期刊
ANALYTICA CHIMICA ACTA
卷 736, 期 -, 页码 85-91出版社
ELSEVIER
DOI: 10.1016/j.aca.2012.05.033
关键词
Ractopamine; Single-chain variable fragment; Fusion protein; Chemiluminescent enzyme immunoassay; Pork
资金
- National Natural Science Foundation of China [30871755]
- China Guangdong Provincial Science and Technology Project [2009A020101004]
A rapid, sensitive chemiluminescent enzyme immunoassay (CLEIA) for ractopamine (RAC) based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein was developed. The scFv gene was prepared by cloning the heavy- and light-chain variable region genes (V-H and V-L) from hybridoma cell line AC2, which secretes antibodies against RAC, and assembling V-H and V-L genes with a linker by means of splicing overlap extension polymerase chain reaction. The resulting scFv gene was inserted into the expression vector pLIP6/GN containing AP to produce the fusion protein in Escherichia coli strain BL21. The purified scFv-AP fusion protein was used to develop a direct competitive CLEIA (dcCLEIA) protocol for detection of RAC. The average concentration required for 50% inhibition of binding and the limit of detection of the assay were 0.25 +/- 0.03 and 0.02 +/- 0.004 ng mL(-1), respectively, and the linear response range extended from 0.05 to 1.45 ng mL(-1). The assay was 10 times as sensitive as the corresponding enzyme-linked immunosorbent assay based on the same fusion protein. Cross-reactivity studies showed that the fusion protein did not cross react with RAC analogs. DcCLEIA was used to analyze RAC spiked pork samples, and the validation was confirmed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS). The results showed a good correlation between the data of dc-CLEIA and HPLC-MS (R-2 > 0.99), indicating that the assay was an efficient analytical method for monitoring food safety. (c) 2012 Elsevier B.V. All rights reserved.
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