Search

An anti-idiotype monoclonal antibody (Mab) able to bind the organophosphate pesticides, chlorfenvinphos (CFV), ethyl paraoxon, tetrachlorfenvinphos and demeton-s-methyl, has been produced using as immunogen a Mab which binds to the active site of cutinase. The principle of using an anti-idiotype antibody as the mimic of a site on a protein able to bind a group of ligands has, therefore, been demonstrated, and may have implications for future research on broad specificity immunoanalysis of groups of compounds.

The application of an inert membrane-based, enzyme-linked immunofiltration assay (ELIFA) to the characterization of immunosorbents suitable for flow immunosensor development is described. For direct assays, eight monoclonal antibodies (MAb) raised against the insecticide carbaryl were immobilized on three sorbents, namely, controlled pore glass (CPG), hydrazide derivatized agarose beads and a hydrophilic polymer with immobilized Protein A/G. The interaction between immobilized antibodies and antigen was directly detected using a carbaryl hapten conjugated to horseradish peroxidase. Immunosorbent characterization was based on both sensitivity and re-usability. Optimal immunosorbent regeneration was achieved using 0.1 M glycine/HCl, pH 2.0 as the desorbent solution. The best covalent immunosorbent was obtained by immobilizing LIB-CNA36 MAb on hydrazide derivatized agarose beads. The best immunosorbent obtained by reversible immobilization was LIB-CNH45 MAb on Protein A/G. Using this support the eventual irreversible denaturation of covalently immobilized MAbs was overcome. For indirect assays, N-hydroxisuccinimide derivatized agarose beads and glutaraldehyde-activated CPG were used as sorbents for hapten immobilization via the amino groups of a carrier protein. In this format, antigen-MAb interactions were detected using a peroxidase-conjugated rabbit anti-mouse immunoglobulin. The highest sensitivity was achieved by LIB-CNH45 MAb in combination with derivatized agarose beads. All these results demonstrated the suitability of ELIFA as a fast, precise and easy-to-use technique for immunosorbent selection.

Monoclonal murine anti-pesticide antibodies were produced by in vitro immunisation (IVI) of cultured splenocytes with the pesticides sulcofuron and flucofuron. The majority of both anti-flucofuron and anti-sulcofuron antibodies obtained were of the IgM isotype, rather than IgG. When used in an indirect enzyme-linked immunosorbent assay (ELISA), the antibodies bound to plate coating antigens which incorporated haptens that mimicked moieties present within the immunising pesticide. The antibodies exhibited a high degree of specificity, with the degree of cross-reactivity related to the structural similarity between the hapten present in the plate coating antigen and the moieties present within the immunising pesticide. These results indicated that antibodies specific to both sulcofuron and flucofuron had been produced by IVI. Synthesis of both hapten analogues and immunogens as required for methods based on in vivo immunisation was avoided, whilst antibody production was also comparatively more rapid than traditional methods and minimised animal discomfort.