Enzyme-Linked Immunosorbent Assay Development for the -Adrenergic Agonist Zilpaterol
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- 作者：Weilin L. Shelver and David J. Smith
- 刊名：Journal of Agricultural and Food Chemistry
- 出版年：2004
- 出版时间：April 21, 2004
- 年：2004
- 卷：52
- 期：8
- 页码：2159 - 2166
- 全文大小：143K
- 年卷期：v.52,no.8(April 21, 2004)
- ISSN：1520-5118
文摘
Zilpaterol is an 
-adrenergic agonist approved for use in cattle in South Africa and Mexico as a growthpromoter. It is not currently approved for use in the EU, USA, or Asia. Here, we report the developmentof an ELISA for zilpaterol. Zilpaterol was reacted with ethyl 4-bromobutyrate followed by refluxing in0.1 M potassium hydroxide. The resulting hapten was reacted with two carrier proteins, bovine serumalbumin (BSA) or keyhole limpet hemocyanin (KLH), using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as an activating agent. Immunization of goats with the zilpaterol-butyrate-KLHresulted in an antibody useful for an ELISA. We utilized zilpaterol-butyrate-BSA as a coating antigenfor ELISA development. The average IC50 derived from the developed zilpaterol immunoassay was3.94 ± 0.48 ng/mL (n = 25). The antibody did not cross react with N-alkyl [bamethane, clenbuterol,(-)-isoproterenol, (+)-isoproterenol, metaproterenol, or salbutamol] or N-arylalkyl (dobutamine,fenoterol, isoxsuprine, ractopamine, or salmeterol) -agonists. The assay was tolerant of up to 10%(v/v) of acetone, ethanol, or methanol, and 15% (v/v) of acetonitrile or DMSO. Salt concentrationsranging from 0.05 to 1.0 M minimally affected B0 or IC50 values. When buffer pH was <7 or >8.8, theIC50 values increased in comparison to those measured at pH 7.4. In conclusion, a sensitive, specificzilpaterol ELISA has been developed that can serve as a rapid screening assay.Keywords: Antibody; analysis; ELISA; assay; zilpaterol; -agonist; growth promoter
-adrenergic agonist approved for use in cattle in South Africa and Mexico as a growthpromoter. It is not currently approved for use in the EU, USA, or Asia. Here, we report the developmentof an ELISA for zilpaterol. Zilpaterol was reacted with ethyl 4-bromobutyrate followed by refluxing in0.1 M potassium hydroxide. The resulting hapten was reacted with two carrier proteins, bovine serumalbumin (BSA) or keyhole limpet hemocyanin (KLH), using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as an activating agent. Immunization of goats with the zilpaterol-butyrate-KLHresulted in an antibody useful for an ELISA. We utilized zilpaterol-butyrate-BSA as a coating antigenfor ELISA development. The average IC50 derived from the developed zilpaterol immunoassay was3.94 ± 0.48 ng/mL (n = 25). The antibody did not cross react with N-alkyl [bamethane, clenbuterol,(-)-isoproterenol, (+)-isoproterenol, metaproterenol, or salbutamol] or N-arylalkyl (dobutamine,fenoterol, isoxsuprine, ractopamine, or salmeterol) -agonists. The assay was tolerant of up to 10%(v/v) of acetone, ethanol, or methanol, and 15% (v/v) of acetonitrile or DMSO. Salt concentrationsranging from 0.05 to 1.0 M minimally affected B0 or IC50 values. When buffer pH was <7 or >8.8, theIC50 values increased in comparison to those measured at pH 7.4. In conclusion, a sensitive, specificzilpaterol ELISA has been developed that can serve as a rapid screening assay.Keywords: Antibody; analysis; ELISA; assay; zilpaterol; -agonist; growth promoter