Development of Robust, Environmental, Immunoassay Formats for the Quantification of Pesticides in Soil

Abstract

The use of antibodies, or more specifically recombinant antibody fragments (scAbs), for the immunodetection of pesticides, could provide a rapid, reliable, and robust method of environmental analysis. The inherent instability of some antibody structures in nonphysiological conditions, however, limits the utility of this approach. We have developed a generic strategy for the stabilization of scAbs in polar and nonpolar solvents, and in a range of other denaturing environments, by the introduction of an interdomain disulfide bond, generating stabilized antibody fragments or stAbs. StAbs specific for the phenoxyacid herbicide mecoprop and the phenylurea herbicide diuron were fully functional when compared to their equivalent unmodified scAbs but significantly more stable in nonphysiological conditions associated with the extraction of organics from environmental samples. Antiatrazine, mecoprop, diuron, and paraquat stAbs were successfully used to accurately identify and quantify pesticides present in solvent and aqueous extracted soil samples. Immunoassay data were validated by HPLC. Recombinant stAbs can be produced readily, cost-effectively, and in quantity from Escherichia coli expression systems and provide robust reagents for immunoassay detection of pollutants present in complex environmental matrices.

Rapid Isolation of a Single-Chain Antibody against the Cyanobacterial Toxin Microcystin-LR by Phage Display and Its Use in the Immunoaffinity Concentration of Microcystins from Water

“…Alternative elution methods could be investigated to determine whether column regeneration is possible. It may also be possible to stabilize the scAb through introduction of an interdomain disulfide bond, which has previously been shown to improve the performance of recombinant antibody fragments in methanol (7,35). However, as it is possible to express large quantities of recombinant scAbs in E. coli at relatively low cost, the development of reusable columns may not be necessary.…”

“…Antibody fragments selected against particular antigens can then be expressed in the bacterial host Escherichia coli in considerably larger amounts than whole antibodies produced by traditional methods. While representing Ͻ20% of the size of an intact antibody, fragments consisting of linked antibody heavy-and lightchain variable domains (scFv) have been shown to retain specificity and sensitivity for the target antigen (35). Furthermore, scFv fragments can be modified to increase their stability under nonphysiological conditions, including methanol (7,35), which is used routinely to extract microcystins from cyanobacterial samples.…”

Immunochemical detection of emerging organic contaminants in environmental waters

“…At the same time, recombinant antibody technology shows many advantages over traditional antibodies in the following aspects: (i) Recombinant antibodies can be adjusted through molecular biology techniques for expression, posttranslational modification, optimization in affinity and specificity according to the application [45]; (ii) Recombinant antibodies can be generated in vitro via phage and bacterial display systems, making the selection and screening procedure flexibly [46]. Furthermore, recombinant antibodies can tolerate a range of conditions in solutions, such as acidic or basic pH, high levels of heavy metal ions, organic solvents at concentrations over 10%, all of which would denature conventional antibodies [47]. The robust nature of recombinant antibodies greatly broadens their application in immunoassays for EOCs analysis, because there might be some EOCs in industrial waste waters containing high concentration of heavy metals or organic wastes.…”

Determination of pesticides by enzyme immunoassay

“…It is now possible to clone not only antibody molecules but also their fragments containing the site of binding with an antigen (Fab fragments, the hypervariable regions of antibodies) in simple cell systems such as E. Coli [122]. Recombinant antibody technologies make it possible to obtain more specific antibodies, to reduce nonspecific protein interactions, and to vary other properties of antibodies: to increase affinity, to change antigenic specificity, and to improve the sensitivity of immunoassay and the stability of antibodies [123,124]. For example, recombinant antibodies have been used in the determination of atrazine [123] and chlorpyrifos [116].…”