抗氯霉素单克隆抗体的制备及化学发光酶免疫分析方法的建立
|
摘要
氯霉素(chloramphenicol, CAP)是一种广谱抗生素,对人体有较强的副作用与毒性,会导致再生障碍性贫血和粒细胞缺乏症,但因其价格便宜且性质稳定,养殖业中仍有违规使用的现象。化学发光酶免疫分析法(CLEIA)具有灵敏度高、线性范围宽等优点。本研究制备了抗CAP单克隆抗体,并建立氯霉素化学发光酶免疫分析方法。
以琥珀氯霉素(HAP)和氯霉素为半抗原,牛血清白蛋白(BSA)为载体,分别采用混合酸酐法和重氮化法合成氯霉素免疫抗原HAP-BSA、CAP-BSA,起始摩尔比分别为50：1、30：1、20：1。以卵清蛋白(OVA)为载体,采用活泼酯法(EDC-NHS法)合成40：1-1：1七个不同起始摩尔比的ELISA检测用包被抗原(HAP-OVA),紫外、红外光谱扫描和SDS-PAGE电泳法鉴定全抗原偶联成功,紫外测定HAP-BSA偶联分别为27：1、18：1、8：1,CAP-BSA偶联比分别为21：1、11:1、5:1, HAP-OVA偶联比26：1-1：3。
将偶联比为18：1的HAP-BSA,偶联比为21：1的CAP-BSA各免疫6只Balb/c小鼠,每种抗原三个剂量梯度25、75、100μμg／只,两个平行。三免后以4#HAP-OVA包被酶标板,ELISA法检测免疫血清效价及其对CAP的敏感性(通过CAP半数抑制浓度IC50值反映),最终选择效价高、IC50值低的6#小鼠冲击免疫。利用杂交瘤技术将6#小鼠脾细胞和SP2/0骨髓瘤细胞进行细胞融合,融合率为84.6%。经过亚克隆筛选出两株产抗CAP单克隆抗体的细胞株,编号为Ⅳ1B3、IV2E7,经扩大培养,用ELISA法检测上清液中抗体效价与敏感性,选择效价高、IC50值低的Ⅳ1B3株细胞注射小鼠腹腔,诱生腹水。辛酸-饱和硫酸铵法进行单克隆抗体纯化。通过优化选择偶联比为3：1的HAP-OVA为最佳包被抗原,此包被抗原下进行ELISA实验测定单克隆抗体效价为2×105,IC50值为15ng/mL。经鉴定,抗CAP单抗与青霉素、庆大霉素、链霉素、四环素药物几乎没有交叉反应,与琥珀氯霉素的交叉反应率为110.3%。
采用混合酸酐法合成不同起始摩尔比的酶标记抗原HAP-HRP, ELISA实验选出最佳酶标记抗原。通过单因素实验确定了化学发光底物液各组分的最佳浓度,发光物质为5×10-4mol/L luminol,发光增强剂为4×10-4mol/L对碘苯酚,氧化剂为3×10-3mol/L H2O2溶液。通过直接竞争CLISA实验优化,确定了单克隆抗体包被浓度为1.5μg/mL, HAP-HRP稀释倍数为1000倍,标准品CAP(待测物)与HAP-HRP竞争时间为30min。建立了氯霉素CLEIA分析方法,标准曲线为Y=-130801gX+35091,线性相关系数r=0.997(其中Y为RLU值,1gX为CAP对数浓度),该方法中CAP半数抑制浓度IC50为2.3ng/mL,检测限为0.018ng/mL,检测范围为0.067ng/mL-36.7ng/mL,批内标准偏差<7%，.批间标准偏差<10%,基围虾的加样回收率在96%-112%该方法重现性和精密度良好。本方法可以为氯霉素化学发光试剂盒提供基础。
Chloramphenicol(CAP), which is considered as a broad-spectrum antibiotic, has serious adverse effect against human bodies, leading to aplastic anemia and granulocytopenia. Due to low price and stable character, Chloramphenicol is still used in aquaculture illegally. Chemiluminescent Enzyme Immunoassay (CLEIA) has the advantage of high specificity, broad linear range, etc. In the study, anti-Chloramphenicol monoclonal antibody was prepared and a method of CLEIA for determination of CAP was developed.
Immunogen was prepared by mixed anhydride method and diazotization method under 3 different molar ratios from 50:1-10:1(hapten to carrier). Both Chloramphenicol Sod Succinate(HAP) and CAP were used as hapten, Bovine Serum Albumin (BSA) was used the carrier. Active ester was also applied under 7 different molar ratios from 40:1-1:1 in the preparation of coating antigen, when Ovalbumin (OVA) as the carrier. UV and IR spectrum and SDS-PAGE was used to assure the conjugation of hapten to carrier. The conjugate ration was 27:1-8:1 for HAP-BAS, 21:1-5:1 for CAP-BAS, and 26:1-1:3 for HAP-OVA through UV detection.
6 Balb/c mice were immunized using 18:1 HAP-BSA and 21:1 CAP-BSA. The immunizing dose was 25,75, 100μg per mouse, respectively. One parallel group was set. After third immunization, the serum titer and sensitivity to Chloramphenicol, which can be reflected by 50% inhibiting concentration (IC50), was detected by ELISA using 4#HAP-OVA as coating antigen. Mouse 6 was chosen for boosted immune. Hybridoma technique was applied for fusing Sp2/0 mouse myeloma cells with splenocytes from Mouse 6. And the fusion rate was 84.6%. The supernatant was detected by ELISA for its titer and sentitive. After subcloning,2 hybridoma cell lines(Ⅳ1B3,Ⅳ2E7), which could secrete the specific anti-CAP antibodies, were established by limiting dilution assay (LDA). After selection by ELISA and culture expansion, cellⅣ1B3 clones, which is more sensitive, and with higher titer and lower IC50, were attained and injected into mice abdomen for anti-CAP monoclonal antibody production, The ascites of the mice was collected and extracted through caprylic acid and equilibrium ammonium sulfate.
After antibodies purification, The conjugation ratio of the coating antigen was optimized at the index of IC50. The molar ratio of HAP:OVA was 3:1. Using the HAP-OVA as coating antigen, the antibody titer was 2×105 and the IC50 was 15ng/mL。The cross reaction rate of with penicillin, gentamicin, streptomycin, and tetracycline was barely zero, and with Chloramphenicol succinate was 110.3%.
Enzyme-labelled antigen was synthesized by conjugating Hap with HRP according the method of mixed anhydride. The best enzyme-labelled antigen was screening by ELISA. Optimum consistency for each ingredient in CLEIA substrate was obtained by single factor experiment. The luminophore was luminal at the concentration of 5×10-4mol/L, enhancer was 4×10-4mol/L 4-iodophenol, and oxidant was 3×10-3 mol/L H2O2 solution. Direct competitive ELISA was developed and optimized. The optimum concentration of coating antigen was 1.5μg/mL, diluted rate of HAP-HRP was 1:1000, reaction time of standard (CAP) or samples with HAP-HRP was 30 min.
The equation of calibration curve was Y=-13080lgX+35091 with the r=0.997 and IC50=2.3ng/mL in the concentration range from 0.067ng/mL to 36.7ng/mL. The coefficients of variation for within and between assays below 7%and 10%,respectively. The average recovery of shrimp was 96%-112% The method showed good repetition and precision and can be the foundation of CLEIS test kits.
以琥珀氯霉素(HAP)和氯霉素为半抗原,牛血清白蛋白(BSA)为载体,分别采用混合酸酐法和重氮化法合成氯霉素免疫抗原HAP-BSA、CAP-BSA,起始摩尔比分别为50：1、30：1、20：1。以卵清蛋白(OVA)为载体,采用活泼酯法(EDC-NHS法)合成40：1-1：1七个不同起始摩尔比的ELISA检测用包被抗原(HAP-OVA),紫外、红外光谱扫描和SDS-PAGE电泳法鉴定全抗原偶联成功,紫外测定HAP-BSA偶联分别为27：1、18：1、8：1,CAP-BSA偶联比分别为21：1、11:1、5:1, HAP-OVA偶联比26：1-1：3。
将偶联比为18：1的HAP-BSA,偶联比为21：1的CAP-BSA各免疫6只Balb/c小鼠,每种抗原三个剂量梯度25、75、100μμg／只,两个平行。三免后以4#HAP-OVA包被酶标板,ELISA法检测免疫血清效价及其对CAP的敏感性(通过CAP半数抑制浓度IC50值反映),最终选择效价高、IC50值低的6#小鼠冲击免疫。利用杂交瘤技术将6#小鼠脾细胞和SP2/0骨髓瘤细胞进行细胞融合,融合率为84.6%。经过亚克隆筛选出两株产抗CAP单克隆抗体的细胞株,编号为Ⅳ1B3、IV2E7,经扩大培养,用ELISA法检测上清液中抗体效价与敏感性,选择效价高、IC50值低的Ⅳ1B3株细胞注射小鼠腹腔,诱生腹水。辛酸-饱和硫酸铵法进行单克隆抗体纯化。通过优化选择偶联比为3：1的HAP-OVA为最佳包被抗原,此包被抗原下进行ELISA实验测定单克隆抗体效价为2×105,IC50值为15ng/mL。经鉴定,抗CAP单抗与青霉素、庆大霉素、链霉素、四环素药物几乎没有交叉反应,与琥珀氯霉素的交叉反应率为110.3%。
采用混合酸酐法合成不同起始摩尔比的酶标记抗原HAP-HRP, ELISA实验选出最佳酶标记抗原。通过单因素实验确定了化学发光底物液各组分的最佳浓度,发光物质为5×10-4mol/L luminol,发光增强剂为4×10-4mol/L对碘苯酚,氧化剂为3×10-3mol/L H2O2溶液。通过直接竞争CLISA实验优化,确定了单克隆抗体包被浓度为1.5μg/mL, HAP-HRP稀释倍数为1000倍,标准品CAP(待测物)与HAP-HRP竞争时间为30min。建立了氯霉素CLEIA分析方法,标准曲线为Y=-130801gX+35091,线性相关系数r=0.997(其中Y为RLU值,1gX为CAP对数浓度),该方法中CAP半数抑制浓度IC50为2.3ng/mL,检测限为0.018ng/mL,检测范围为0.067ng/mL-36.7ng/mL,批内标准偏差<7%，.批间标准偏差<10%,基围虾的加样回收率在96%-112%该方法重现性和精密度良好。本方法可以为氯霉素化学发光试剂盒提供基础。
Chloramphenicol(CAP), which is considered as a broad-spectrum antibiotic, has serious adverse effect against human bodies, leading to aplastic anemia and granulocytopenia. Due to low price and stable character, Chloramphenicol is still used in aquaculture illegally. Chemiluminescent Enzyme Immunoassay (CLEIA) has the advantage of high specificity, broad linear range, etc. In the study, anti-Chloramphenicol monoclonal antibody was prepared and a method of CLEIA for determination of CAP was developed.
Immunogen was prepared by mixed anhydride method and diazotization method under 3 different molar ratios from 50:1-10:1(hapten to carrier). Both Chloramphenicol Sod Succinate(HAP) and CAP were used as hapten, Bovine Serum Albumin (BSA) was used the carrier. Active ester was also applied under 7 different molar ratios from 40:1-1:1 in the preparation of coating antigen, when Ovalbumin (OVA) as the carrier. UV and IR spectrum and SDS-PAGE was used to assure the conjugation of hapten to carrier. The conjugate ration was 27:1-8:1 for HAP-BAS, 21:1-5:1 for CAP-BAS, and 26:1-1:3 for HAP-OVA through UV detection.
6 Balb/c mice were immunized using 18:1 HAP-BSA and 21:1 CAP-BSA. The immunizing dose was 25,75, 100μg per mouse, respectively. One parallel group was set. After third immunization, the serum titer and sensitivity to Chloramphenicol, which can be reflected by 50% inhibiting concentration (IC50), was detected by ELISA using 4#HAP-OVA as coating antigen. Mouse 6 was chosen for boosted immune. Hybridoma technique was applied for fusing Sp2/0 mouse myeloma cells with splenocytes from Mouse 6. And the fusion rate was 84.6%. The supernatant was detected by ELISA for its titer and sentitive. After subcloning,2 hybridoma cell lines(Ⅳ1B3,Ⅳ2E7), which could secrete the specific anti-CAP antibodies, were established by limiting dilution assay (LDA). After selection by ELISA and culture expansion, cellⅣ1B3 clones, which is more sensitive, and with higher titer and lower IC50, were attained and injected into mice abdomen for anti-CAP monoclonal antibody production, The ascites of the mice was collected and extracted through caprylic acid and equilibrium ammonium sulfate.
After antibodies purification, The conjugation ratio of the coating antigen was optimized at the index of IC50. The molar ratio of HAP:OVA was 3:1. Using the HAP-OVA as coating antigen, the antibody titer was 2×105 and the IC50 was 15ng/mL。The cross reaction rate of with penicillin, gentamicin, streptomycin, and tetracycline was barely zero, and with Chloramphenicol succinate was 110.3%.
Enzyme-labelled antigen was synthesized by conjugating Hap with HRP according the method of mixed anhydride. The best enzyme-labelled antigen was screening by ELISA. Optimum consistency for each ingredient in CLEIA substrate was obtained by single factor experiment. The luminophore was luminal at the concentration of 5×10-4mol/L, enhancer was 4×10-4mol/L 4-iodophenol, and oxidant was 3×10-3 mol/L H2O2 solution. Direct competitive ELISA was developed and optimized. The optimum concentration of coating antigen was 1.5μg/mL, diluted rate of HAP-HRP was 1:1000, reaction time of standard (CAP) or samples with HAP-HRP was 30 min.
The equation of calibration curve was Y=-13080lgX+35091 with the r=0.997 and IC50=2.3ng/mL in the concentration range from 0.067ng/mL to 36.7ng/mL. The coefficients of variation for within and between assays below 7%and 10%,respectively. The average recovery of shrimp was 96%-112% The method showed good repetition and precision and can be the foundation of CLEIS test kits.
引文
[1]王自良,赵坤,王改平.氯霉素的毒性及其在动物性食品中的残留[J].河南科技学院学报,2005,33(2)：101-105.
[2]邢健.2001氯霉素事件-中国遭遇欧盟伏击战[J].WTO经济导刊,2001,26-28.
[3]张远,王永强,高世君等.动物性食品中氯霉素残留的定量分析研究[J].食品研究与开发,2006,27(4)：116-118.
[4]孙俐,郑文杰.动物源性食品的氯霉素ELISA检测及分析[J].天津大学学报,2007,22(2)：76-79.
[5]郝俊虎,王仙琴,闫永利等.氯霉素对出口畜产品的危害[J].饲料研究,2005,(5)：8-10.
[6]陈志明.氯霉素引起铁粒幼细胞性贫血一例报告[J].中华内科杂志,1980,19(4)：251.
[7]王自良,赵坤,张改平.氯霉素的毒性及其在动物性食品中的残留与检测[J].河南科技学院学报(自然科学版),2005,33(2)：101-105.
[8]季长虹.抗菌素临床使用的若干问题[J].中级医刊,1981,(10)：542.
[9]杜升东.药物过敏性休克[J].中级医刊,1980,(2)：60.
[10]Commission Decision 2003/181/EC of 13March 2003 amending Decision 2002/657/EC as regards the setting of minimum required performance limits (MRPLs) for certain residues in food of animal origin1Official Journal, L 71,15/03/2003:17-181.
[11]张宇.氯霉素在动物体内的残留与检测[J].农业论坛,2007,(1)：68.
[12]王自良,张海棠,李军民.氯霉素在动物性食品中的检测[J].中国饲料,2005,(4)：34-36.
[13]Keukens Beck. High performance liquid chromatographic screening and confirmation methods for chloramphenicol residues in meat with offline cartridge samp le cleanup and online diode array UV-V IS detection[J]. Journal of Chromatography,1986,352:445-453.
[14]邹月利,赵李霞,张永忠.奶中氯霉素残留量的高效液相色谱方法研究[J].食品工业,2006,50-52.
[15]李俊锁.兽药残留分析研究进展[J].中国农业科学,1997,30(5)：81-87.
[16]吉文亮,马永建,仓公敖.高效液相色谱串联质谱法测定蜂蜜中残留氯霉素[J].药物分析杂志,2006,26(12)：1814-1817.
[17]陈小霞,岳振峰,吉彩霓等.高效液相色-电喷雾电离三级四极杆质谱测定鸡肉中氯霉素、 甲砜霉素和氟甲砜霉素的残留量[J].色谱,2005,23(1)：92-95.
[18]王丙涛.气质联用检测饲料中氯霉素的方法研究[J].现代畜牧兽医.
[19]康金国,刘国燕,柴春彦.牛乳中氯霉素残留的聚乙烯亚胺-纳米金修饰电极测定[J].中国兽医科学,2008,38(5)：431-437.
[20]赵芸,余霞奎,柳爱春.水产品中氯霉素检测方法的比较与探讨[J].杭州农业科技,2006,1：22-25.
[21]高荣,赵一泽,赵建增.化学发光免疫分析技术应用研究进展[J].中国生物制品学杂志,2008,21：338-342.
[22]林斯.氯霉素酶促化学发光免疫分析方法的建立[D].中国原子能科学研究所,2005.
[23]高彬文,张素霞,沈建忠等.化学发光酶联免疫分析法检测鱼虾中氯霉素残留[J].中国兽医杂志,2007,43(5)：68-69.
[24]李美佳.当代免疫学技术与应用[M].北京：北京医科大学中国协和医科大学联合出版社,1998549-561.
[25]Rilo I, Takashi H. Direct determination of plasma endothelin21 by chemiluminescence enzyme immunoassay.Clin. Chem.1996,42(8):1155-1158.
[26]Helen L, Larry J K. Influence of different luminols on the characteristics of the chemilumine scence reaction in human neutropjils.J.Biolumin. Chemilumin.1995,10:353-359.
[27]Metelitxa D I, Eryomin A N. Enhanced chemiluminescence in the oxidation of luminol and an isoluminol cortisol conjugate by hydrogen peroxide in reversed micelles.J.Biolumin. Chemilu min.1992,7:21-26.
[28]Mayer A. Neuenhofer S. Luninescent labels More than just an alternative to radioisotopes Angew.Chem.Int.Ed. Engl.1994,33 (10):1044-1072.
[29]赵利霞,李振甲,魏彦林等.化学发光免疫分析[J].世界科技研究与发展,2004,26(4)：24-33.
[30]Vidziunaite R, Mikulskis P, Kulys J. Chemiluminescence im-munoassay (CLIA) for the det-ection of brucellosis and tularaemia antigens. J. Biolumin,Chemilumin.1995,10 (4):199-203.
[31]Sasamoto K, Deng G., Ushijima T, Ohkura Y, Ueno K,Benzothiazole Derivatives as substrat es for alkaline phosphatase assay with fluorescence and chemiluminescence detection. Analyst,1995,120(6):1709-1714.
[32]Ito H, Suzuki H, Miki Y, Hayashi T, Iwata M, Yamaki M, Jpn. Patent,08,038,196 (1996).
[33]Mitoma Y., Hara K., Kumakura, S. Jpn. Patent,07,311,197(1995) Thorpe G H., Kricka L J, Gillespie E, Moseley S, Amess R.Baggett N, Whitehead T P. Enhanced of the horseradish peroxidase-catalyzed chemiluminescent oxidation of cyclic diacyl hy-drazides by 62 hydroxybenzothiazoles. Anal. Biochem.,1985,145:96-100.
[34]Thorpe G H., Kricka L J., Gillespie E., Moseley S., Amess R.Baggett N., Whitehead T P. Enhanced of the horseradish peroxidase-catalyzed chemiluminescent oxidation of cyclic diacyl hy-drazides by 62hydroxybenzothiazoles. Anal. Biochem.,1985,145:96-100.
[35]Kricka L J., US Patent,5,512,451 (1996).
[36]Nozaki O, Kricka L J, Stanley PE.Bioluminescence and chemiluminescence literature-
immunoassays and protein blotting assays.Journal of Bioluminescence and Chemiluminescence, 1992,7(4):263-298.
[37]Kissel T R, Friedman A E., Fingar S A, Chemiluminescent reagent and assay using a substituted acetanilide for light generation[P]. US patent,5705357,1998-01-06.
[38]Bronstein I, McGrath P., Chemiluminescence lights up. Nature,1989,338:599-600.
[39]Bronstein I, Voyta J C., Edwards B.A comparision of chemiluminescent and colorimetric substrates in a hepatitis B virus DNA hybridization assay. Clin Chim,1990,194(1):73-90.
[40]张丽民.化学发光标记及发光免疫分析[J].基础医学与临床,1995,15,4.
[41]Schaap A P, Akhavan H, Romano L J. Chemiluminescent Substrates for Alkaline Phosphatase:Application to Ultrasensitive Enzyme-linked Immunoassays and DNA Probes[J].Clin Chem,1989,35(9):1853-1854.
[42]Nishizonol,etal. Rapid and sensitive chemiluminescent enzyme immunoassay for measuring tumor markers[J].Clin Chem,1991,37(9):1639-1644.
[43]Zhao L, Lin J M, Li Z. Comparison and development of two different solid phase chemiluminescence ELISA for the determination of albumin in urine[J].Anal Chim Acta, 2005,41:199-207.
[44]任世奇,王栩,唐宝军等.微板式化学发光酶免疫分析法临床测定人血清中孕酮.分析化学研究报告.2008,36(6)：729-734.
[45]靳辉,王栩,辛天兵等.微孔板化学发光酶免疫分析法定量检测肿瘤标志物CA72-4的方法学研究[J].科学通报.2008,53(13)：1538-1543.
[46]王栩,林金明.化学发光免疫分析技术新进展[J].分析实验室,2007,26(6)：111-122.
[47]Xu Wang, Qian-Yun Zhang, Zhen-Jia Li,et. Development of high-performance magnetic chemiluminescence enzyme immunoassay for a-fetoprotein (AFP) in human serum[J].Clinica Chimica Acta.2008,393:90-94.
[48]吴丹凝,王栩,林金明等.磁性微粒子化学发光免疫分析法测定人血清中雌三醇[J].化学学报,2007,65(23)：2755-2760.
[49]赵利霞,林金明,屈锋.微板式磁化学发光酶免疫分析法对人绒毛膜促性腺激素(HCG)的灵敏快速测定[J].化学学报,2004,62(1)：71-77.
[50]Zhao L, Lin J M. Development of a micro-plate magnetic chemiluminescence enzyme immunoassay (MMCLEIA) for rapid and high-throughput analysis of 17-estradiol in water samples[J]. J Biotechnol,2005,118:177-186.
[51]Dungchai W, Siangproh W, Lin J M etal. Development of a sensitive micro-magnetic chemiluminescence enzyme immunoassay for the determination of carcinoembryonic antigenAnal Bioanal Chem,2007,387:1965-1971.
[52]Hermanson G T.Bioconjugate techniques[M],San Diego:Academic Press,1996,785.
[53]Stabler TV,Siegel AL.Clin Chem,1990,36(6):906.
[54]朱承谟.标记免疫分析技术进展[J].标记免疫分析与临床,2002,9(3)：176-179.
[55]尹东光,贺佑丰,刘一兵等.标记免疫分析技术的发展点评[J].标记免疫分析与临床,2003,10(1)：40-42.
[56]Van Huystee R B, Roig M G, Shnyrov V L etal. Peroxidase stability related to its calcium and glycans[J]. Phytochem Rev,2004,3:19-28.
[57]Sakharov I Y,Alpeeva I S, Efremov E E.Use of soybean peroxidase in chemilum inescent enzyme-linked immunosorbent assay[J].Journal of Agricultural and Food Chemistry,2006, 54(5):1584-1587.
[58]Russell, J;Colpitts,T; Holets-McCormack, S, et al. Defined protein conjugates as signaling agents in immunoassays[J]. Clinical Chemistry,2004,50(10):1921-1929.
[59]Russell,JC;Colpitts, TL; Holets-McCormack,SR, etal. Solid phase assembly of defined protein conjugates[J].Bioconjugate Chemistry,2002,13(5):958-96.
[60]Stern E, Klemic J F, Routenberg D A etal. Label-free immunodetection with CMOS-compati ble semiconducting nanowires[J].Nature,2007,445:519-522.
[61]Fan A, Lau C, Lu J. Magnetic Bead-Based Chemiluminescent Metal Immunoassay with a Colloidal Gold Label[J].Anal Chem,2005,77:3238-3242.
[62]Wang Z, Hu J, Jin Y etal. In Situ Amplified Chemiluminescent Detection of DNA and Immunoassay of IgG Using Special-Shaped Gold Nanoparticles as Label[J].Clin Chem,2006,52:1958-1961.
[63]Chumbimuni-Torres K Y, Dai Z, Rubinova N etal. Potentiometric Biosensing of Proteins with Ultrasensitive Ion-Selective Microelectrodes and Nanoparticle Labels[J].J Am Chem Soc,2006,128:13676.
[64]Li Z P, Liu C H, Fan Y S, etal.A chemiluminescent metalloimmunoassay based on silver deposition on colloidal gold labels[J]. Analytical Biochemistry,2006,359(2):247-252.
[65]王延华,李官成,Xin-Fu Zhou.抗体理论与技术[M].科学出版社,卿5,74-75.
[66]石德时,王桂枝,毕丁仁等.氯霉素抗体的制备[J].华中农业大学学报,2001,20(5)：463-465.
[67]杨艳艳,张改平,王自良等.氯霉素单克隆抗体的研制及其免疫学特性鉴定[J].中国农学通报,2006,5(22)：7-12.
[68]刘岩,王云龙,张鑫等.抗氯霉素单克隆抗体的制备及其特性[J].中国兽药杂志,2006,40(5)：29-31.
[69]魏书林,曹振,沈建忠等.抗氯霉素单克隆抗体的制备与鉴定[J].中国兽医杂志,2004,8(40)：62-64.
[70]王桂枝,殷斌烈.氯霉素免疫原的合成与鉴定[J].中国兽医学,1998,2(18)：163-167.
[71]吴媛媛,包永明,王(?)等.氯霉素全抗原的合成与鉴定[J].化学通报,2005,6：458-463.
[72]王琰.基因工程抗体研究进展[J].中国免疫学杂志,1999,19(5)：193-195.
[73]冯仁青,郭振泉,宓捷波.现代抗体技术及其应用[M].北京：北京大学出版社,2006,75-76.
[74]刘岩,王艳鸽等.抗氯霉素单克隆抗体的制备及其特性[J].中国兽药杂志，2006,40(5)：29-31.
[75]吕琦,张爱玲等.抗盐酸克伦特罗单克隆抗体的制备及鉴定[J].云南大学学报,2006,28(1)：88-92.
[76]覃雅丽,石德时等.抗氯霉素单克隆抗体的制备及鉴定[J].中国预防兽医学报(增刊),2000,22(9)：63-65.
[77]A. Gao, Q. Chen, Y. Cheng, J. Lei, L. Zeng, Preparation of monoclonal antibodies against a derivative of semicarbazide as a metabolic target of nitrofurazone[J].Anal.'Chim. Acta 2007,592:58-63.
[78]M.P. Bruce, V.Boyd, C.Duch, J.R.White, Dialysis-based bioreactor systems for the production of monoclonal antibodies alternatives to ascites production in mice[J].J.Immunol. Methods,2002,264:59-68.
[79]K.Y.F.Yau, H.Lee, J.C.Hall.Emerging trends in the synthesis and improvement of hapten-specific recombinant antibodies[J].Biotechnol. Adv.2003,21,599-637.
[80]J. Brichta, M.Hnilova,T.Viskovic, Vet. Med.Czech 50 (2005)231.
[81]袁明秀,陈丽蓉,陈雅蕙等.生物化学实验原理和方法(第二版)[M].北京大学出版社,2005(8)：262-270.
[82]吴定,张羽航,姚汝华.乳中磺胺甲基嘧啶残留酶联免疫测定[J].食品科学,1998,19(6)：42-45.
[83]江湖,熊勇华,许扬等.EDC法制备黄曲霉毒素B1人工抗原的研究[J].食品科学,2005,26(7)：125-128.
[84]杨利国,胡少昶,魏平华等.酶免疫测定技术[M].南京：南京大学出版社,1998：281-393.
[85]沈关心,周汝麟.现代免疫学实验技术[M].湖北科学技术出版社,1998：119.
[86]金晶.ELISA法和胶体金免疫层析法检测猪尿样中的莱克多巴胺[D].南昌大学硕士论文.2007.
[87]VEN WEEMEN B K, SCHUURS A H W M. The influence of heterologous combinations of antiserum and enzyme-Labeled estrogen on the characteristics of estrogen enzyme immuno-assay[J]. Immunochemistry,1975(12):667-670.
[88]Kohler G,Milstein C.Continuous cultures of fused cells.secreting antibody of Predefined specificity[J].Nature,1975,256:495-497.
[89]司徒镇强,吴军正.细胞培养[M].西安：世界图书出版公,2007.1.
[90]黄丽.抗AMP单抗的制备及牛奶中AMP残留ELISA检测方法的建立[D].南昌大学硕士论文,2007.
[91]柳中辉,吕昌龙.免疫学常用实验技术[M].北京：科学出版社,2002.8：11-13.
[92]陆彦.氨苄青霉素单克隆抗体制备及其残留检测试剂盒的初步研究[D].新疆农业大学硕士论文,2005.
[93]章静波.组织和细胞培养技术[M].北京：人民卫生出版社,2002,261-263.
[94]钟秋芳.腐马素B1单克隆抗体的制备[D].中国农业大学硕士学位论文,2000,15-18.
[95]胥传来,彭池方,郝凯等.化学发光酶免疫法测定水产品中残留氯霉素[J].分析化学,2005,33(12)：1809.
[2]邢健.2001氯霉素事件-中国遭遇欧盟伏击战[J].WTO经济导刊,2001,26-28.
[3]张远,王永强,高世君等.动物性食品中氯霉素残留的定量分析研究[J].食品研究与开发,2006,27(4)：116-118.
[4]孙俐,郑文杰.动物源性食品的氯霉素ELISA检测及分析[J].天津大学学报,2007,22(2)：76-79.
[5]郝俊虎,王仙琴,闫永利等.氯霉素对出口畜产品的危害[J].饲料研究,2005,(5)：8-10.
[6]陈志明.氯霉素引起铁粒幼细胞性贫血一例报告[J].中华内科杂志,1980,19(4)：251.
[7]王自良,赵坤,张改平.氯霉素的毒性及其在动物性食品中的残留与检测[J].河南科技学院学报(自然科学版),2005,33(2)：101-105.
[8]季长虹.抗菌素临床使用的若干问题[J].中级医刊,1981,(10)：542.
[9]杜升东.药物过敏性休克[J].中级医刊,1980,(2)：60.
[10]Commission Decision 2003/181/EC of 13March 2003 amending Decision 2002/657/EC as regards the setting of minimum required performance limits (MRPLs) for certain residues in food of animal origin1Official Journal, L 71,15/03/2003:17-181.
[11]张宇.氯霉素在动物体内的残留与检测[J].农业论坛,2007,(1)：68.
[12]王自良,张海棠,李军民.氯霉素在动物性食品中的检测[J].中国饲料,2005,(4)：34-36.
[13]Keukens Beck. High performance liquid chromatographic screening and confirmation methods for chloramphenicol residues in meat with offline cartridge samp le cleanup and online diode array UV-V IS detection[J]. Journal of Chromatography,1986,352:445-453.
[14]邹月利,赵李霞,张永忠.奶中氯霉素残留量的高效液相色谱方法研究[J].食品工业,2006,50-52.
[15]李俊锁.兽药残留分析研究进展[J].中国农业科学,1997,30(5)：81-87.
[16]吉文亮,马永建,仓公敖.高效液相色谱串联质谱法测定蜂蜜中残留氯霉素[J].药物分析杂志,2006,26(12)：1814-1817.
[17]陈小霞,岳振峰,吉彩霓等.高效液相色-电喷雾电离三级四极杆质谱测定鸡肉中氯霉素、 甲砜霉素和氟甲砜霉素的残留量[J].色谱,2005,23(1)：92-95.
[18]王丙涛.气质联用检测饲料中氯霉素的方法研究[J].现代畜牧兽医.
[19]康金国,刘国燕,柴春彦.牛乳中氯霉素残留的聚乙烯亚胺-纳米金修饰电极测定[J].中国兽医科学,2008,38(5)：431-437.
[20]赵芸,余霞奎,柳爱春.水产品中氯霉素检测方法的比较与探讨[J].杭州农业科技,2006,1：22-25.
[21]高荣,赵一泽,赵建增.化学发光免疫分析技术应用研究进展[J].中国生物制品学杂志,2008,21：338-342.
[22]林斯.氯霉素酶促化学发光免疫分析方法的建立[D].中国原子能科学研究所,2005.
[23]高彬文,张素霞,沈建忠等.化学发光酶联免疫分析法检测鱼虾中氯霉素残留[J].中国兽医杂志,2007,43(5)：68-69.
[24]李美佳.当代免疫学技术与应用[M].北京：北京医科大学中国协和医科大学联合出版社,1998549-561.
[25]Rilo I, Takashi H. Direct determination of plasma endothelin21 by chemiluminescence enzyme immunoassay.Clin. Chem.1996,42(8):1155-1158.
[26]Helen L, Larry J K. Influence of different luminols on the characteristics of the chemilumine scence reaction in human neutropjils.J.Biolumin. Chemilumin.1995,10:353-359.
[27]Metelitxa D I, Eryomin A N. Enhanced chemiluminescence in the oxidation of luminol and an isoluminol cortisol conjugate by hydrogen peroxide in reversed micelles.J.Biolumin. Chemilu min.1992,7:21-26.
[28]Mayer A. Neuenhofer S. Luninescent labels More than just an alternative to radioisotopes Angew.Chem.Int.Ed. Engl.1994,33 (10):1044-1072.
[29]赵利霞,李振甲,魏彦林等.化学发光免疫分析[J].世界科技研究与发展,2004,26(4)：24-33.
[30]Vidziunaite R, Mikulskis P, Kulys J. Chemiluminescence im-munoassay (CLIA) for the det-ection of brucellosis and tularaemia antigens. J. Biolumin,Chemilumin.1995,10 (4):199-203.
[31]Sasamoto K, Deng G., Ushijima T, Ohkura Y, Ueno K,Benzothiazole Derivatives as substrat es for alkaline phosphatase assay with fluorescence and chemiluminescence detection. Analyst,1995,120(6):1709-1714.
[32]Ito H, Suzuki H, Miki Y, Hayashi T, Iwata M, Yamaki M, Jpn. Patent,08,038,196 (1996).
[33]Mitoma Y., Hara K., Kumakura, S. Jpn. Patent,07,311,197(1995) Thorpe G H., Kricka L J, Gillespie E, Moseley S, Amess R.Baggett N, Whitehead T P. Enhanced of the horseradish peroxidase-catalyzed chemiluminescent oxidation of cyclic diacyl hy-drazides by 62 hydroxybenzothiazoles. Anal. Biochem.,1985,145:96-100.
[34]Thorpe G H., Kricka L J., Gillespie E., Moseley S., Amess R.Baggett N., Whitehead T P. Enhanced of the horseradish peroxidase-catalyzed chemiluminescent oxidation of cyclic diacyl hy-drazides by 62hydroxybenzothiazoles. Anal. Biochem.,1985,145:96-100.
[35]Kricka L J., US Patent,5,512,451 (1996).
[36]Nozaki O, Kricka L J, Stanley PE.Bioluminescence and chemiluminescence literature-
immunoassays and protein blotting assays.Journal of Bioluminescence and Chemiluminescence, 1992,7(4):263-298.
[37]Kissel T R, Friedman A E., Fingar S A, Chemiluminescent reagent and assay using a substituted acetanilide for light generation[P]. US patent,5705357,1998-01-06.
[38]Bronstein I, McGrath P., Chemiluminescence lights up. Nature,1989,338:599-600.
[39]Bronstein I, Voyta J C., Edwards B.A comparision of chemiluminescent and colorimetric substrates in a hepatitis B virus DNA hybridization assay. Clin Chim,1990,194(1):73-90.
[40]张丽民.化学发光标记及发光免疫分析[J].基础医学与临床,1995,15,4.
[41]Schaap A P, Akhavan H, Romano L J. Chemiluminescent Substrates for Alkaline Phosphatase:Application to Ultrasensitive Enzyme-linked Immunoassays and DNA Probes[J].Clin Chem,1989,35(9):1853-1854.
[42]Nishizonol,etal. Rapid and sensitive chemiluminescent enzyme immunoassay for measuring tumor markers[J].Clin Chem,1991,37(9):1639-1644.
[43]Zhao L, Lin J M, Li Z. Comparison and development of two different solid phase chemiluminescence ELISA for the determination of albumin in urine[J].Anal Chim Acta, 2005,41:199-207.
[44]任世奇,王栩,唐宝军等.微板式化学发光酶免疫分析法临床测定人血清中孕酮.分析化学研究报告.2008,36(6)：729-734.
[45]靳辉,王栩,辛天兵等.微孔板化学发光酶免疫分析法定量检测肿瘤标志物CA72-4的方法学研究[J].科学通报.2008,53(13)：1538-1543.
[46]王栩,林金明.化学发光免疫分析技术新进展[J].分析实验室,2007,26(6)：111-122.
[47]Xu Wang, Qian-Yun Zhang, Zhen-Jia Li,et. Development of high-performance magnetic chemiluminescence enzyme immunoassay for a-fetoprotein (AFP) in human serum[J].Clinica Chimica Acta.2008,393:90-94.
[48]吴丹凝,王栩,林金明等.磁性微粒子化学发光免疫分析法测定人血清中雌三醇[J].化学学报,2007,65(23)：2755-2760.
[49]赵利霞,林金明,屈锋.微板式磁化学发光酶免疫分析法对人绒毛膜促性腺激素(HCG)的灵敏快速测定[J].化学学报,2004,62(1)：71-77.
[50]Zhao L, Lin J M. Development of a micro-plate magnetic chemiluminescence enzyme immunoassay (MMCLEIA) for rapid and high-throughput analysis of 17-estradiol in water samples[J]. J Biotechnol,2005,118:177-186.
[51]Dungchai W, Siangproh W, Lin J M etal. Development of a sensitive micro-magnetic chemiluminescence enzyme immunoassay for the determination of carcinoembryonic antigenAnal Bioanal Chem,2007,387:1965-1971.
[52]Hermanson G T.Bioconjugate techniques[M],San Diego:Academic Press,1996,785.
[53]Stabler TV,Siegel AL.Clin Chem,1990,36(6):906.
[54]朱承谟.标记免疫分析技术进展[J].标记免疫分析与临床,2002,9(3)：176-179.
[55]尹东光,贺佑丰,刘一兵等.标记免疫分析技术的发展点评[J].标记免疫分析与临床,2003,10(1)：40-42.
[56]Van Huystee R B, Roig M G, Shnyrov V L etal. Peroxidase stability related to its calcium and glycans[J]. Phytochem Rev,2004,3:19-28.
[57]Sakharov I Y,Alpeeva I S, Efremov E E.Use of soybean peroxidase in chemilum inescent enzyme-linked immunosorbent assay[J].Journal of Agricultural and Food Chemistry,2006, 54(5):1584-1587.
[58]Russell, J;Colpitts,T; Holets-McCormack, S, et al. Defined protein conjugates as signaling agents in immunoassays[J]. Clinical Chemistry,2004,50(10):1921-1929.
[59]Russell,JC;Colpitts, TL; Holets-McCormack,SR, etal. Solid phase assembly of defined protein conjugates[J].Bioconjugate Chemistry,2002,13(5):958-96.
[60]Stern E, Klemic J F, Routenberg D A etal. Label-free immunodetection with CMOS-compati ble semiconducting nanowires[J].Nature,2007,445:519-522.
[61]Fan A, Lau C, Lu J. Magnetic Bead-Based Chemiluminescent Metal Immunoassay with a Colloidal Gold Label[J].Anal Chem,2005,77:3238-3242.
[62]Wang Z, Hu J, Jin Y etal. In Situ Amplified Chemiluminescent Detection of DNA and Immunoassay of IgG Using Special-Shaped Gold Nanoparticles as Label[J].Clin Chem,2006,52:1958-1961.
[63]Chumbimuni-Torres K Y, Dai Z, Rubinova N etal. Potentiometric Biosensing of Proteins with Ultrasensitive Ion-Selective Microelectrodes and Nanoparticle Labels[J].J Am Chem Soc,2006,128:13676.
[64]Li Z P, Liu C H, Fan Y S, etal.A chemiluminescent metalloimmunoassay based on silver deposition on colloidal gold labels[J]. Analytical Biochemistry,2006,359(2):247-252.
[65]王延华,李官成,Xin-Fu Zhou.抗体理论与技术[M].科学出版社,卿5,74-75.
[66]石德时,王桂枝,毕丁仁等.氯霉素抗体的制备[J].华中农业大学学报,2001,20(5)：463-465.
[67]杨艳艳,张改平,王自良等.氯霉素单克隆抗体的研制及其免疫学特性鉴定[J].中国农学通报,2006,5(22)：7-12.
[68]刘岩,王云龙,张鑫等.抗氯霉素单克隆抗体的制备及其特性[J].中国兽药杂志,2006,40(5)：29-31.
[69]魏书林,曹振,沈建忠等.抗氯霉素单克隆抗体的制备与鉴定[J].中国兽医杂志,2004,8(40)：62-64.
[70]王桂枝,殷斌烈.氯霉素免疫原的合成与鉴定[J].中国兽医学,1998,2(18)：163-167.
[71]吴媛媛,包永明,王(?)等.氯霉素全抗原的合成与鉴定[J].化学通报,2005,6：458-463.
[72]王琰.基因工程抗体研究进展[J].中国免疫学杂志,1999,19(5)：193-195.
[73]冯仁青,郭振泉,宓捷波.现代抗体技术及其应用[M].北京：北京大学出版社,2006,75-76.
[74]刘岩,王艳鸽等.抗氯霉素单克隆抗体的制备及其特性[J].中国兽药杂志，2006,40(5)：29-31.
[75]吕琦,张爱玲等.抗盐酸克伦特罗单克隆抗体的制备及鉴定[J].云南大学学报,2006,28(1)：88-92.
[76]覃雅丽,石德时等.抗氯霉素单克隆抗体的制备及鉴定[J].中国预防兽医学报(增刊),2000,22(9)：63-65.
[77]A. Gao, Q. Chen, Y. Cheng, J. Lei, L. Zeng, Preparation of monoclonal antibodies against a derivative of semicarbazide as a metabolic target of nitrofurazone[J].Anal.'Chim. Acta 2007,592:58-63.
[78]M.P. Bruce, V.Boyd, C.Duch, J.R.White, Dialysis-based bioreactor systems for the production of monoclonal antibodies alternatives to ascites production in mice[J].J.Immunol. Methods,2002,264:59-68.
[79]K.Y.F.Yau, H.Lee, J.C.Hall.Emerging trends in the synthesis and improvement of hapten-specific recombinant antibodies[J].Biotechnol. Adv.2003,21,599-637.
[80]J. Brichta, M.Hnilova,T.Viskovic, Vet. Med.Czech 50 (2005)231.
[81]袁明秀,陈丽蓉,陈雅蕙等.生物化学实验原理和方法(第二版)[M].北京大学出版社,2005(8)：262-270.
[82]吴定,张羽航,姚汝华.乳中磺胺甲基嘧啶残留酶联免疫测定[J].食品科学,1998,19(6)：42-45.
[83]江湖,熊勇华,许扬等.EDC法制备黄曲霉毒素B1人工抗原的研究[J].食品科学,2005,26(7)：125-128.
[84]杨利国,胡少昶,魏平华等.酶免疫测定技术[M].南京：南京大学出版社,1998：281-393.
[85]沈关心,周汝麟.现代免疫学实验技术[M].湖北科学技术出版社,1998：119.
[86]金晶.ELISA法和胶体金免疫层析法检测猪尿样中的莱克多巴胺[D].南昌大学硕士论文.2007.
[87]VEN WEEMEN B K, SCHUURS A H W M. The influence of heterologous combinations of antiserum and enzyme-Labeled estrogen on the characteristics of estrogen enzyme immuno-assay[J]. Immunochemistry,1975(12):667-670.
[88]Kohler G,Milstein C.Continuous cultures of fused cells.secreting antibody of Predefined specificity[J].Nature,1975,256:495-497.
[89]司徒镇强,吴军正.细胞培养[M].西安：世界图书出版公,2007.1.
[90]黄丽.抗AMP单抗的制备及牛奶中AMP残留ELISA检测方法的建立[D].南昌大学硕士论文,2007.
[91]柳中辉,吕昌龙.免疫学常用实验技术[M].北京：科学出版社,2002.8：11-13.
[92]陆彦.氨苄青霉素单克隆抗体制备及其残留检测试剂盒的初步研究[D].新疆农业大学硕士论文,2005.
[93]章静波.组织和细胞培养技术[M].北京：人民卫生出版社,2002,261-263.
[94]钟秋芳.腐马素B1单克隆抗体的制备[D].中国农业大学硕士学位论文,2000,15-18.
[95]胥传来,彭池方,郝凯等.化学发光酶免疫法测定水产品中残留氯霉素[J].分析化学,2005,33(12)：1809.