Location: Food and Feed Safety ResearchTitle: Production and characterization of a single-chain variable fragment linked alkaline phosphatase fusion protein for detection of O,O-diethyl organophosphorus pesticides in a one-step enzyme-linked immunosorbent assay) Author
Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer reviewed journal
Publication Acceptance Date: 4/25/2012
Publication Date: 4/25/2012
Publication URL: http://handle.nal.usda.gov/10113/57270
Citation: Xu, Z., Dong, J., Wang, H., Li, Z., Beier, R.C., Jiang, Y., Lei, H., Shen, Y., Yang, J., Sun, Y. 2012. Production and characterization of a single-chain variable fragment linked alkaline phosphatase fusion protein for detection of O,O-diethyl organophosphorus pesticides in a one-step enzyme-linked immunosorbent assay. Journal of Agricultural and Food Chemistry. 60:5076-5083. Interpretive Summary: This work demonstrates a unique way to produce an enzyme-linked immunosorbent assay (ELISA) (similar to a pregnancy test strip) that uses the variable portions of antibodies combined with a reporter system to determine O, O-diethyl organophosphorus pesticides in cabbage and river water samples. Antibodies are substances that are produced by the immune system in response to foreign substances which enter the body. Once the antibodies to a foreign substance are isolated, they can be used in a method to detect the presence of that foreign substance. Antibodies are defensive proteins that are made up of two main types of regions. Antibodies have a conserved region that is very much the same for all antibodies and a variable region that allows antibodies to specifically bind other chemicals. The work here targeted producing only the variable region of antibodies, combined with the required reporter system, which were then used to develop an ELISA for the detection of O,O-diethyl organophosphorus pesticides that exhibited good sensitivity and reproducibility.
Technical Abstract: A single-chain variable fragment (scFv) and alkaline phosphatase (AP) fusion protein for detection of O, O-diethyl organophosphorus pesticides (OPs) was produced and characterized. The scFv gene was prepared by cloning VL and VH genes from a hybridoma cell secreting monoclonal antibody with broad-specificity for O, O-diethyl OPs. The amplified VL and VH regions were assembled using linker (Gly4Ser) by means of splicing overlap extension polymerase chain reaction to obtain the scFv gene, which was cloned into the expression vector pLIP6/GN containing an AP gene to produce the scFv-AP fusion protein in Escherichia coli strain BL21. The protein was purified by antigen-conjugated immunoaffinity chromatography and characterized by SDS-PAGE, western blotting. and competitive direct enzyme-linked immunosorbent assay (cdELISA). The fusion protein is bi-functional, retaining both antigen binding capacity and AP enzymatic activity. Recovery studies from spiked river water and Chinese cabbage samples demonstrated that the fusion protein-based cdELISA exhibited good sensitivity and reproducibility.