Cloning, Expression, and Characterization of Recombinant Fab Antibodies against Dioxin

Using two hybridoma cell lines (DD1 and DD3) secreting anti-dioxin monoclonal antibodies as a source for messenger RNA and cDNA, light and heavy chain gene fragments of Fab domains were amplified by the polymerase chain reaction (PCR). The amplified gene fragments were cloned into the pFabUSDAI vector for expression of recombinant Fab antibodies in Escherichia coli. Expression of the soluble and functional recombinant Fab antibodies (designated rFab1-1 and rFab3-3) was confirmed by an indirect immunoassay using dioxin conjugated to rabbit serum albumin. On the basis of these rFabs, two competitive inhibition immunoassays using 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) as a competitor were developed. The concentration of 2,3,7,8-TCDD required to inhibit color development by 50% (IC50) determined from the dose response curves for rFAB1-1 and rFAB3-3 were 10.4 ± 2.4 and 12.2 ± 6.0 ng/mL, respectively. The binding properties of both rFab antibodies for other chemically related compounds were relatively similar to those of their respective monoclonal antibodies and enzymatically derived Fab fragments. Keywords: Dioxin; recombinant antibody; cloning antibody genes; Fab; expression vector; Escherichia coli; immunoassay