Toxicity assessment and on‐line monitoring: Immunoassays

Abstract

Pesticides and environmental pollutants are traditionally analyzed by high performance liquid chromatography, gas chromatography, or gas chromatography/mass spectrometry. The rigorous drinking water ordinances of various European countries enforce the development of fast screening methods. Immunoassays (IAs) gain growing importance as a screening tool in pesticide residue analysis. Improvements of antibody quality over the last decade has enabled the construction of highly sensitive enzyme immunoassays (EIAs) for pesticides with detection limits in the lower ppt range. The feature of an IA depends upon the selectivity and affinity of the antibodies (Ab) applied for these tests. Polyclonal and monoclonal systems can both be used to produce high-quality Ab. The choice of the method depends on the scope and intention for Ab application. Polyclonal antibodies (pAb) may provide excellent reagents for detection systems such as immunoassays. Problems arising from the limited amounts can be overcome to a certain degree by using animals providing larger amounts of sera, e.g., sheep or goats. The pAb, however, are heterogeneous with respect to affinity and analyte recognition. A wide distribution of IAs in residue analysis can profit from the monoclonal antibody (mAb) technology. Apart from the greater expense connected to mAb production, it offers the desired supply of antibodies with defined biological properties during a prolonged period of time. This technique is based on the fusion of antibody-secreting Blymphocytes from immunized mice with myeloma cells. The resulting fusion products (hybridomas) will grow in culture and may continue to secrete Ab. After a careful screening and subcloning process, stable hybridoma cell lines can usually be selected that secrete the desired mAb. All IAs depend upon the measurement of binding site occupancy by the analyte as the common basic principle because the occupancy of Ab binding sites by the analyte reflects the analyte concentration in the medium. Since the binding reaction itself does not produce a signal, a helper reagent or tracer is added in order to estimate Ab occupancy by measuring the tracer signal. Enzyme tracers benefit from signal amplification, because of the catalytic activity of their enzymes. Competitive IAs can be applied for the quantification of pesticides and environmental pollutants. They determine the unoccupied sites and use limiting Ab concentrations. Examples are given for the assay of s-triazines. Cross-reactivities and matrix effects are the most common sources of error. A significant step during the establishment of IAs for routine analyses is validation by independent methods. Interlaboratory tests play a decisive role. Under certain conditions, such as the participation of trained personnel in certified laboratories, IAs represent rapid, relatively simple, and reliable screening procedures, which make a valuable contribution to the analysis of pesticides. A practical guide specifies the steps required for atrazine determinations with mAb and pAb. It also includes details for the production of pAb for atrazine. © 1994 by John Wiley & Sons, Inc..