射免疫分析及其在诊断毛地黄中毒中的价值[J].竞争放射分析，1981，127.
[37]肖祥熊，邹颂海，沈得恩,等.实用放射免疫分析及其临床意义[M].上海：同济大学出版社，1985，18-20.
[38] 朱剑锋，邓芳，陈常庆,等.抗FMOC-苯丙氨酰氨基己酸的单克隆抗体的制备[J]. 细胞与分子免疫学杂志 ,1997，13(1)：34-38.
[39] Dodds E C, Goldberg W, Lawson P, et al. Oestrogenic activity of certain synthetic compounds[J]. Nature(Lond.), 1938, 141:247-248.
[40] Noller K L，Fish C R. Diethylstilbestrol usage: its interesting past, important present, and questionable future[J]. Med clin North Am, 1974, 58:793-810.
[41] Newbold R R. Gender-related behavior in women exposed prenatally to diethylstilbestrol[J]. Env Health Perspect, 1993, 101:208-213.
[42] Herbst A L，Scully R E. Adenocarcinoma of the vagina. Association of maternal stilbestrol therapy with tumor appearance in young women [J]. New Engl J Med, 1971, 284: 878-881.
[43] Herbst A L, Scully R E. Adenocarcinoma of the vagina in adolescence: A report of 7 cases including 6 clear cell carcinomas[J]. Cancer, 1970, 25, 745-757.
[44] Ferguson J H. Effect of stilbestrol on pregnancy compared to the effect of a placebo[J]. American Journal of Obstetrics and Gynecology.1953, 65:592-601.
[45] Dieckmann W J, Davis M E, Rynkiewicz L M, et al. Dose the administration of diethylstilbestrol during have therapeutic value [J]. American Journal of Obstetrics and Gynecology, 1953, 66:1062-1081.
[46] Bibbo M, Haenszel W M, Wied G L, et al. A twenty-five follow-up study of women exposed to diethylstilbestrol during pregnancy [J]. New England Journal of medicine, 1978, 298:763-767.
[47] Beral V, Colwell L. Randomised trial of high doses of stilbestrol and ethisterone in pregnancy long-term follow-up of mothers [J]. British medical Journal, 1980, 281:1008-1011.
[48] Vessy M P, Fairweather D V, Norman-Smith B, et al. A randomized double-bind controlled of the value of stilbestrol therapy in pregnancy: long-term follow-up of mothers and offsprings [J]. Br J Obstet Gynecol , 1983, 90:1007-1017.
[49] Greenberg E R, Barnes A B, Resseguie L, et al. Breast cancer in mothers given diethylstilbestrol in pregnancy [J]. New Engl J Med, 1984, 311:1393-1398.
[50] Hadjimichael O C, Meigs J W, Falcier F W, et al. Cancer risk among women exposed to exogenous estrogens during pregnancy [J]. J Natl Cancer Inst, 1984, 73:831-834.
[51] Colton T, Greenberg E R, Noller K, et al. Brest cancer in mothers prescribed diethylstilbestrol in pregnancy [J]. J Am Med Assoc, 1993, 269:2096-2100.
[52] Calle E E, Mervis C A, Thun M J, et al. diethylstilbestrol and risk of fatal breast cancer in a prospective cohort of US women[J]. Am J Epidmiol, 1996, 144: 645-652.
[53] Herbst A L, Poskanzer D C, Robboy S J, et al. Prenatal exposure to stilbestrol: A prospective comparison of exposed female offspring with uneposed controls N [J]. ew England Journal of Medicine, 1975, 292:334-339.
[54] Bibbo M, Gill W B, Azizi F, et al. Follow-up study of male and female offspring of
DES-exposed Mothers [J]. Obstetrics and Gynecology, 1977, 49:1-8.
[55] O’Brien P C, Noller K I, Robboy S T, et al. Viginal epithelial changes in young women enrolled in national cooperative diethylstilbestrol adenosis(DESAD)project [J]. Obstetrics and Gynecology, 1979, 58:300-308.
[56] Antonioli D A, Burke L, Friedman E A. Natural history of diethylstilbestrol associated tract lesions cervical ectopy and cervicovaginal hood [J].American Journal of Obstetrics and Gynecology,1980, 137:847-853.
[57] Schmidt G, Fowler W C. Gynecologic operative experience in women exposed to DES in utero [J]. South Med J, 1982, 75(3):260-263.
[58] Claman P, Berger M J. Phenotypic differences in upper genital tract abnormalities and reproductive history in dizygotic twins exposed to diethylstilbestrol in utero[J]. J Reprod Med, 1990, 35(4):431-433.
[59] Hnederson N D. Genetic analysis of acquisition and retention of a conditioned fear in mice [J]. J Comp Physiol Psychol,1968, 65(2):325-30.
[60] Gill W B, Schumacher G F, Bibbo M. Pathological semen and anatomical abnormal of the genital tract in human male subjects Exposed to diethylstilbestrol in utero [J]. Journal of Urology, 1977, 117:477-480.
[61] Newbold R. Cellular and molecular effects of developmental exposure to diethylstilbestrol: implications for other environmental estrogens [J]. Environ Health Perspect, 1995, 103 (Suppl 7):83-87.
[62] Barnes A B, Colton T, Gundersen I, et al. Fertility and outcome of pregnancy in women exposed in utero to diethylstilbestrol [J]. New England Journal of medicine, 1980, 302:609-613.
[63] Mangan C E, Borow L, Burtnett-Rubin M M, et al. Pregnancy outcome in 98 women exposed to diethylstilbestrol in utero, their mothers, and unexposed siblings [J]. Obstet Gynecol, 1982, 59(3):315-319.
[64] Linn S, Lieberman E, Schoenbaum S C, et al. Adverse outcomes of pregnancy in women exposed to diethylstilbestrol in utero [J]. J Reprod Med, 1988, 33(1):3-7.
[65] Thorp J M, Fowler W C, Donehoo R, et al. Antepartum and intrapartum events in women exposed in utero to diethylstilbestrol [J]. Obstet Gynecol, 1990, 76(5 Pt 1):828-832.
[66] Senekjian E K, Potkul R K, Frey K, et al. Infertility among daughters either exposed or not exposed to diethylstilbestrol [J]. Am J Obstet Gynecol, 1988, 158(3 Pt 1):493-498.
[67] Karande V C, Lester R G, Muasher S J, et al. Are implantation and pregnancy outcome impaired in diethylstilbestrol-exposed women after in vitro fertilization and embryo transfer [J]. Fertil Steril, 1990,54(2):287-291.
[68] Greenwald P, Barlow I L, Nasca P C, et al. Vaginal cancer after maternal treatment with synthetic estrogen [J]. New England Journal of Medicine,1971, 285:390-392.
[69] Noller K I, Decker D G, Lanier A P, ea al. Clear cell adenocarcinoma of the cervix after maternal treatment with synthetic estrogens [J]. Mayo Clinic Proceedings, 1972, 47: 629-630.
[70] Herbst A L, Coll P, Robboy S J, et al. Age-incidence and risk of Diethylstilbestrol-related clear celladenocerinoma of the vagina and Cervix [J]. American Journal of Obstetrics and Gynecology, 1977, 128(1):43-50
[71] Kelsey J L. A review of the epidemiology of human breast cancer[J]. Epidemiology Review, 1979, 1:74-109.
[72] Hoover R, Gray L A, Fraument I F. Stilbestrol and the risk of ovarian cancer [J]. The Lancet, 1977, 2:533-534.
[73] Beral V, Colwell L. Randomised trial of high doses of stilbestrol and ethisterone in pregnancy long-term follow-up of mothers [J]. British medical Journal, 1980, 281:1008-1011.
[74] Scheirs J G, Vingerhoets A J. Handedness and other laterality indices in women prenatally exposed to DES [J]. J Clin Exp Neuropsychol, 1995 , 17(5):725-30.
[75] Pillard R C, Rosen L R, Meyer-Bahlburg H, et al. Psychopathology and social functioning in men prenatally exposed to diethylstilbestrol (DES) [J]. Psychosom Med, 1993, 55(6):485-491.
[76] Saunders E J. Physical and psychological problems associated with exposure to diethylstilbestrol (DES) [J]. Hosp Community Psychiatry, 1988 , 39(1):73-77.
[77] Swan S H. Intrauterine exposure to diethylstilbestrol: long-term effects in humans [J]. APMIS, 2000 , 108(12):793-804.
[78] Windmill K F, Meade B J, Lee V W. Effect of prepubertal gonadectomy and sex steroid treatment on the growth and lymphocyte populations of the rat thymus [J]. Reprod Fertil Dev, 1993, 5(1):73-81.
[79] Gould K A, Shull J D, Gorski J. DES action in the thymus: inhibition of cell proliferation and geneti variation [J]. Mol Cell Endocrinol, 2000, 170(1-2):31-39.
[80] Calemine J B, Gogal R M, Lengi A, et al. Immunomodulation by diethylstilbestrol is dose and gender related: effects on thymocyte apoptosis and mitogen-induced proliferation [J]. Toxicology , 2002, 178(2):101-118.
[81] Forsberg J G.. Neonatal estrogen treatment and its consequences for thymus development, serum level of autoantibodies to cardiolipin, and the delayed-type hypersensitivity response [J]. J Toxicol Environ Health A, 2000, 60(3):185-221.
[82] Smith B J, Holladay S D. Immune alterations in geriatric mice dosed subacutely with diethylstilbestrol (DES) [J]. J Appl Toxicol, 1997, 17(5):265-271.
[83] Onsrud M, Sander S. Influence of in vivo diethylstilbosterol phosphate on some human blood lymphocyte sub-populations [J]. Acta Pathol Microbiol Immunol Scand, 1982 , 90(5):271-276.
[84] Haukaas S, Hoisaeter P A, Kalland T. In vitro and in vivo effects of diethylstilbestrol and estramustine phosphate (Estracyt) on the mitogen responsiveness of human peripheral blood lymphocytes [J]. Prostate, 1982, 3(4):405-414.
[85] Ablin R J, Bartkus J M, Gonder M J. Vitro effects of diethylstilboestrol and the LHRH analogue leuprolide on natural killer cell activity [J]. Immunopharmacology, 1988, 15(2):95-101.
[86] Birnboim H C. DNA clastogenic activity of diethylstilbestrol [J]. Biochem Pharmacol, 1995, 34(18):3251-3257.
[87] Khan M Z, Hashimoto Y, Iwami Y, et al. Hormonal regulation of T-cell subsets in the oviduct: an immunohistochemical study using sex-hormone-treated chicken [J]. Vet Med Sci, 1996, 58(12):1161-1167.
[88] Ford C D, Johnson G H, Smith W G.. Natural killer cells in in utero diethylstilbesterol-exposed patients [J]. Gynecol Oncol, 1983, 16(3):400-404.
[89] Kalland T, Campbell T. Effects of diethylstilbestrol on human natural killer cells in vitro[J]. Immunopharmacology, 1984,8(1):19-25.
[90] Kalland T. Exposure of neonatal female mice to diethylstilbestrol persistently impairs NK activity through reduction of effector cells at the bone marrow level [J]. Immunopharmacology, 1984, 7(2):127-134.
[91] Kalland T, Haukaas SA. Effects of diethylstilbestrol and estramustine phosphate (estracyt) on natural killer cell activity and tumor susceptibility in male mice [J]. Prostate, 1984, 5(6):649-660.
[92] Heizman R J.Veterinary Drug Residues [M]. Report Eur.15126-EN Published on behalf of European Communities.1994:4-6
[93] Zondek B, Marx L. Paemia and calcaemia in the cock induced by DES [J]. Nature(Lond.) 1939, 143:378-379.
[94] Lorenz F W. Fattening cockerels by stilbestrol administration [J]. Poult. Sci, 1943,22:190-19.
[95] Dinusson W E, Andrew W M,Beeson T. The effects of stilbestrol, testosterone, and thyroid alternation on growth and fattening of beef heifers [J]. J Anim Sci, 1948:321-330.
[96] Andrews F N, Beeson W M, Harper C. The effects of stilbestrol and testosterone on the
growth and fattening of lambs [J]. J Anim Sci, 1949, 8:578-582.
[97] Preston R L, Cheng C, Stoty D, et al. The influence of oral adminstration of diethylstilbestrol upon estrogenic residues in the tissues of beef cattle [J]. Sci, 1056, 15:3-12.
[98] Preston R. Possible role of DES on mature size in steers [J]. Sci,1978, 47(Supply .1):436(Aster).
[99] Preston R. Influence of diethylstilbestrol on body water space in ruminans [J] .Proc Soc Exp Biol Med , 1969, 132:401-404.
[100] Wade N. DES: a case study of regulatory abidication [J]. Science, 1972, 177: 335-337.
[101] Jukes T. Diethylstilbestrol in beef production:What is the risk to consumers [J] Preventive Med , 1976, 5:438-453.
[102] Marcus A. Cancer from beef: DES, Federal food regulation and consumer confidence [M]. The Johns Hopkins University Press , 1994, Baltimore, MD.
[103] Cole G H, Gass R J. On the safety of estrogenic residues in edible animal products [J] . Biosvience, 1975, 25:19-25.
[104] Burroughs W, Culbertson E , Cheng , et al. The influence of oral administration of diethylstilbestrol to beef steers [J]. J Anim Sci, 1955, 14:1015-1024。
[105] Raun A P. A skill study of the effects of two levels of stilbestrol from dry and liquid premixes on feedlot performance of fattening steers [M]. Expt, 1965, CA-108, Eli Lilly and Co, Inc, Greenfield IN.
[106] Burroughs W, Christianson R. Regular, double and triole levels of stibestrol for finishing yearling cattle [M]. A.S Leaflet 1963, R49, Anim.
[107] Andrews F N. DES history (Letter to the editor) [J]. Science, 1995, 268:16
[108] IARC. International Agency for Research on Cancer. IARC.Monography on the Evalution of the Carinogenic Risk of Chemical to Man. Sex Hormones. Vol.6.243pp.Lyon, France:IARC, 1974.
[109] IARC. International Agency for Research on Cancer. IARCMonography on the Evalution of the Carinogenic Risk of Chemical to Humans. Sex Hormones(Ⅱ). Vol.21.583pp.Lyon, France:IARC, 1979.
[110] Council of European communities, Directive 81/602/EEC, Off J Eur Commun, 1981, L222, 32.
[111] Council of European communities, Directive 88/146EEC,Off J Eur Commun, 1988, L70:16.
[112] Donoho A L, Johnson W S, Sieck R F, et al. Gas chromatographic determination of diethylstilbestrol and its glucuronide in cattle tissue [J]. Journal of the AOAC, 1973,
56(4):785-792.
[113] Coffin D E, Pilon J C. Gas chromatographic determination of diethylstilbestrol residues in animal tissues [J]. Journal of the AOAC, 1973, 56(2):352－357.
[114] Tirpenou A E, Kilkidis S D, Kamarianos A P. Modified method for eletron capture gas-liquid chromatographic determination of diethylstilbestrol residues in urine of fattened bulls [J]. Journal of the AOAC, 1983, 66(5):1230-1233
[115] Busico F, Moretti G, Cartoni G P. Determination of estrogens in animal tissues by GC-MS with negative ion chemical ionization [J]. Journal of High Resolution Chromatography, 1992, 15(2)94-98.
[116] Reuvers T H, Perogordo E, Jimenez R. Rapid screening method for the determination of diethylstilbestrol in edible animal tissue by column liquid chromatographiy with eletrochemical detection [J]. Journal of chromatography, 1991, 564, 477-484.
[117] Lagana A. General and selective isolation procedure for high-performance liquid chromatographic detemination of anabolic steroids in tissues [J]. Journal of Chromatography, 1991, 588, 89-98.
[118] Leindsteiner K. The specificity of the serological reactions [M]. Cambridge: Harward University Press, 1945, 293.
[119] Campfied L A. Mathematical analysis of competitive protein binding assays [M]. In: Odell W,Franchimont P Eds. Principles of Competicie Protein-binding assays，2nd ed. New York: John Wiley and Sons, 1983, 125.
[120] Jeffcoate S L, White N, Fraser H M. Proceedings: Radioimmunoassay of thyrotrophin releasing hormone in blood and urine [J]. N Engl J Med,1974, 291(2):181.
[121] Kennedy D G, Blanchflower W J, O,Dornan B C. Development of an ELISA for maduramicin and determination of the depletion kinetics of maduramicin residues in poultry [J]. Food Addit Contam,1997, 14(1):27-33.
[122] Engvall E, Perlman P. Enzyme-linked immunosorbent assay (ELISA):Quantitative assay of immunoglobulin G.Immunochemistry [J]. 1971 , 8(9):871-874.
[123] Van Weemen B K, Schuurs A H. Immunoassay using hapten--enzyme conjugates [J]. FEBS Lett, 1972, 24(1):77-81.
[124] Harlow E, Lane D. Antibodies: a Laboratory Manual [J].Cold Spring Harbor, NY, 1998.
[125] Wainer B H, Fitch F W, Rothberg R M, et al. Morphine-3-succinyl-bovine serum albumin: an immunogenic hapten-protein conjugate [J]. Science,1972 , 176(39):1143-1145.
[126] Dong Min, Li Guofu, Li Jin, et al. Immunological Detection of Arbutin [J]. Tsinghua Science and Technology, 1999, 4(3):311-316.
[127] Erlanger B F, Borek E, Beiser S M et al. Steroid protein conjugates I: Preparation and characterization of bovine serum albumin with testosterone and with cortisone [J]. J chem., 1957, 228:713-727 .
[128] O'Neill S P, Robb T E. Hapten concentrations in the circulation of animals actively immunized with hapten-protein conjugates [J]. J. Immunol. 1976 , 116(2):363-366.
[129] Sambrook J，Russel D W. 分子克隆实验指南[M]，第三版，科学出版社，1713－1721.
[130] Collins W P, Hennam J F. Molec Cleavage of structural proteins during the assembly of the head of bacteriophage T4 [J]. Aspects Med, 1976, 1:27.
[131] Rombauts P, Pierdet A, Jouquey A. Preparation and properties of anti-diethylstilbestrol immune sera [J]. C R Acad Sci Hebd Seances Acad Sci D. 1973 , 277(18):1921-1924.
[132] Erlanger B F, Borek O F, Beiser S, et al. Steroid-protein conjugated II: Preparation and characterization of conjugates of bovine serum albumin with progesterone, deoxycorticosterone and oestrone [J]. J Biol Chem. 1959, 243:1090-1094.
[133] Erlanger B F, Langone J. Preparation of antigenic hapten-carrier conjugate:A survey. In Methods in enzymology, Eds.:Academic press: New York, 1980,Vol.70, Part A.
[134] Li Q X, Zhao M S, Gee S L, et al. Development of enzyme-linked immunosorbent assays for 4-nitrophenol and substituted 4-nitrophenols [J]. J Agri Food Chem, 1991, 39:1685-1692.
[135]Schneider P, Hammock B D. Influence of the ELISA for S-triazine herbicides using monoclonal antibodies [J]. J Agric Food Chem,1992, 40:525.
[136] Faraj B A, Ali F M. Devlopment and application of a radioimmunoassay for tetracycline [J]. J Pharmacol Exp Ther, 1981, 217:10.
[137] Feng P C, Horton S R, Sharp R. A general method for developing immunoassays to chloroacetailide herbicides [J]. J Agric Food Chem, 1992, 40:211.
[138] Jung F, Meyer H D, Hamm R T. Development of a sensitive enzyme-linked immunosorbent assay for the fungicid fenpropimorph [J]. Agric Food Chem, 1989, 37:1183.
[139] Pallardy M, Alberici G F, Goodman A, et al. Antibody recognition of substituted ammonium ions:Modulation by the counterion [J]. J Immunol Methods,1987, 99:179.
[140] Buenafe A C, Rittenberg M B. Combinding site specificity of monoclonal antibodies to the organophosphate hapten soman [J]. Mol Immunol. 1987, 24:401.
[141] Fenton II, Singer S J. Affinity labeling of antibodies to the p-azophyltrimethylammonnium hapten, and a comparison of affinity-labeled antibodies of two different species [J]. Biochemistry, 1971, 10:1429.
[142] Flurkey B S, Bolger M B, Linthicum D S. Preparation and characterization of antisera
and monoclonal antibodies to seratonergic and dopaminegric ligands [J]. J Neuroimmunol, 1985, 8:115.
[143] Groopman J D, Trudel L J, Donshue P R，et al. High affinity monoclonal antibodies for aflatoxins and their application to solid-phase immunoassays [J]. Proc Natl Acad Sci USA, 1984, 81:7728.
[144] Leudtke P R, Korner M, Neve K A, et al. Monoclonal antibodies with high affinity for spiroperidol [J]. J Neurochem, 1988, 50:1253.
[145] Li Q X, Zhao M S, Gee S L, et al. Development of enzyme-linked immunosorbent assays for 4-nitrophenol and substituted 4-nitrophenols [J]. J Agric Food Chem, 1991, 39:1685-1692.
[146] Mcadam D P, Hill A S, Beasley H L, et al. Mono- and polyclonal antibodies to the organophosphate fenitrothion I:Approaches to hapten-protein conjugation [J]. J Agri Food Chem,1992, 40:1466.
[147 ] Schmidt D J, Clarkson C E, Swanson T A. Monoclonal antibodies for immunoassay of avermectins [J]. J Agric Food Chem, 1990, 38:1763.
[148] Queffelec Anne-Laurence, Nodet P, Haelters Jean-Pierre, et al. Hapten synthesis for a monoclonal antibody based ELISA for Deltamethrin [J]. J Agric Food Chem, 1998, 46:1670-1676.
[149] Sawada J, Janejal N, Nagamatsu K, et al. Production and characterization of high-affinity monoclonal antibodies against morphine [J]. Mol Immunol, 1991, 25:937.
[150] Bull J P, Serreqi A N, Gamboa H R, et al. Improved Enzyme-linked immunosorbent assay to detect didecyldimethylammonium chloride, a quaternary ammonium compound [J]. J Agric Food Chem, 1998, 467:4779-4786.
[151] Tristram G P, Daniel P S, Abba I T, et al. Medical immunology [M].北京：科学出版社，2002,72-81.
[152] R. M. Sharpe. Environmental oestrogens and male infertility [J]. Pure & Appl Chem 1998, 70(9):1685-1701.
[2] 李俊锁，邱月明，王超. B-激动剂残留的高灵敏免疫测定法研究[J] .兽药残留分析，2002，2：106-107.
[3] 郭志峰，安秋荣，刘鹏岩.鸡蛋中DES的GC-MS法分析测定[J].质谱学报，1997，18(3)：36-40.
[4] 林奕芝，张世英，梁伟，等. HPLC法同时测定肉与肉制品中五种激素残留量[J]. 中国卫生检验杂志，2002，12(4)：411-412.
[5] 李薇，张娟. 高效液相色谱法测定儿童保健品中DES的方法[J].江苏预防医学，2001，12（1）：65.
[6] 陈永红，鲁长豪，陶锐，等.用HPLC法测定人尿中20种20种甾体激素[J] .中国公共卫生学报，1995，14(6)：372-375.
[7] 陈永红，鲁长豪，陶锐，等.反相高效液相色谱法同时测定保健口服液中20种甾体激素的研究 [J] .色谱，1996，14(5)：342-345.
[8] 陈杏琴，任永红，刘俊荣，等.石家庄市鸡肉中DES含量调查与分析[J] .中华预防医学杂志，2000，6(34)：332.
[9] 常碧影，张萍，袁汉成. LC/MC测定饲料中DES方法的研究[J] .现代科学仪器，2003，1：38－41.
[10] 林培英，武向锋，刘荔荔，等.毛细管区带电泳法测定DES片剂中DES的含量[J] .药学实践杂志，2002，20（5）：289－290.
[11] 李俊锁，邱月明，王超. 兽药残留分析[M] .上海：上海科学技术出版社，2002，106－107.
[12] 徐英宏，孙亚欣. 薄层色谱法同时鉴别复方DES栓的四种成分[J] .辽宁药物与临床，2002，5（增刊）：9.
[13] 潘祖亭，姚礼峰，马红燕. DES荧光性质的研究及其分析应用[J] . 武汉大学学报(自然科学版)，1997，43(6)：701－705.
[14] 尹伯元主编.发射免疫分析在医学中的应用[M] .北京： 原子能出版社，1991，1.
[15] 肖祥熊，邹颂海，沈德恩，等.实用放射免疫分析及其临床意义[M] .上海：同济大学出版社，1985，23－28.
[16] 胡鲲，杨先乐，张菊. 酶联免疫法检测中华鳖肌肉中DES[J] .上海水产大学学报，2002，11（3）：199-202.
[17] 何方洋，邱阳生，杨根海，等.克伦特罗单克隆抗体的制备与鉴定 [J] .中国兽医科技，2001，31(6)：30-31.
[18] 王永成，常文保，郭振泉. 雌三醇单克隆抗体的制备与表征[J] . 北京大学学报(自然科学版)，2002，38(4)：459-465.
[19] 覃雅丽，石德时，王桂枝，等.抗氯霉素单克隆抗体的制备及鉴定[J] . 中国兽医学报，2001，21（6）：556-558.
[20] 洪孝庄，孙曼荠.蛋白质连接技术[M].北京：中国医药科技出版社,1993，52-56.
[21] 姜玲，章文才，柯云，等. 抗槲皮素抗体的研制[J] .免疫学杂志，2000，16(5)：383-386.
[22] 黄世乐，陈祖义.混合酸酐法合成DES免疫原[J] .核农学通报, 1990，11(4)：183－184 .
[23] 黄世乐，陈祖义，成冰.RIA测定畜产品中的DES[J] .核农学报, 2001，27(1):20-24.
[24]吴颂如,万寅生. 植物小分子物质的免疫测定技术[J].植物生理通讯.1989,5:68-72.
[25] 荣康泰，龚雄麒.对氧磷-血蓝蛋白免疫家兔的抗毒试验[J] .中华预防医学杂志.1980，14(3)：301-304.
[26] 荣康泰，徐勤惠，李根令，等.半抗原与载体的最佳分子结合比[J] .生物化学杂志，1985，1(2)：27-31.
[27] 朱立平，陈学清.免疫学常用实验方法，人民军医出版社，2000.
[28] 李成文.免疫化学研究进展[M].北京：中国科学技术出版社，1992，1-5.
[29] 马宝骊，肖祥熊.医学免疫学[M].上海：同济大学出版社，1987，238-241.
[30] 朱力平，陈学清.免疫学常用实验方法[M].北京：人民军医出版社，2000,120-138.
[31] 龚非力.医学免疫学[M].北京：科学出版社，2000，187-193.
[32] 王振国，宋广义，王君双.食品农药残留免疫学检测技术[J].吉林农业大学学报，1997,19(3):113-120.
[33] 李银生, 曾振灵. 兽药残留的现状与危害[J].中国兽药杂志,2002,1：25-28.
[34] 焦彦朝.动物性食物中的抗生素残留对人体的影响[J].当代畜牧.1994,3:37.
[35] 刘忠，陈定中，吕植.用于放射免疫分析的胰岛素抗体的制备[J].王世真.竞争放射分析[M].北京：原子能出版社，1981，127.
[36]朱承漠，史爱兰，许叔祥，等.血清地高辛放射免疫分析及其在诊断毛地黄中毒中的价值[J].竞争放射分析，1981，127.
[37]肖祥熊，邹颂海，沈得恩,等.实用放射免疫分析及其临床意义[M].上海：同济大学出版社，1985，18-20.
[38] 朱剑锋，邓芳，陈常庆,等.抗FMOC-苯丙氨酰氨基己酸的单克隆抗体的制备[J]. 细胞与分子免疫学杂志 ,1997，13(1)：34-38.
[39] Dodds E C, Goldberg W, Lawson P, et al. Oestrogenic activity of certain synthetic compounds[J]. Nature(Lond.), 1938, 141:247-248.
[40] Noller K L，Fish C R. Diethylstilbestrol usage: its interesting past, important present, and questionable future[J]. Med clin North Am, 1974, 58:793-810.
[41] Newbold R R. Gender-related behavior in women exposed prenatally to diethylstilbestrol[J]. Env Health Perspect, 1993, 101:208-213.
[42] Herbst A L，Scully R E. Adenocarcinoma of the vagina. Association of maternal stilbestrol therapy with tumor appearance in young women [J]. New Engl J Med, 1971, 284: 878-881.
[43] Herbst A L, Scully R E. Adenocarcinoma of the vagina in adolescence: A report of 7 cases including 6 clear cell carcinomas[J]. Cancer, 1970, 25, 745-757.
[44] Ferguson J H. Effect of stilbestrol on pregnancy compared to the effect of a placebo[J]. American Journal of Obstetrics and Gynecology.1953, 65:592-601.
[45] Dieckmann W J, Davis M E, Rynkiewicz L M, et al. Dose the administration of diethylstilbestrol during have therapeutic value [J]. American Journal of Obstetrics and Gynecology, 1953, 66:1062-1081.
[46] Bibbo M, Haenszel W M, Wied G L, et al. A twenty-five follow-up study of women exposed to diethylstilbestrol during pregnancy [J]. New England Journal of medicine, 1978, 298:763-767.
[47] Beral V, Colwell L. Randomised trial of high doses of stilbestrol and ethisterone in pregnancy long-term follow-up of mothers [J]. British medical Journal, 1980, 281:1008-1011.
[48] Vessy M P, Fairweather D V, Norman-Smith B, et al. A randomized double-bind controlled of the value of stilbestrol therapy in pregnancy: long-term follow-up of mothers and offsprings [J]. Br J Obstet Gynecol , 1983, 90:1007-1017.
[49] Greenberg E R, Barnes A B, Resseguie L, et al. Breast cancer in mothers given diethylstilbestrol in pregnancy [J]. New Engl J Med, 1984, 311:1393-1398.
[50] Hadjimichael O C, Meigs J W, Falcier F W, et al. Cancer risk among women exposed to exogenous estrogens during pregnancy [J]. J Natl Cancer Inst, 1984, 73:831-834.
[51] Colton T, Greenberg E R, Noller K, et al. Brest cancer in mothers prescribed diethylstilbestrol in pregnancy [J]. J Am Med Assoc, 1993, 269:2096-2100.
[52] Calle E E, Mervis C A, Thun M J, et al. diethylstilbestrol and risk of fatal breast cancer in a prospective cohort of US women[J]. Am J Epidmiol, 1996, 144: 645-652.
[53] Herbst A L, Poskanzer D C, Robboy S J, et al. Prenatal exposure to stilbestrol: A prospective comparison of exposed female offspring with uneposed controls N [J]. ew England Journal of Medicine, 1975, 292:334-339.
[54] Bibbo M, Gill W B, Azizi F, et al. Follow-up study of male and female offspring of
DES-exposed Mothers [J]. Obstetrics and Gynecology, 1977, 49:1-8.
[55] O’Brien P C, Noller K I, Robboy S T, et al. Viginal epithelial changes in young women enrolled in national cooperative diethylstilbestrol adenosis(DESAD)project [J]. Obstetrics and Gynecology, 1979, 58:300-308.
[56] Antonioli D A, Burke L, Friedman E A. Natural history of diethylstilbestrol associated tract lesions cervical ectopy and cervicovaginal hood [J].American Journal of Obstetrics and Gynecology,1980, 137:847-853.
[57] Schmidt G, Fowler W C. Gynecologic operative experience in women exposed to DES in utero [J]. South Med J, 1982, 75(3):260-263.
[58] Claman P, Berger M J. Phenotypic differences in upper genital tract abnormalities and reproductive history in dizygotic twins exposed to diethylstilbestrol in utero[J]. J Reprod Med, 1990, 35(4):431-433.
[59] Hnederson N D. Genetic analysis of acquisition and retention of a conditioned fear in mice [J]. J Comp Physiol Psychol,1968, 65(2):325-30.
[60] Gill W B, Schumacher G F, Bibbo M. Pathological semen and anatomical abnormal of the genital tract in human male subjects Exposed to diethylstilbestrol in utero [J]. Journal of Urology, 1977, 117:477-480.
[61] Newbold R. Cellular and molecular effects of developmental exposure to diethylstilbestrol: implications for other environmental estrogens [J]. Environ Health Perspect, 1995, 103 (Suppl 7):83-87.
[62] Barnes A B, Colton T, Gundersen I, et al. Fertility and outcome of pregnancy in women exposed in utero to diethylstilbestrol [J]. New England Journal of medicine, 1980, 302:609-613.
[63] Mangan C E, Borow L, Burtnett-Rubin M M, et al. Pregnancy outcome in 98 women exposed to diethylstilbestrol in utero, their mothers, and unexposed siblings [J]. Obstet Gynecol, 1982, 59(3):315-319.
[64] Linn S, Lieberman E, Schoenbaum S C, et al. Adverse outcomes of pregnancy in women exposed to diethylstilbestrol in utero [J]. J Reprod Med, 1988, 33(1):3-7.
[65] Thorp J M, Fowler W C, Donehoo R, et al. Antepartum and intrapartum events in women exposed in utero to diethylstilbestrol [J]. Obstet Gynecol, 1990, 76(5 Pt 1):828-832.
[66] Senekjian E K, Potkul R K, Frey K, et al. Infertility among daughters either exposed or not exposed to diethylstilbestrol [J]. Am J Obstet Gynecol, 1988, 158(3 Pt 1):493-498.
[67] Karande V C, Lester R G, Muasher S J, et al. Are implantation and pregnancy outcome impaired in diethylstilbestrol-exposed women after in vitro fertilization and embryo transfer [J]. Fertil Steril, 1990,54(2):287-291.
[68] Greenwald P, Barlow I L, Nasca P C, et al. Vaginal cancer after maternal treatment with synthetic estrogen [J]. New England Journal of Medicine,1971, 285:390-392.
[69] Noller K I, Decker D G, Lanier A P, ea al. Clear cell adenocarcinoma of the cervix after maternal treatment with synthetic estrogens [J]. Mayo Clinic Proceedings, 1972, 47: 629-630.
[70] Herbst A L, Coll P, Robboy S J, et al. Age-incidence and risk of Diethylstilbestrol-related clear celladenocerinoma of the vagina and Cervix [J]. American Journal of Obstetrics and Gynecology, 1977, 128(1):43-50
[71] Kelsey J L. A review of the epidemiology of human breast cancer[J]. Epidemiology Review, 1979, 1:74-109.
[72] Hoover R, Gray L A, Fraument I F. Stilbestrol and the risk of ovarian cancer [J]. The Lancet, 1977, 2:533-534.
[73] Beral V, Colwell L. Randomised trial of high doses of stilbestrol and ethisterone in pregnancy long-term follow-up of mothers [J]. British medical Journal, 1980, 281:1008-1011.
[74] Scheirs J G, Vingerhoets A J. Handedness and other laterality indices in women prenatally exposed to DES [J]. J Clin Exp Neuropsychol, 1995 , 17(5):725-30.
[75] Pillard R C, Rosen L R, Meyer-Bahlburg H, et al. Psychopathology and social functioning in men prenatally exposed to diethylstilbestrol (DES) [J]. Psychosom Med, 1993, 55(6):485-491.
[76] Saunders E J. Physical and psychological problems associated with exposure to diethylstilbestrol (DES) [J]. Hosp Community Psychiatry, 1988 , 39(1):73-77.
[77] Swan S H. Intrauterine exposure to diethylstilbestrol: long-term effects in humans [J]. APMIS, 2000 , 108(12):793-804.
[78] Windmill K F, Meade B J, Lee V W. Effect of prepubertal gonadectomy and sex steroid treatment on the growth and lymphocyte populations of the rat thymus [J]. Reprod Fertil Dev, 1993, 5(1):73-81.
[79] Gould K A, Shull J D, Gorski J. DES action in the thymus: inhibition of cell proliferation and geneti variation [J]. Mol Cell Endocrinol, 2000, 170(1-2):31-39.
[80] Calemine J B, Gogal R M, Lengi A, et al. Immunomodulation by diethylstilbestrol is dose and gender related: effects on thymocyte apoptosis and mitogen-induced proliferation [J]. Toxicology , 2002, 178(2):101-118.
[81] Forsberg J G.. Neonatal estrogen treatment and its consequences for thymus development, serum level of autoantibodies to cardiolipin, and the delayed-type hypersensitivity response [J]. J Toxicol Environ Health A, 2000, 60(3):185-221.
[82] Smith B J, Holladay S D. Immune alterations in geriatric mice dosed subacutely with diethylstilbestrol (DES) [J]. J Appl Toxicol, 1997, 17(5):265-271.
[83] Onsrud M, Sander S. Influence of in vivo diethylstilbosterol phosphate on some human blood lymphocyte sub-populations [J]. Acta Pathol Microbiol Immunol Scand, 1982 , 90(5):271-276.
[84] Haukaas S, Hoisaeter P A, Kalland T. In vitro and in vivo effects of diethylstilbestrol and estramustine phosphate (Estracyt) on the mitogen responsiveness of human peripheral blood lymphocytes [J]. Prostate, 1982, 3(4):405-414.
[85] Ablin R J, Bartkus J M, Gonder M J. Vitro effects of diethylstilboestrol and the LHRH analogue leuprolide on natural killer cell activity [J]. Immunopharmacology, 1988, 15(2):95-101.
[86] Birnboim H C. DNA clastogenic activity of diethylstilbestrol [J]. Biochem Pharmacol, 1995, 34(18):3251-3257.
[87] Khan M Z, Hashimoto Y, Iwami Y, et al. Hormonal regulation of T-cell subsets in the oviduct: an immunohistochemical study using sex-hormone-treated chicken [J]. Vet Med Sci, 1996, 58(12):1161-1167.
[88] Ford C D, Johnson G H, Smith W G.. Natural killer cells in in utero diethylstilbesterol-exposed patients [J]. Gynecol Oncol, 1983, 16(3):400-404.
[89] Kalland T, Campbell T. Effects of diethylstilbestrol on human natural killer cells in vitro[J]. Immunopharmacology, 1984,8(1):19-25.
[90] Kalland T. Exposure of neonatal female mice to diethylstilbestrol persistently impairs NK activity through reduction of effector cells at the bone marrow level [J]. Immunopharmacology, 1984, 7(2):127-134.
[91] Kalland T, Haukaas SA. Effects of diethylstilbestrol and estramustine phosphate (estracyt) on natural killer cell activity and tumor susceptibility in male mice [J]. Prostate, 1984, 5(6):649-660.
[92] Heizman R J.Veterinary Drug Residues [M]. Report Eur.15126-EN Published on behalf of European Communities.1994:4-6
[93] Zondek B, Marx L. Paemia and calcaemia in the cock induced by DES [J]. Nature(Lond.) 1939, 143:378-379.
[94] Lorenz F W. Fattening cockerels by stilbestrol administration [J]. Poult. Sci, 1943,22:190-19.
[95] Dinusson W E, Andrew W M,Beeson T. The effects of stilbestrol, testosterone, and thyroid alternation on growth and fattening of beef heifers [J]. J Anim Sci, 1948:321-330.
[96] Andrews F N, Beeson W M, Harper C. The effects of stilbestrol and testosterone on the
growth and fattening of lambs [J]. J Anim Sci, 1949, 8:578-582.
[97] Preston R L, Cheng C, Stoty D, et al. The influence of oral adminstration of diethylstilbestrol upon estrogenic residues in the tissues of beef cattle [J]. Sci, 1056, 15:3-12.
[98] Preston R. Possible role of DES on mature size in steers [J]. Sci,1978, 47(Supply .1):436(Aster).
[99] Preston R. Influence of diethylstilbestrol on body water space in ruminans [J] .Proc Soc Exp Biol Med , 1969, 132:401-404.
[100] Wade N. DES: a case study of regulatory abidication [J]. Science, 1972, 177: 335-337.
[101] Jukes T. Diethylstilbestrol in beef production:What is the risk to consumers [J] Preventive Med , 1976, 5:438-453.
[102] Marcus A. Cancer from beef: DES, Federal food regulation and consumer confidence [M]. The Johns Hopkins University Press , 1994, Baltimore, MD.
[103] Cole G H, Gass R J. On the safety of estrogenic residues in edible animal products [J] . Biosvience, 1975, 25:19-25.
[104] Burroughs W, Culbertson E , Cheng , et al. The influence of oral administration of diethylstilbestrol to beef steers [J]. J Anim Sci, 1955, 14:1015-1024。
[105] Raun A P. A skill study of the effects of two levels of stilbestrol from dry and liquid premixes on feedlot performance of fattening steers [M]. Expt, 1965, CA-108, Eli Lilly and Co, Inc, Greenfield IN.
[106] Burroughs W, Christianson R. Regular, double and triole levels of stibestrol for finishing yearling cattle [M]. A.S Leaflet 1963, R49, Anim.
[107] Andrews F N. DES history (Letter to the editor) [J]. Science, 1995, 268:16
[108] IARC. International Agency for Research on Cancer. IARC.Monography on the Evalution of the Carinogenic Risk of Chemical to Man. Sex Hormones. Vol.6.243pp.Lyon, France:IARC, 1974.
[109] IARC. International Agency for Research on Cancer. IARCMonography on the Evalution of the Carinogenic Risk of Chemical to Humans. Sex Hormones(Ⅱ). Vol.21.583pp.Lyon, France:IARC, 1979.
[110] Council of European communities, Directive 81/602/EEC, Off J Eur Commun, 1981, L222, 32.
[111] Council of European communities, Directive 88/146EEC,Off J Eur Commun, 1988, L70:16.
[112] Donoho A L, Johnson W S, Sieck R F, et al. Gas chromatographic determination of diethylstilbestrol and its glucuronide in cattle tissue [J]. Journal of the AOAC, 1973,
56(4):785-792.
[113] Coffin D E, Pilon J C. Gas chromatographic determination of diethylstilbestrol residues in animal tissues [J]. Journal of the AOAC, 1973, 56(2):352－357.
[114] Tirpenou A E, Kilkidis S D, Kamarianos A P. Modified method for eletron capture gas-liquid chromatographic determination of diethylstilbestrol residues in urine of fattened bulls [J]. Journal of the AOAC, 1983, 66(5):1230-1233
[115] Busico F, Moretti G, Cartoni G P. Determination of estrogens in animal tissues by GC-MS with negative ion chemical ionization [J]. Journal of High Resolution Chromatography, 1992, 15(2)94-98.
[116] Reuvers T H, Perogordo E, Jimenez R. Rapid screening method for the determination of diethylstilbestrol in edible animal tissue by column liquid chromatographiy with eletrochemical detection [J]. Journal of chromatography, 1991, 564, 477-484.
[117] Lagana A. General and selective isolation procedure for high-performance liquid chromatographic detemination of anabolic steroids in tissues [J]. Journal of Chromatography, 1991, 588, 89-98.
[118] Leindsteiner K. The specificity of the serological reactions [M]. Cambridge: Harward University Press, 1945, 293.
[119] Campfied L A. Mathematical analysis of competitive protein binding assays [M]. In: Odell W,Franchimont P Eds. Principles of Competicie Protein-binding assays，2nd ed. New York: John Wiley and Sons, 1983, 125.
[120] Jeffcoate S L, White N, Fraser H M. Proceedings: Radioimmunoassay of thyrotrophin releasing hormone in blood and urine [J]. N Engl J Med,1974, 291(2):181.
[121] Kennedy D G, Blanchflower W J, O,Dornan B C. Development of an ELISA for maduramicin and determination of the depletion kinetics of maduramicin residues in poultry [J]. Food Addit Contam,1997, 14(1):27-33.
[122] Engvall E, Perlman P. Enzyme-linked immunosorbent assay (ELISA):Quantitative assay of immunoglobulin G.Immunochemistry [J]. 1971 , 8(9):871-874.
[123] Van Weemen B K, Schuurs A H. Immunoassay using hapten--enzyme conjugates [J]. FEBS Lett, 1972, 24(1):77-81.
[124] Harlow E, Lane D. Antibodies: a Laboratory Manual [J].Cold Spring Harbor, NY, 1998.
[125] Wainer B H, Fitch F W, Rothberg R M, et al. Morphine-3-succinyl-bovine serum albumin: an immunogenic hapten-protein conjugate [J]. Science,1972 , 176(39):1143-1145.
[126] Dong Min, Li Guofu, Li Jin, et al. Immunological Detection of Arbutin [J]. Tsinghua Science and Technology, 1999, 4(3):311-316.
[127] Erlanger B F, Borek E, Beiser S M et al. Steroid protein conjugates I: Preparation and characterization of bovine serum albumin with testosterone and with cortisone [J]. J chem., 1957, 228:713-727 .
[128] O'Neill S P, Robb T E. Hapten concentrations in the circulation of animals actively immunized with hapten-protein conjugates [J]. J. Immunol. 1976 , 116(2):363-366.
[129] Sambrook J，Russel D W. 分子克隆实验指南[M]，第三版，科学出版社，1713－1721.
[130] Collins W P, Hennam J F. Molec Cleavage of structural proteins during the assembly of the head of bacteriophage T4 [J]. Aspects Med, 1976, 1:27.
[131] Rombauts P, Pierdet A, Jouquey A. Preparation and properties of anti-diethylstilbestrol immune sera [J]. C R Acad Sci Hebd Seances Acad Sci D. 1973 , 277(18):1921-1924.
[132] Erlanger B F, Borek O F, Beiser S, et al. Steroid-protein conjugated II: Preparation and characterization of conjugates of bovine serum albumin with progesterone, deoxycorticosterone and oestrone [J]. J Biol Chem. 1959, 243:1090-1094.
[133] Erlanger B F, Langone J. Preparation of antigenic hapten-carrier conjugate:A survey. In Methods in enzymology, Eds.:Academic press: New York, 1980,Vol.70, Part A.
[134] Li Q X, Zhao M S, Gee S L, et al. Development of enzyme-linked immunosorbent assays for 4-nitrophenol and substituted 4-nitrophenols [J]. J Agri Food Chem, 1991, 39:1685-1692.
[135]Schneider P, Hammock B D. Influence of the ELISA for S-triazine herbicides using monoclonal antibodies [J]. J Agric Food Chem,1992, 40:525.
[136] Faraj B A, Ali F M. Devlopment and application of a radioimmunoassay for tetracycline [J]. J Pharmacol Exp Ther, 1981, 217:10.
[137] Feng P C, Horton S R, Sharp R. A general method for developing immunoassays to chloroacetailide herbicides [J]. J Agric Food Chem, 1992, 40:211.
[138] Jung F, Meyer H D, Hamm R T. Development of a sensitive enzyme-linked immunosorbent assay for the fungicid fenpropimorph [J]. Agric Food Chem, 1989, 37:1183.
[139] Pallardy M, Alberici G F, Goodman A, et al. Antibody recognition of substituted ammonium ions:Modulation by the counterion [J]. J Immunol Methods,1987, 99:179.
[140] Buenafe A C, Rittenberg M B. Combinding site specificity of monoclonal antibodies to the organophosphate hapten soman [J]. Mol Immunol. 1987, 24:401.
[141] Fenton II, Singer S J. Affinity labeling of antibodies to the p-azophyltrimethylammonnium hapten, and a comparison of affinity-labeled antibodies of two different species [J]. Biochemistry, 1971, 10:1429.
[142] Flurkey B S, Bolger M B, Linthicum D S. Preparation and characterization of antisera
and monoclonal antibodies to seratonergic and dopaminegric ligands [J]. J Neuroimmunol, 1985, 8:115.
[143] Groopman J D, Trudel L J, Donshue P R，et al. High affinity monoclonal antibodies for aflatoxins and their application to solid-phase immunoassays [J]. Proc Natl Acad Sci USA, 1984, 81:7728.
[144] Leudtke P R, Korner M, Neve K A, et al. Monoclonal antibodies with high affinity for spiroperidol [J]. J Neurochem, 1988, 50:1253.
[145] Li Q X, Zhao M S, Gee S L, et al. Development of enzyme-linked immunosorbent assays for 4-nitrophenol and substituted 4-nitrophenols [J]. J Agric Food Chem, 1991, 39:1685-1692.
[146] Mcadam D P, Hill A S, Beasley H L, et al. Mono- and polyclonal antibodies to the organophosphate fenitrothion I:Approaches to hapten-protein conjugation [J]. J Agri Food Chem,1992, 40:1466.
[147 ] Schmidt D J, Clarkson C E, Swanson T A. Monoclonal antibodies for immunoassay of avermectins [J]. J Agric Food Chem, 1990, 38:1763.
[148] Queffelec Anne-Laurence, Nodet P, Haelters Jean-Pierre, et al. Hapten synthesis for a monoclonal antibody based ELISA for Deltamethrin [J]. J Agric Food Chem, 1998, 46:1670-1676.
[149] Sawada J, Janejal N, Nagamatsu K, et al. Production and characterization of high-affinity monoclonal antibodies against morphine [J]. Mol Immunol, 1991, 25:937.
[150] Bull J P, Serreqi A N, Gamboa H R, et al. Improved Enzyme-linked immunosorbent assay to detect didecyldimethylammonium chloride, a quaternary ammonium compound [J]. J Agric Food Chem, 1998, 467:4779-4786.
[151] Tristram G P, Daniel P S, Abba I T, et al. Medical immunology [M].北京：科学出版社，2002,72-81.
[152] R. M. Sharpe. Environmental oestrogens and male infertility [J]. Pure & Appl Chem 1998, 70(9):1685-1701.