Production of scFv recombinant fragments against 2,4-dichlorophenoxyacetic acid hapten using naďve phage library
J. Brichta, H. Vesela, M. Franekhttps://doi.org/10.17221/5776-VETMEDCitation:Brichta J., Vesela H., Franek M. (2003): Production of scFv recombinant fragments against 2,4-dichlorophenoxyacetic acid hapten using naďve phage library. Veterinarni Medicina, 48: 237-247.
Three single chain variable fragment (scFv) antibodies against 2,4-dichlophenoxyacetic acid (2,4-D) herbicide were produced by the Griffin1.library. The selection of the scFv from the phage library was carried out by 2,4-D-protein coated tubes with different levels of hapten substitution in the conjugate. The scFv phage clones were isolated within the five round library panning and the antibodies were expressed in Escherichia coli HB2151. The recombinant products were purified by metal affinity chromatography yielding 200 g of pure scFv per 1 liter of bacterial culture. The antibody fragments provided steep curves in conventional indirect ELISA having the IC50 values from 10.2 to 14.5 ng/ml established for 2,4-D standard. Interestingly enough, the recombinant ScFv E1 antibody exhibited 68% cross-reactivity with 2,4-dichlorphenol (2,4-D = 100%), and 38.0% with methylchlorophenoxyacetic acid (MCPA) whereas reaction with other phenoxyacetic compounds was low. Similar characteristics were obtained for other two recombinant products. Low stability for the isolated scFv antibodies was found in storage buffer even in the presence of stabilizers and protease inhibitors. Factors influencing stability of the recombinant antibodies are discussed.Keywords:
naďve phage library; selection; phage antibody; 2,4-D hapten; antibody stability