盐酸克伦特罗单克隆抗体的制备及酶免疫残留检测方法的建立
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摘要
克伦特罗(Clenbuterol，CL)是一种常被非法用作促生长添加剂的β_2-受体激动剂，彻底查禁CL的非法滥用，必须要有高效、灵敏、特异的残留检测方法。本研究利用偶氮化法制备出CL完全抗原，用杂交瘤技术制备出CL单克隆抗体，建立了CL固相抗体ELISA残留检测方法，试验结果如下：
在阴暗避光4℃的条件下，用亚硝酸钠使CL发生偶氮化反应，偶氮化CL与载体蛋白酪氨酸残基上的酚羟基反应，从而生成“CL-载体蛋白”偶联物。分别制备出CL-KLH、CL-BSA和CL-OVA三种偶联物，CL-KLH与CL-BSA分别免疫小鼠制备抗血清，用CL-OVA包被酶标板，检测到的抗体可以和游离的CL发生特异性的结合反应。
用CL-KLH免疫BALB／c小鼠，连续5次，间隔3周。最后一次免疫后的第3d，无菌采取脾脏，脾细胞用聚乙二醇与SP2／O骨髓瘤细胞融合，用96孔细胞培养板，HAT 1640选择性培养基，在37℃，5％CO_2条件下半量换液培养。第11d换液时对有细胞克隆生长的培养孔，以CL-OVA包被酶标板，间接ELISA检测阳性孔，阳性孔有限稀释法连续3次克隆，最后获得了四株稳定分泌CL-McAb的杂交瘤细胞株，分别是1D6、1H5、1H7和2F10，四株杂交瘤培养上清液效价分别达到1：1100、1：1200、1：900和1：700。腹水效价都在6×10~5以上，对CL亲和力从强至弱依次为1D6＞1H5＞1H7＞2F10。对沙丁胺醇交叉反应性测定，结果最小的是1D6，只有2.32％的交叉反应件。1H5、1H7和2F10交叉反应性依次为12.9％、26％和＜5％。抗原识别位点分析结果表明，这四株McAb至少识别两个不同的抗原位点。
用辣根过氧化物酶标记CL，得到了酶标抗原HRP-CL，用CL单克隆抗体包被酶标反应板，10％兔血清封闭，侍检测样品中的CL与HRP-CL竞争CL-McAb上的结合位点，建立起固相抗体竞争ELISA，CL-McAb最佳包被浓度为1.25μg／ml，酶标抗原的最佳稀释倍数为1：400，最适检测范围为1～100ng／ml，从而建立了快速检测CL的固相抗体竞争ELISA方法。
This paper reported the preparation of CL-McAb and the development of enzyme immunoassay for the detection of clenbuterol residues.
β2- agonist clenbuterol has been used as bronchiodilators in human medicine for over 30 years. Unfortunately, this agent has also been used illegally now as growth-promoter in livestock production. Its illegal use will led to toxic effects after human consumption of meat products. The analytical method for clenbuterol residues detection is urgently needed.
Clenbuterol was a hapten. To prepare antigen, clenbuterol was conjugated to carrier proteins KLH, BSA and OVA by diazotization. CL-KLH and CL-BSA were used for the immunization of mice and CL-OVA was used as the coating antigen for the ELISA study. Both antiserums of CL-KLH and CL-BSA could bind specifically with CL. The anti-clenbuterol antibody titer of antiserum immunized with CL-KLH was higher than that with CL-BSA.
Fore hybridoma secreting monoclonal antibodies against CL were achieved by injection of BALB/c mice with CL-KLH. These four McAbs were characterized for the cross reactivities, specificities, neutralizing abilities vs. CL and relative affinity. The titer degree of ascites fluid of these four McAb was 1 : 3×106~1 : 6×105. 1D6 showed 3.98% cross reaction with Salbutamol. Relative affinity showed 1D6>1H5>1H7>2F10.
Clenbuterol was diazotized to be labelled with HRP. HRP-CL could bind with CL-McAb specifically. The samples was added in the ELISA plat wells
which were coated with CL-McAb(lD6) and blocked with 10% rabbit serum. CL in the samples could bind with CL-McAb. After washing, the HRP-CL was added and incubated for 15min at 37℃. After the plat was washed again, OPD-H2O2 was added and the reaction was terminated by adding 2M H2SO4 in 15 min. The OD490nm values was read at 490nm in a microplat reader. The contraction of CL-McAb and dilution of HRP-CL were determined. The standard curve was obtained. This immunoassay provided a analytical method for the detection of clenbuterol residues and could be use in preparation of clenbuterol ELISA kit.
在阴暗避光4℃的条件下，用亚硝酸钠使CL发生偶氮化反应，偶氮化CL与载体蛋白酪氨酸残基上的酚羟基反应，从而生成“CL-载体蛋白”偶联物。分别制备出CL-KLH、CL-BSA和CL-OVA三种偶联物，CL-KLH与CL-BSA分别免疫小鼠制备抗血清，用CL-OVA包被酶标板，检测到的抗体可以和游离的CL发生特异性的结合反应。
用CL-KLH免疫BALB／c小鼠，连续5次，间隔3周。最后一次免疫后的第3d，无菌采取脾脏，脾细胞用聚乙二醇与SP2／O骨髓瘤细胞融合，用96孔细胞培养板，HAT 1640选择性培养基，在37℃，5％CO_2条件下半量换液培养。第11d换液时对有细胞克隆生长的培养孔，以CL-OVA包被酶标板，间接ELISA检测阳性孔，阳性孔有限稀释法连续3次克隆，最后获得了四株稳定分泌CL-McAb的杂交瘤细胞株，分别是1D6、1H5、1H7和2F10，四株杂交瘤培养上清液效价分别达到1：1100、1：1200、1：900和1：700。腹水效价都在6×10~5以上，对CL亲和力从强至弱依次为1D6＞1H5＞1H7＞2F10。对沙丁胺醇交叉反应性测定，结果最小的是1D6，只有2.32％的交叉反应件。1H5、1H7和2F10交叉反应性依次为12.9％、26％和＜5％。抗原识别位点分析结果表明，这四株McAb至少识别两个不同的抗原位点。
用辣根过氧化物酶标记CL，得到了酶标抗原HRP-CL，用CL单克隆抗体包被酶标反应板，10％兔血清封闭，侍检测样品中的CL与HRP-CL竞争CL-McAb上的结合位点，建立起固相抗体竞争ELISA，CL-McAb最佳包被浓度为1.25μg／ml，酶标抗原的最佳稀释倍数为1：400，最适检测范围为1～100ng／ml，从而建立了快速检测CL的固相抗体竞争ELISA方法。
This paper reported the preparation of CL-McAb and the development of enzyme immunoassay for the detection of clenbuterol residues.
β2- agonist clenbuterol has been used as bronchiodilators in human medicine for over 30 years. Unfortunately, this agent has also been used illegally now as growth-promoter in livestock production. Its illegal use will led to toxic effects after human consumption of meat products. The analytical method for clenbuterol residues detection is urgently needed.
Clenbuterol was a hapten. To prepare antigen, clenbuterol was conjugated to carrier proteins KLH, BSA and OVA by diazotization. CL-KLH and CL-BSA were used for the immunization of mice and CL-OVA was used as the coating antigen for the ELISA study. Both antiserums of CL-KLH and CL-BSA could bind specifically with CL. The anti-clenbuterol antibody titer of antiserum immunized with CL-KLH was higher than that with CL-BSA.
Fore hybridoma secreting monoclonal antibodies against CL were achieved by injection of BALB/c mice with CL-KLH. These four McAbs were characterized for the cross reactivities, specificities, neutralizing abilities vs. CL and relative affinity. The titer degree of ascites fluid of these four McAb was 1 : 3×106~1 : 6×105. 1D6 showed 3.98% cross reaction with Salbutamol. Relative affinity showed 1D6>1H5>1H7>2F10.
Clenbuterol was diazotized to be labelled with HRP. HRP-CL could bind with CL-McAb specifically. The samples was added in the ELISA plat wells
which were coated with CL-McAb(lD6) and blocked with 10% rabbit serum. CL in the samples could bind with CL-McAb. After washing, the HRP-CL was added and incubated for 15min at 37℃. After the plat was washed again, OPD-H2O2 was added and the reaction was terminated by adding 2M H2SO4 in 15 min. The OD490nm values was read at 490nm in a microplat reader. The contraction of CL-McAb and dilution of HRP-CL were determined. The standard curve was obtained. This immunoassay provided a analytical method for the detection of clenbuterol residues and could be use in preparation of clenbuterol ELISA kit.
引文
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[9] Guy PA, Savoy MC, Stadler RH. Quantitative analysis of cienbuterol in meat products using liquid chromatography-electrospray ionisation tandem mass spectrometry. J Chromatogr B Biomed Sci Appl, 1999, 24,736(1-2):209～219.
[10] 魏德成，郭俊峰，杨冀州．供港活猪“瘦肉精”残留检测方法及其重要意义．河南畜牧兽医．2000，21(2)：35～36．
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[3] Vela J, Yanes EG, Stalcup AM. Quantitative determination of clenbuterol, salbutamol and tulobuterol enantiomers by capillary electrophoresis. Fresenius J Anal Chem, 2001,369(3-4):212～219.
[4] Toussaint B, Hubert PH, Tjaden UR, et al. Enantiomeric separation of clenbuterol by transient isotachophoresis-capillaryzone electrophoresis-UV detection new optimization technique for transient isotachophoresis. J Chromatogr A 2000,25,871(1-2):173～180.
[5] Tsai CE, Kondo F. Liquid chromatographic determination of salbutamol and clenbuterol residues inswine serum and muscle. Microbios, 1994, 80(325): 251～258.
[6] 林海丹，江庆娣，伍绍登．饲料中盐酸克伦特罗含量的测定．饲料工业，2000，21(1)：25～26．
[7] Doerge DR, Bajic S, Lowes S. Analysis of clenbuterol in human plasma using liquid chromatography/atmospheric-pressure chemical-ionization mass spectrometry. Rapid Commun Mass Spectrom, 1993,7(6):462～464.
[8] Crescenzi C, Bayoudh S, Cormack PA, et al. Determination of clenbuterol in bovine liver by combining matrix solid-phase dispersion and molecular imprinted solid-phase extraction followed by liquid chromatography/electrospray ion trap multiple-stage mass spectrometry. Anal them,2001,15,73(10):2171～2177.
[9] Guy PA, Savoy MC, Stadler RH. Quantitative analysis of cienbuterol in meat products using liquid chromatography-electrospray ionisation tandem mass spectrometry. J Chromatogr B Biomed Sci Appl, 1999, 24,736(1-2):209～219.
[10] 魏德成，郭俊峰，杨冀州．供港活猪“瘦肉精”残留检测方法及其重要意义．河南畜牧兽医．2000，21(2)：35～36．
[11] PloumME, Haasnoot W, Paulussen RJ, et al. Test strip enzyme immunoassays
and the fast screening of nortestosterone and clenbuterol residues in urine samples at the parts per billion level. J Chromatogr, 1991, 5, 564 (2):413～427.
[12] Haasnoot W, Streppel L, Cazemier G, et al. Development of a tube enzyme immunoassay for "on-site' screening of urine samples in the presence of beta-agonists. Analyst, 1996,121(8):1111～1114.
[13] Shelver WL, Smith DJ. Evaluation of commercial immunoassays for cross-reactivity to clenbuterol stereoisomers and bovine metabolites. Food Addit Contam, 2000,17(lO):837～845.
[14] Bacigalupo MA, Ius A, Meroni G, et al. Comparison of time-resolved fluoroimmunoassay and immunoenzymometric assay for clenbuterol. Analyst, 1995, 120(8):2269～2271.
[15] Adam A, Ong H, Sondag D, et al. Radioimmunoassay for albuterol using a monoclonal antibody: application for direct quantification in horse urine. J Immunoassay, 1990,11(3):329～345.
[16] Collins S, O'Keeffe M, Smyth MR. Multi-residue analysis for beta-agonists in urine and liver samples using mixed phase columns with determination by radioimmunoassay. Analyst, 1994,119(12):2671～2674.
[17] Haasnoot W, Stouten P, Schilt R, et al. A fast immunoassay for the screening of beta-agonists in hair. Analyst, 1998,123(12):2707～2710.
[18] Lawrence JF, Menard C.Determination of clenbuterol in beef liver and muscle tissue using immunoaffinity chromatographic cleanup and liquid chromatography with ultraviolet absorbance detection. J Chromatogr B Biomed Sci Appl, 1997, 29, 696(2):291～297.
[19] Haughey SA, Baxter GA, Elliott CT, et al. Determination of clenbuterol residues in bovine urine by optical immunobiosensor assay. J AOAC Int, 2001, 84(4): 1025～1030.
[20] 白雪帆、杨为松等．ELISA方法用于McAb亲和常数的测定。免疫学杂志，1992，8(2)：126～128。
[21] J. P. Briand, S. Muller and M.H.V. Van Regenmortel. Symthetic peptides as antigens: pitfalls of conjugation methods. Journal of immunological methods. 1985, 78: (59～69).
[22] 万文徽．单克隆抗体亲和常数的测定。单克隆抗体通迅，1993，9(2)：(72～75)。
[23] 陈继明，韩正康，龚晓明等。两种β_2-受体激动剂快速免疫检测方法。中国兽医
科技，1998，28(9)：5～7。
[24] 聂国荣。β-兴奋剂正副作用及检测方法。中国动物检疫，2001，18(6)：43～44。
[25] 王选年，康晓迪，潘耀谦。盐酸克伦特罗的残留及检测。动物医学进展，2002，23(3)：41～46。
[26] 方柄虎，陈杖榴，黄显会。克伦特罗在猪体内的生物利用度及药物动力学研究。畜牧兽医学报，1997，28(3)：233～237
[27] 陈茹，吴时清，林志雄。克伦特罗及其检测技术。动植物检疫，1999(2)：29～32。
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