Page Banner

United States Department of Agriculture

Agricultural Research Service

Title: Chain Shuffling: Improved Sensitivity of Dioxin Competitive Immunoassay

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

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: December 11, 1997
Publication Date: N/A

Technical Abstract: Immunoassays for major agrochemicals have proven to be very useful analytical tools and screening methods. Some exhibit high sensitivity and the desired specificity, while others require much improvement, mainly due to the difficulties in synthesizing the "correct" hapten. Antibody engineering technology has the potential to improve the affinity of existing antibodies via molecular manipulations, thus providing an alternative to the conventional hapten synthesis approach for altering the binding properties of an antibody. We expressed two recombinant Fab fragments in Escherichia coli. The recombinant Fabs were derived from two hybridoma cell lines, DD1 and DD3, that secrete antibodies against the environmental pollutant dioxin. Using competitive inhibition ELISAs (cELISA), where 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) was used as a competitor, IC50 values of 10.4 +/- 2.4 ng/mL and 12.2 +/- 6.0 ng/mL for the rFab1-1 and rFab3-3 (derived from Mab DD1 and DD3, respectively) were observed. Experiments were performed in which the heavy (H) and light (L) chains of the rFabs were shuffled. Combining the H-chain of DD3 with the light-chain of DD1 (rFab3-1) resulted in an improved (three-fold) relative affinity as measured in our cELISA. These experiments demonstrate that chain shuffling offers an alternative for creating an antibody with altered affinity. The characterization of rFabs for sensitivity and specificity against dioxin congeners and related chlorinated compounds, and comparison of their binding profiles with those of parent antibodies and proteolytic Fab fragments also were studied and will be discussed.

Last Modified: 9/21/2014
Footer Content Back to Top of Page