title: Development of a single-chain variable fragment-alkaline phosphatase fusion protein and a sensitive direct competitive chemiluminescent enzyme immunoassay for detection of ractopamine in pork.
abstract: 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.
authors: Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou 510642, China.
authors: Jie-Xian
authors: JX
authors: Dong
authors: Zhen-Feng
authors: ZF
authors: Li
authors: Hong-Tao
authors: HT
authors: Lei
authors: Yuan-Ming
authors: YM
authors: Sun
authors: Frédéric
authors: F
authors: Ducancel
authors: Zhen-Lin
authors: ZL
authors: Xu
authors: Jean-Claude
authors: JC
authors: Boulain
authors: Jin-Yi
authors: JY
authors: Yang
authors: Yu-Dong
authors: YD
authors: Shen
authors: Hong
authors: H
authors: Wang
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry*; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D005506:Food Contamination / Q000032:analysis*; D007124:Immunoenzyme Techniques; D007128:Immunoglobulin Fragments / Q000737:chemistry; D008460:Meat / Q000032:analysis*; D010627:Phenethylamines / Q000032:analysis*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013552:Swine; D053719:Tandem Mass Spectrometry
chemical_list: D007128:Immunoglobulin Fragments; D010627:Phenethylamines; D011993:Recombinant Fusion Proteins; C060897:ractopamine; D000469:Alkaline Phosphatase