Immunoassay for wheat processing quality: utilization of a sandwich assay incorporating an immobilized single-chain fragment.

A single-chain fragment (scFv) was engineered from a monoclonal antibody to high molecular weight glutenin subunits (HMW-GS), wheat flour polypeptides that play a major role in determining the mixing- and extension strength-related properties of dough and its subsequent baking performance. The scFv was expressed in a thioredoxin mutant Escherichia coli strain that allows disulfide bond formation in the cytoplasm and incorporated into a diagnostic test for wheat quality. Although the scFv lacks the more highly conserved antibody constant regions usually involved with immobilization, it was able to be directly immobilized to a polystyrene microwell solid phase without chemical or covalent modification of the protein or solid phase and utilized as a capture antibody in a double-antibody (two-site) immunoassay. In the sandwich assay, increasing HMW-GS concentrations produced increasing assay color, and highly significant correlations were obtained between optical densities obtained in the ELISA using the scFv and the content of large glutenin polymers in flours as well as measures of dough strength as measured by resistance to dough extension in rheological testing. The assay using the scFv was able to be carried out at lower flour sample extract dilutions than that required for a similar assay utilizing a monoclonal capture antibody. This research shows that engineered antibody fragments can be utilized to provide superior assay performance in two-site ELISAs over monoclonal antibodies and is the first application of an engineered antibody to the analysis of food processing quality.

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.

Continuous flow immunosensor for atrazine detection.

The hapten atrazine was detected under continuous flow conditions using a micro-column which contained immobilized monoclonal antibodies (Ab) against atrazine and atrazine labeled with alkaline phosphatase (An*). The equilibrium of the antibody-hapten system, was achieved by a continuous flow of the tracer An* through the micro-column containing the immobilized antibodies. The activity of the tracer was monitored continuously, after the micro-column, by an amperometric detector using p-hydroquinone phosphate as substrate. When pulses of unlabeled atrazine (An) were added to the An* flowing continuously through the micro-column, An* bound to the antibody was displaced, with a consequent change of the detector signal. By this method atrazine concentrations in the range 9-180 micrograms/l were monitored under conditions of continuous operation. Since the equilibrium condition for the system Ab-An* was continuously restored by the flow of An* through the micro-column the regeneration of the antibody was not required.

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.