Thomas Giersch

Production of monoclonal antibodies for the determination of s‐triazines with enzyme immunoassays

Two triazine derivatives, ametryn sulphoxide (2‐ethylamino‐4‐isopropylamino‐6‐methyl‐sulphoxide‐1,3,5‐triazine) and dichloroatrazine (2,6‐dichloro‐4‐isopropylamino‐1,3,5‐triazine) were conjugated to bovine serum albumin (BSA) and used for the immunization of BALB/c mice. Hybridomas were produced by cell fusion of immune spleen and mouse myeloma cells (PAI‐B3AG8.I). After screening with a competitive enzyme‐linked immunosorbent assay (ELISA), four anti‐triazine monoclonal antibodies from permanent hybridoma cell lines were selected for further characterization. Cross‐reaction studies with the antibodies developed against the ametryn sulphoxide conjugate showed strong affinities to terbutryn and prometryn. ELISAs with antibodies from clone AS‐K1F4 reached a detection limit of approximately 0.1 μg/l for terbutryn, and with AS‐K1A11 antibodies a limit of 0.3 μg/l for prometryn was attained. Cell lines from mice immunized with the dichloroatrazine‐conjugate produced antibodies with the highest affinities to az...

Assessment of affinity constants by rapid solid phase detection of equilibrium binding in a flow system

We present a method for the determination of affinity constants based on equilibrium binding between an analyte and an antibody in liquid phase by a heterogeneous phase detection scheme. Equilibrium concentration of free antibody binding sites was probed kinetically by direct optical detection of specific binding to an immobilised analyte derivative. The additional binding signal due to dissociation of the analyte-antibody complex during detection was minimised by the use of fast flow-through conditions. The concentration of free antibody binding sites was titrated by adding increasing analyte concentrations. The affinity constant was derived from the titration curve by a non-linear least square fit of a model function. The affinity of monoclonal triazine antibodies to several s-triazine pesticides and a relevant metabolite was investigated. Kinetic determination of equilibrium concentration of free binding sites was carried out by reflectometric interference spectroscopy (RIfS) using flow injection analysis. The capabilities of the model were investigated using different analyte-antibody pairs and various antibody concentrations. Both bivalent IgG and monovalent Fab fragments were used to compare different binding models. The applied model corresponds well to the titration curves for affinity constants of 10(7) M(-1) and higher. For lower affinity constants significant deviations due to dissociation of the analyte-antibody complex during detection were observed.

Development and evaluation of a dipstick immunoassay format for the determination of atrazine residues on-site

On the basis of a semi-quantitative dipstick immunoassay (IA) for atrazine with visual detection (Giersch, T., J. Agric. Food Chem., 1993, 41, 1006), a quantitative format suitable as a field assay for the analysis of pesticide residues in water and liquid food samples on-site is described. For antibody immobilization, different membranes and immobilization techniques were investigated. The measuring range for atrazine was 0.3-10 micrograms l-1 using reflectance detection. The total assay time was about 25 min with dipsticks previously coated with antibody. Atrazine-spiked water and liquid food samples were selected for assay evaluation. The samples could be measured directly without the need for any prior enrichment or clean-up steps. A satisfactory agreement was found between the results of the dipstick IA and HPLC or GC measurements of both the original and spiked samples.

Optimization of a monoclonal antibody-based enzyme immunoassay for the detection of terbuthylazine

The application of immunoassays in pesticide residue analysis is of increasing interest due to the sensitivity, simple handling and fast throughput of samples. For a wide application of these assays, a sufficient supply of standardized antibodies over a long period of time is necessary. The monoclonal antibody technology is solving this problem with an increasing number of cell lines which produce antibodies against different pesticides. Hybridomas were produced by cell fusion of spleen cells from mice immunized with dichloroatrazine conjugated to bovine serum albumin and mouse myeloma cells (PAI-B3 Ag8I). After screening with a competitive enzyme immunoassay, a monoclonal antibody that was specific for terbuthylazine and was produced by a permanent hybridoma cell line was selected for immunoassay development and optimization. For this purpose, an antigen- and antibody-immobilized ELISA technique was improved by varying the test parameters. Comparing both methods, the latter turned out to be superior (50% binding = 0.8 microgram/l, detection limit = 0.14 microgram/l).

Dextran, a hapten carrier in immunoassays for s-triazines. A comparison with ELISAs based on hapten-protein conjugates.

A conjugate of 2-aminohexylamino-4-ethylamino-6-isopropylamino-1,3,5-triazine (AHA), a derivative of the herbicide atrazine, with dextran as carrier has been synthesized and used as the coating antigen in ELISA procedures. The quantification of terbutryn, atrazine and prometryn in ELISA formats using monoclonal antibodies and the AHA-dextran conjugate was at least as sensitive as ELISAs using protein conjugates as immobilized antigens (sensitivity at 50% B/B0 was 0.4-0.6 micrograms/l for terbutryn). Formats with immobilized antibody and enzyme labelled AHA proved to be less sensitive (1.5 micrograms/l for terbutryn). The observed differences in sensitivity do not apparently result from structural effects of the carrier bound hapten since all conjugates were prepared with one form of the hapten, 2-aminohexylamino-atrazine, which was covalently linked via its amino function to the carriers or enzymes.

Affinity characterization of monoclonal and recombinant antibodies for multianalyte detection with an optical transducer

The selectivity of immunoassay is limited by the cross-reactivity of antibodies to structurally related analytes. This becomes a drawback for applications that require discrimination of slightly different analytes. An approach to overcoming this problem is the application of antibody arrays that show differences in their affinity patterns. We have investigated this method using systematic modelling of multianalyte systems based on test-independent affinity parameters. A model system of anti-s-triazine antibodies and s-triazine derivatives has been investigated. The immunoassay is carried out in an indirect test format using an optical transducer for label-free monitoring of antibody binding at an immobilized hapten. The concentration of free antibody in equilibrium with the analyte is probed in a flow-through system. This format allows simple modelling of the response and assessment of the affinity constant from the calibration curve. The affinity patterns of five monoclonal antibodies and a recombinant single-chain fragment with respect to five s-triazine derivatives are determined by this method. An array of three antibodies is selected and the response pattern to mixtures of three analytes determined. Measured and calculated pattern correspond in principle, but systematic deviations are observed due to the perturbation of equilibrium during detection. The correlation of the true analyte concentration and the analyte concentrations predicted from the signal pattern using the affinity constants strongly depend on the selectivity and the affinity of the antibodies.

Magnetic bead selection of hybridomas producing pesticide antibodies

Standardized immunoassays for environmental monitoring require monoclonal antibodies (MAbs) with defined selectivities and affinities. An important step in the production of MAbs is the development of efficient screening procedures to identify suitable cell lines. We demonstrate the usefulness of immunomagnetic cell separation for the screening of hybridomas. It is based on the expression of surface receptors resembling the secreted antibodies on the membrane surface of hybridoma cells. Producers are tagged by the target molecule covalently linked to magnetic beads and subsequently removed by permanent magnet. Several examples are given surpassing conventional techniques for the screening of s‐triazine antibodies.

Immunoenzymatic determination of atrazine in rat tissue samples

In the present study our objective was to adapt a competitive enzyme immunoassay (EIA) for the determination of atrazine in animal tissues (liver and kidney). Female Wistar rats were administered through gavage with a single dose of atrazine (500 or 1000 mg/kg body weight) and sacrificed 24, 48 h and 7 days following the exposure. Using liver tissue samples the extraction procedure was improved. The atrazine concentrations measured in the liver were higher than those found in the kidney, but both can be ranked as low compared with the amount of the administered doses. These data confirm that tissue retention is minimal. Further studies are necessary in order to make an overall evaluation concerning the amount of atrazine retained in the organism. We consider that the competitive EIA may be a promising technique for epidemiological screening of pesticides.

Monoclonal antibody-based enzyme immunoassays for the sensitive detection of s-triazines in water

Immunoassays in pesticide residue analysis significantly profit from the monoclonal antibody (mAb) technology because a sufficient supply of standardized antibodies can be provided. For the production of atrazine-specific mAbs hybridoma cells were produced by fusion of mouse myeloma cells and spleen cells from mice which were immunized with 4-chloro-6-ethylamino- 1,3,5-triazine-2-(6-aminohexanoic acid) coupled to keyhole limped hemocyanin. After screening with a competitive enzyme immunoassay (EIA) a mAb with high binding affinity towards atrazine was selected. A sensitive EIA was developed detecting atrazine with a range from 0.05 to 1 (mu) g/l with a test midpoint of 0.1 (mu) g/l. The mAb cross-reacts predominantly with propazine (136%). Since this herbicide is not used in most European countries, the test allows a rapid and inexpensive screening for atrazine in the ppt range. Another EIA has been constructed for the detection of terbuthylazine. The limiting factor in EIA development is the screening for cell lines secreting mAbs with high affinity and selectivity towards the analyte. Super paramagnetic beads being coated with suitable immonoconjugates are shown to bind to hybridomas presenting hapten-specific receptors on their surface. Hybridomas secreting hapten-specific mAbs can be removed by a magnet and be cloned subsequently by standard procedures. A considerable demand of mAbs is expected in the future due to new emerging techniques such as immunosensors.