鸡肌肉组织中氯霉素残留的酶联免疫吸附检测法研究
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摘要
本研究采用活化酯法制备氯霉素(Chloranmphenicol,CAP)免疫抗原，分别免疫家兔和Balb／c小鼠获得抗CAP抗血清，并用间接酶联免疫吸附检测法(ELISA)对抗血清效价进行了检测，分别建立了检测CAP残留的间接竞争ELISA方法，可用于检测鸡肌肉组织中CAP的残留量。
CAP免疫抗原(CAP-BSA)的偶联比率为8∶1，包被抗原(CAP-OA)的偶联比率为2∶1。
在以多克隆抗体建立的ELISA方法中，用方阵滴定法确定包被抗原最适包被浓度为1.7μg／ml，抗血清最高效价为1∶640000，抗血清纯化后最适工作浓度为1∶20000。采用间接竞争ELISA法建立检测CAP的标准曲线，线性检测范围为0.1ng／ml～36.45ng／ml，检测限为0.05ng／ml。以0.5、1、2.5、5ng／g的浓度添加时，鸡肌肉组织中的回收率为42.00％～92.80％，变异系数为4.76％～26.29％。
在以单克隆抗体建立的ELISA方法中，用方阵滴定法确定包被抗原最适包被浓度为0.85μg／ml，腹水最高效价为1∶10~8，纯化后最适工作浓度为1∶10000。采用间接竞争ELISA法建立检测CAP的标准曲线，线性检测范围为0.1ng／ml～25ng／ml，检测限为0.1ng／g。以0.5、1、2．5、5ng／g的浓度添加时，鸡肌肉组织中回收率为71.01％～127.86％，变异系数为2.68％～16.16％。
上述2种ELISA方法快速、灵敏、方便，满足了氯霉素残留检测的要求，适用于残留大规模的快速筛选检测。
In this study, one kind of immunogen was prepared: chloramphenicol(CAP) was coupled to carrier protein (BSA) using the N-hydroxysuccinimide activated ester method. Then the anti-CAP serum were raised in rabbits and in Balb/c mice respectively. After monitoring the titer of anti-serum by the indirect Enzyme Linked ImmunoSorbent Assay (ELIS A), the indirect competivive ELISA (ciELISA) detecting residues of CAP in chicken muscle tissues was developed.
The coupling ratio of chloramphenicol to BSA was 8:1 and chloramphenicol to OVA was 2:1.
In the ciELISA of polyclonal antibody(PcAb), the optimal concentration of coating antigen (CAP-OVA) was 1.7 u g /ml and the titer of anti-serum was 1:640000. The optimal working dilution of the purified PcAb was 1:20000. The linear range of calibration curves to detect CAP was 0.1ng/ml~36.45ng/ml and the limit of detection was 0.05ng/ml. The recoveries ranged from 42% to 92.80% for detecting CAP residues in chicken muscle tissues and the coefficients of variation was 4.76%~26.29%.
In this study, two hybridomas against CAP were obtained and designated ID10 and 5E6. The monoclonal antibody (McAb) was obtained by the production of ascite. Some characteristics of the McAb were detected, the results were as following: the subclass was IgG1, the titer of supernatant was 1:512, the titer of ascitic fluid was 1 :1×108, the molecular weight was 150KD and 156 KD, the chromosomal numbers were 94-105, the affinity constant was 1.26×1010L/M. Except the chloramphenicol succinate salt, no other CAP's analogues and the two carrier proteins had cross reactivity with the antibody. The antibody had excellent stability in this study. The ciELISA of McAb was developed after the purification of the ascite fluid. In the ciELISA the optimal concentration of coating antigen (CAP-OVA) was 0.85 u g /ml , the optimal working dilution of the McAb was 1:10000, the limit of detection was 0.1ng/ml, the linear range of calibration curves to detect CAP was 0.1ng/ml~25 ng/ml, the recoveries ranged from 71.01% to 127.86% for detecting CAP residues in chicken muscle tissues, and the coefficients of variation was 2.68%~16.16%.
CAP免疫抗原(CAP-BSA)的偶联比率为8∶1，包被抗原(CAP-OA)的偶联比率为2∶1。
在以多克隆抗体建立的ELISA方法中，用方阵滴定法确定包被抗原最适包被浓度为1.7μg／ml，抗血清最高效价为1∶640000，抗血清纯化后最适工作浓度为1∶20000。采用间接竞争ELISA法建立检测CAP的标准曲线，线性检测范围为0.1ng／ml～36.45ng／ml，检测限为0.05ng／ml。以0.5、1、2.5、5ng／g的浓度添加时，鸡肌肉组织中的回收率为42.00％～92.80％，变异系数为4.76％～26.29％。
在以单克隆抗体建立的ELISA方法中，用方阵滴定法确定包被抗原最适包被浓度为0.85μg／ml，腹水最高效价为1∶10~8，纯化后最适工作浓度为1∶10000。采用间接竞争ELISA法建立检测CAP的标准曲线，线性检测范围为0.1ng／ml～25ng／ml，检测限为0.1ng／g。以0.5、1、2．5、5ng／g的浓度添加时，鸡肌肉组织中回收率为71.01％～127.86％，变异系数为2.68％～16.16％。
上述2种ELISA方法快速、灵敏、方便，满足了氯霉素残留检测的要求，适用于残留大规模的快速筛选检测。
In this study, one kind of immunogen was prepared: chloramphenicol(CAP) was coupled to carrier protein (BSA) using the N-hydroxysuccinimide activated ester method. Then the anti-CAP serum were raised in rabbits and in Balb/c mice respectively. After monitoring the titer of anti-serum by the indirect Enzyme Linked ImmunoSorbent Assay (ELIS A), the indirect competivive ELISA (ciELISA) detecting residues of CAP in chicken muscle tissues was developed.
The coupling ratio of chloramphenicol to BSA was 8:1 and chloramphenicol to OVA was 2:1.
In the ciELISA of polyclonal antibody(PcAb), the optimal concentration of coating antigen (CAP-OVA) was 1.7 u g /ml and the titer of anti-serum was 1:640000. The optimal working dilution of the purified PcAb was 1:20000. The linear range of calibration curves to detect CAP was 0.1ng/ml~36.45ng/ml and the limit of detection was 0.05ng/ml. The recoveries ranged from 42% to 92.80% for detecting CAP residues in chicken muscle tissues and the coefficients of variation was 4.76%~26.29%.
In this study, two hybridomas against CAP were obtained and designated ID10 and 5E6. The monoclonal antibody (McAb) was obtained by the production of ascite. Some characteristics of the McAb were detected, the results were as following: the subclass was IgG1, the titer of supernatant was 1:512, the titer of ascitic fluid was 1 :1×108, the molecular weight was 150KD and 156 KD, the chromosomal numbers were 94-105, the affinity constant was 1.26×1010L/M. Except the chloramphenicol succinate salt, no other CAP's analogues and the two carrier proteins had cross reactivity with the antibody. The antibody had excellent stability in this study. The ciELISA of McAb was developed after the purification of the ascite fluid. In the ciELISA the optimal concentration of coating antigen (CAP-OVA) was 0.85 u g /ml , the optimal working dilution of the McAb was 1:10000, the limit of detection was 0.1ng/ml, the linear range of calibration curves to detect CAP was 0.1ng/ml~25 ng/ml, the recoveries ranged from 71.01% to 127.86% for detecting CAP residues in chicken muscle tissues, and the coefficients of variation was 2.68%~16.16%.
引文
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[2] Mildred C. Rebstoek, Harry M. Crooks, Jr., John Controulis and Quentin R. Bartz. Chloramphenicol.Ⅳ. Chemical studies. J.Am.Chem.Soc., 1949.,71:2458-2462
[3] Loren M.Long&H.D.Troutman.Chloramphenicol(chloromycetin):Ⅵ.a synthetic approach.J.Am.Chem.Soc, 1949(71):2469-2472
[4] Loren M.Long&H.D.Troutman.Chloramphenicol(chloromycetin):Ⅶ.synthesis throuth p-Nitroacetophenone. J.Am.Chem.Soc, 1949(71):2473-2475
[5] John Controulis,Mildred C.Restock&happy M.Crooks,JR. Chloramphenicol(chloromycetin):Ⅴ.synthesis. J.Am.Chem.Soc, 1949(71):2463-2468
[6] Bartz,Q.R.,J. Isolation and characterization of chloromycetin.Bioi.Chem,1948,172:445-447
[7] 李正化主编．1993，药物化学．人民卫生出版社．
[8] 沈建忠，谢联金主编．兽医药理学．中国农业大学出版社，2000
[9] 杨藻宸主编．医学药理学．人民卫生出版社，2000
[10] 张德昌主编．医学药理学．人民卫生出版社，1998
[11] 汪明性主编．新编实用药物学．科学技术出版社，2000
[12] 袁玉花．鸡蛋中氯霉素残留的检测方法及消除规律研究,2002
[13] Anadon A, Bringas f, Martinez I.arranaga M R, et al. 1994. Bioavailability pharmaeokineties and residues of chloramphenicol in the chicken. Journal of Veterinary Pharmacology and Therapeutics,17(1): 5258.
[14] 付太银．肉仔鸡氯霉素中毒．中国兽医杂志1999 25(12)：50．
[15] 施为国，汪力宽．氯霉素引起种鸡受精率下降．中国家禽，1999(21)4：30．
[16] 李端．药理学．人民卫生出版社，第四版。
[17] 杜向党，阎若潜，沈建忠．动物医学进展，2004，25(2)：27-29
[18] Cecile V B, M arie-Claude L D, Jean Louis M, et ai CAT Ⅲchloramphenicol resistance in Pasteurella haemolytica and Pasteurella multocida isolated from calves [J].J of Anti Chem, 1996, 38:205-213
[19] Mosher R H, Camp D J, Yang K Q, et al Inactivation of ehioramphenicol byO-phosphorylation[J].J ofB iol Chem, 1995,270(45):27000-27006
[20] Shaw W V,BaM D.Bacterial resistance to chloramphenicolBritish [J] I MedicalBulletin,1984,40(1):36-4L
[21] Luc B. Characterization of the nonezymatic ehloramphenicol resistance (can 1A) gene of the In4 integron of T n 1696: sffn ilarity of the product to transmembrane transport proteins.J of Bacteriology, 1991, 17304): 4493-4502
[22] George A M, Hall R M. Effiux of chloramphenicol by the Cm IA 1 protein [J] FEM SM ierob L ett,
2002, 209: 209-213.
[23] 焦彦朝，何家香，梅先芝．用微生物鉴定法测定鲤鱼肌肉组织残留氯霉素．中国饲料，2000(5)22-24．
[24] Keukens Beck.High performance liquid chromatographic screening and conformation methods for chloramphenicol residues in meat with off-line cartridge sample clean-up and on-line diode array U V-VIS detection. J. ofCh ramatogr,1986(352):445～453
[25] Nagata, Saeki. Simultaneous determination of thiampenicol, florfenieol, and ehloramphenicol residues in muscles of animals and cultured fish by liquid chromarography. J. liq chranatogr. 1992,15(12):2045-2056
[26] Degroodt J M.,W yhow ski deBukanski B.. CAP and nitrofuran residue analysis by HPLC and photodiode array detection in meat and fish.L iq Chramatogr, 1992,15(13): 2355～2371
[27] JacobsonWC,Allen EH,Wisemcn HG. Determination of Chloramphenieol in liver, kidney, muscle,and whole blood. Presented at 88th AnnualMeeting of AOAC, 1974(10): 14-17
[28] Robert K.Munns,David C.Holland,jose E.Roybal,Joseph M.Storey, and AustinR.Long, GC Determination of Chloramphenicol Residues in Shrimp:Interlaboratory Study, 1994,77:3,596～601.
[29] Allen P.Pfenning,Jose E.Roybal,Heidis Rupp,Sherri B.Tumipseed,Steve A.Gonzales,and JeffreyA.Hurlbut,Simultaneous Determination of Residues of Chloramphenicol, Florfenicol,Florfenicol Amine,and Thiamphenicol in Shrimp Tissue by GC with ECD.,J.AOAC.2000,83:26～30.
[30] 王建华，陈世山．同时测定鱼肉中氯霉素和甲砜霉素残留量的毛细管气相色谱法．分析测试学报，2001，20(3)：89～91
[31] Delepine B,Sanders P.J Chroma.,1992,582:113.
[32] Victor Hormazabal and Magne Yndestand. Simultaneous Determination of Chloramphenieol and Ketoprofen in Meat and Milk and Chloramphenicol in Egg ,Honey ,and Urine Using LC-MS.J.LC.& Rel. Technol.,2001,24:16,2477～2486.
[33] Tomoko NagaTa and Hisao Oka, Determination of Residual Chioramphenicol,Florfenicoll,and Thiamphenicol in Yellowtail Fish Muscles by Capillary GC-MS.,1996,44: 1280～1284.
[34] L.A.Ginkel,H.J.Van. Rossum,P.W.Zoontjes,H.Van Blitlersuijk, G.Ellen,E.Van.Der.Heeft,A.P.J.M.De.Jong and G.Zomer, Development and Validation of a GC-MS Procedure for the Identification and Quantification of Residues of Chlorarnphenieol, Analytical Chimica Acta.,1990,237: 61～69.
[35] Kathleen P.Holland,A.Carolyn Herry, Roger T.Wilson,and John S.Dreas.Four-laboratory Validation Study of the Determination of Chloramphenicoi in Veal CalfUrine.J AOAC.,1995,78:2,483～491.
[36] 李俊锁、邱月明、王超著兽药残留分析．上海科学技术出版社，2002
[37] Robert N. Hamburger. Chloramphenicoi-specific antibody. Science, 1966.8,152:203-205
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