Page Banner

United States Department of Agriculture

Agricultural Research Service

Title: Cloning and Expression of Recombinant Fab Antibodies and Immunoassays for Dioxin

Authors
item Lee, Nan Ju
item Holtzapple, Carol
item Stanker, Larry

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 16, 1998
Publication Date: N/A

Interpretive Summary: Dioxin is an environmental pollutant that can get into the food chain through contaminated raw materials. For example, chickens were contaminated with dioxin because the anti-caking agent (clay) used in the feed contained dioxin. In order to prevent dioxin from being in the food that goes to market we have developed a simple and rapid test which can be used to quickly screen many samples for the presence of dioxin residues. This test is an improvement over previous tests because one of the components of the test (an antibody) can be easily altered. Therefore, in the future, tests that can detect dioxin at even lower levels can be developed using this innovative antibody. The test developed in this study will help food manufacturers and government agencies to screen and analyze for the presence of dioxin residues in foods and environmental samples.

Technical Abstract: 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 E coli. Expression of the soluble and functional recombinant Fab antibodies (designated as rFab1-1 and rFab3-3) was confirmed by an indirect immunoassay using dioxin conjugated to rabbit serum albumin. Based on 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 their respective monoclonal antibodies and proteolytic Fab fragments.

Last Modified: 10/25/2014