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ARS Home » Research » Publications at this Location » Publication #139685
Title: DEVELOPMENT OF RECOMBINANT ANTI-DIOXIN ANTIBODIES AND EFFECTS OF LIGHT CHAIN REARRANGEMENTS ON LIGAND BINDING

Author
item Stanker, Larry
item NANGU, LEE - XXXXXX
item Holtzapple, Carol

Submitted to: Analytica 2000
Publication Type: Abstract Only
Publication Acceptance Date: 9/11/2002
Publication Date: N/A
Citation: N/A

Interpretive Summary: Since the development of hybridoma technology in 1975, monoclonal antibodies (Mabs) have been successfully used in the development of numerous assays for detection and measurement of small molecules such as pesticides, drugs, and toxic chemicals. Previously we developed Mab-based for polychlorinated dibenzo-p-dioxins. The Mabs bound selected di-, tri-, tetra- (including 2,3,7,8-TCDD), and pentachloro Dioxi isomers. Using the DNA sequence of the Mabs (previously determined), we generated specific PCR primers with desired restriction sites and used these to amplify and clone the Fab portion of these antibodies into the expression vector pfabUSDA1. Recombinant Fab (rFAB) for both DD-1 and DD-3 were expressed and DNA sequences were identical to earlier sequences determined from cDNA libraries. Parental DD-1, rFAB-DD1, and enzymatically derived DD-1 Fab fragments showed comparable binding (IC50 values in a competition ELISA using 2,3,7,8-TCDD were 0.01,) .015, and 0.025 g TCDD/ml respectively). Similar results were obtained with the recombinant DD-3 antibodies. Likewise, the parental and recombinant molecules have similar reactivity profiles.

Technical Abstract: Since the development of hybridoma technology in 1975, monoclonal antibodies (Mabs) have been successfully used in the development of numerous assays for detection and measurement of small molecules such as pesticides, drugs, and toxic chemicals. Previously we developed Mab-based for polychlorinated dibenzo-p-dioxins. The Mabs bound selected di-, tri-, tetra- (including 2,3,7,8-TCDD), and pentachloro Dioxi isomers. Using the DNA sequence of the Mabs (previously determined), we generated specific PCR primers with desired restriction sites and used these to amplify and clone the Fab portion of these antibodies into the expression vector pfabUSDA1. Recombinant Fab (rFAB) for both DD-1 and DD-3 were expressed and DNA sequences were identical to earlier sequences determined from cDNA libraries. Parental DD-1, rFAB-DD1, and enzymatically derived DD-1 Fab fragments showed comparable binding (IC50 values in a competition ELISA using 2,3,7,8-TCDD were 0.01,) .015, and 0.025 g TCDD/ml respectively). Similar results were obtained with the recombinant DD-3 antibodies. Likewise, the parental and recombinant molecules have similar reactivity profiles.