对氯甲基苯甲酸链接的莱克多巴胺人工抗原的合成与鉴定

食品科学 ›› 2013, Vol. 34 ›› Issue (1): 180-184.

对氯甲基苯甲酸链接的莱克多巴胺人工抗原的合成与鉴定

陈臣1，罗顺菁1,*，刘成梅1，邹常春2，汪志宇1，王文飞1

1.南昌大学食品科学与技术国家重点实验室 2.南昌大学资产管理处

收稿日期:2011-10-09 修回日期:2012-12-25 出版日期:2013-01-15 发布日期:2013-01-07
通讯作者: 罗舜菁 E-mail:luoshunjing@yahoo.com.cn
基金资助:
江西省科技支撑计划项目

Synthesis and Characterization of Ractopamine Antigen Linked by 4-Methylbenzoic Acid

Received:2011-10-09 Revised:2012-12-25 Online:2013-01-15 Published:2013-01-07

摘要/Abstract

摘要： 采用对氯甲基苯甲酸(CBA)为链接臂，用混合酸酐法将莱克多巴胺(RAC)与牛血清白蛋白(BSA)偶联制备人工抗原(RAC-CBA-BSA)，通过紫外、红外、电泳鉴定抗原合成成功；再以卵清蛋白(OVA)为载体蛋白合成包被抗原(RAC-CBA-OVA)，测定其与抗体的亲和力和抑制率。间接竞争ELISA结果以对CBA为链接臂合成的包被抗原对应的抗体滴度为7047.3、IC50为17.05ng/mL，结果表明，CBA可以用于RAC人工抗原的合成，使用RAC-CBA-OVA作为包被抗原可以提高ELISA检测灵敏度。

关键词: 莱克多巴胺, 对氯甲基苯甲酸, ELISA灵敏度, 人工抗原

Abstract: The mixed anhydride method was used to prepare artificial antigen through the conjugation between ractopamine (RAC) and bovine serum albumin (BSA) in the presence of 4-chloromethyl benzoic acid (CBA). The prepared artificial antigen was identified by IR, UV and SDS-PAGE. RAC-CBA was also conjugated with OVA to form a coating antigen. The affinity and inhibitory activity of the coating antigen were measured. This study suggests that CBA can be used to prepare artificial RAC antigens. Meanwhile, CBA conjugated with RAC to form coating agent improves the sensitivity of ELISA.

Key words: ractopamine, 4-chloromethyl benzoic acid, ELISA sensitivity, artificial antigens

中图分类号:

S 859.84

陈?臣1，罗顺菁1,*，刘成梅1，邹常春2，汪志宇1，王文飞1. 对氯甲基苯甲酸链接的莱克多巴胺人工抗原的合成与鉴定[J]. 食品科学, 2013, 34(1): 180-184.

参考文献

[1]SHELVER W L,SIMINTH D J,BERRY E S.Production and charac-terization of a monoclonal antibody against theβ-adrenergic agonist ractopamine[J].Journal of Agricultural and Food Chemistry,2000(48):4020-4026.
[2]GABIOLA C,CALONGE M A G,PORTILLO M P,et al.Validation of a method for the determination of salbutamol in animal urine by gas chromatography-mass spectrometry and its application to treated lamb samples[J].J Microcolumn Separations,1996(8):361-364.
[3]VANOOSTHUYZE K E I,ARTS C J M,PETEGHEM C H V.Devel-opment of a fast and simple method for determination of β-agonists in urine by extraction on empore membranes and detection by a test strip immunoassay[J].Journal of Agricultural and Food Chemistry,1997(45):3129-3137.
[4]SHELVER W L,SMITH D J.Determination of ractopamine in cattle and sheep urine samples using an optical biosensor analysis:compara-tive study with HPLC and ELISA[J].Journal of Agricultural and Food Chemistry,2003(51):3751-3721.
[5]SHEN S H,OYANG J,BAEYENS W R G,et al.Determination of β-agonists by ion chromatography with direct conductivity detection[J].J Pharmaceutical and Biomedical Analysis,2005(38):166-172.
[6]REGUEIRO J A G,PEREZ B,CASADMONT G.Determination of clenbuterol and salbutamol in urine by capillary gas chromatography with capillary columns of 100μm[J].J Chromatogr A,1993(655):73-76.
[7]POLETTINIA,MONTAGNAM,HONGENDOORNEA,etal.Applica- bility of coupled-column liquid chromatography to the analysis of β-agonists in urine by direct sample injection 1.Development of a single-residue reversed-phase liquid chromatography-UV method for clenbuterol and selection of chromatographic conditions suitable for multi-residue analysis[J].J Chromatogr A,1995(695):19-31.
[8]JOSEFSSON M,SABANOVIC A.Sample preparation on polymeric solid phase extraction sorbents for liquid chromatographic-tandem mass spectrometric analysis of human whole blood-A study on a number of beta-agonists and beta-antagonists[J].J Chromatogr A,2006(1120):1-12.
[9]SPYRIDAKI M H,VONAPARTI P K A,VALAVIANI P.et al.Doping control analysis in human urine by liquid chromatography-electrospray ionization ion trap mass spectrometry for the Olympic Games Athens 2004:Determination of corticosteroids and quantification of ephedrines, salbutamol and morphine[J].Anal Chim Acta,2006(573):242-249.
[10] KIM Y J, CHO Y A, LEE H-S, et al. Investigation of the effect of hapten heterology on immunoassay sensitivity and development of an enzyme-linked immunosorbent assay for the organophosphorus insecticide fenthion [J]. Analytica Chimica Acta, 2003, 494(1-2): 29-40.
[11] Tijssen P.Practice and theory of enzyme immunoassays [M].New York:Elsevier Amsterdam,1985.27.
[12] Sambrook J,Russell D W.Molecular cloning:A laboratory manul(3rd ed.)[M].New York:Cold Spring Harbor Labora-toryPress,2002.
[13]陈奎治，彭亦如，林伟，等.光谱分析法研究八羧酸酞菁铝配合物与牛血清白蛋白的结合作用［J］.光谱学与光谱分析，2007(27):84.
[14]ZHANG Q，WANG L，AHN K C，et al.Hapten heterology for a specific and sensitive indirect enzyme－linked immunosorbent assay for organophosphorus insecticide fenthion［J］.Analytica Chimica Acta，2007，596(2):303－311.
[15]BUTLER J E.Enzyme－Linked Immunosorbent Assay［J］. Journal of Immunoassay，2000，21(2):165－209.
[16]Landsteiner K. The specificity of serological reactions [M]. Boston:Harvard University Press, 1945: 78-86.
[17]Schneider P,Hammock B D[J].A g ric.Food Chem.,1992,40(3):525～530
[18]罗舜菁，钟寒燕，刘成梅，等.氯霉素免疫抗原及包被抗原的制备［J］.食品科学，2010，383(10):91－94.