Stephen D. Garrett

A nonisotopic estrogen receptor-based assay to detect estrogenic compounds

We have used the ligand binding domain of the recombinant human estrogen receptor (hER) to develop a nonisotopic assay for detection of estrogenic compounds. The assay is based on competition of the estrogenic ligand with 17β-estradiol for binding to the receptor, which leaves 17β-estradiol free to bind to an anti–17β-estradiol antibody. Unbound anti–17β-estradiol antibody then binds to immobilized 17β-estradiol–protein conjugate (to which hER is unable to bind for steric reasons), and is detected by an enzyme-labeled anti-rabbit IgG antibody. We used the assay to detect estrogenic compounds (mainly members of the flavonoid group of plant polyphenols) in a variety of commonly consumed plant foods.

Characterization of the interactions between immobilized parathion and the corresponding recombinant scfv antibody using a piezoelectric biosensor

A piezoelectric quartz crystal sensor with immobilized parathion was used for real‐time kinetic characterization of the interactions with recombinant anti‐parathion single chain Fv antibody IFRN AA01 (scFv). Parathion was linked to the sensor gold electrodes modified with a self‐assembled layer of aminothiophenol using either bovine serum albumin (BSA) or dextran as spacer molecules. The kinetic dissociation rate constant kd was 8 × 10−4 s−1 for both types of sensors, and the association rate constants ka, were 590 and 260 mol−1 1 s−1 for BSA and dextran‐linked parathion, respectively. The regeneration of BSA‐parathion coated crystals was not successful, however. Those crystals with bound scFv were used to study the formation of scFv dimers. Dextran‐parathion modified crystals were successfully regenerated using proteinase K. The affinity of scFv to dextran‐parathion was 50 X lower when compared with the affinity of the anti‐parathion monoclonal antibody (IgG, IFRN 1701) the sequence of which served for p...

Alteration of the Binding Characteristics of a Recombinant scFv Anti-parathion Antibody - 1. Mutagenesis Targeted at the V H CDR 3 Domain

A single-chain variable fragment (scFv) recombinant antibody against the organophosphorous insecticide parathion has been used to generate a set of mutants. Single point mutations were introduced into positions 97-100b of the third complementarity-determining region (CDR) of the heavy chain using oligonucleotide-directed mutagenesis. The mutant scFvs were characterised in an ELISA for parathion and related pesticides. Three groups of clones with altered properties were identified: those which lost all ability to bind to the solid-phase pesticide conjugate in the ELISA; those with reduced binding to the solid phase conjugate, and those with changed ELISA sensitivity to free pesticide. supstitutions into positions 100a and 100b were particularly represented in the first group, indicating that these are either important residues for binding of parathion or that they adversely affect folding of the scFv. The mutation tyrosine to histidine had very different effects depending on the position of the tyrosine in...

Alteration of the Binding Characteristics of a Recombinant scFv Anti-parathion Antibody - 2. Computer Modelling of Hapten Docking and Correlation with ELISA Binding

Recombinant antibody technology and the ability to manipulate antibody binding characteristics offers much potential to agri-food analysis, and in particular to the generation of antibodies against pesticides. As part of the development of a mutagenesis strategy aimed at changing antibody specificity, the sequence of an scFv recombinant antibody specific to ethyl-parathion has been used to generate a molecular model using ProMod software. Docking studies showed the involvement in the binding pocket of CDR H 3 , as expected, but also of CDR L 2 and of small areas of the framework region of the light chain, based on the Kabat definition of CDRs. The majority of the L 2 contacts do, however, fall within a recently proposed re-definition of L 2 . The validity of the model was supported by comparing docking studies of structurally related pesticides with cross-reaction data from an ELISA.