Monika Wortberg

An immunoarray for the simultaneous determination of multiple triazine herbicides

An immunochemical method for simultaneous analysis of cross-reacting analytes is presented. We demonstrate the general principle using triazine herbicides as the model system. The analysis is based on a combination of individual enzyme immunoassays (immunoarray) for triazine herbicides using antibodies with different cross-reactivity patterns towards the selected analytes. The assay signals obtained can be mathematically evaluated to estimate concentrations of each analyte out of a ternary or quaternary mixture. The mathematical model utilizes an extension of the empirical four parameter log-logistic fit. Using mono- and polyclonal antibodies it was possible to quantify the four analytes atrazine, simazine, cyanazine, and prometon in the low to sub-ppb range simultaneously.

Flow-injection immunosensor for triazine herbicides using Eu( III) chelate label fluorescence detection

An immunochemical flow-injection system for the determination of triazine herbicides based on principles of immunoaffinity chromatography was developed. Triazine herbicide derivatives immobilized on oxirane acrylic beads serve as the affinity column. They are saturated with fluorescently labelled monoclonal anti-herbicide antibodies prior to the assay. The label used is the fluorescent Eu(III) chelate W8044-Eu. A fraction of the fluorescent antibodies is replaced when exposed to analyte and this fluorescence is detected in a postcolumn mode by means of a special laser-based fluorimeter. With the reusable affinity column a detection limit of μg l−1 for the herbicide atrazine was obtained.

Extension of the four-parameter logistic model for ELISA to multianalyte analysis

The standard implementation of enzyme-linked immunosorbent assay (ELISA) for single analytes can lead to false conclusions if cross reacting compounds are present in the sample. This paper discusses the extension of the usual four-parameter logistic model for ELISA to the case of multiple cross-reacting analytes. The use of the extended model in multianalyte analysis (MELISA) is illustrated and compared with a more simplistic approach. Data on the analysis of a binary mixture of s-triazines suggests the superiority of the proposed model. This model is also suitable for other forms of immunoassay that use the four-parameter logistic curve.

An approach to the construction of an immunoarray for differentiating and quantitating cross reacting analytes

Abstract We demonstrate the construction of an immunoarray to categorize, identify and quantitate different triazine herbicides or their environmental metabolites in water. In a first step cluster analysis is used to examine the performance of a small subset of antibodies out of a larger library, and hence to select a small array that is capable of categorizing the triazine herbicides into different groups. At the 1 ppb level it is possible to categorize an analyte as a chloro-s-triazine, a hydroxy metabolite, or as a methoxy/methylthio substituted triazine when using only 2–4 antibodies. At higher concentrations even the identity of the triazine can be determined with the same number of antibodies. A selected combination of antibodies is then used to identify and quantify unknowns by comparing their immunoassay responses to an array of calibration curves and using the maximum likelihood criterion.

Rapid assays for environmental and biological monitoring.

Rapid, inexpensive, sensitive, and selective enzyme-linked immunosorbent assays (ELISAs) now are utilized in environmental science. In this laboratory, many ELISAs have been developed for pesticides and other toxic substances and also for their metabolites. Compounds for which ELISAs have recently been devised include insecticides (organophosphates, carbaryl, pyrethroids, and fenoxycarb), herbicides (s-triazines, arylureas, triclopyr, and bromacil), fungicides (myclobutanil), TCDD, and metabolites of naphthalene and toluene. New rapid assays have been developed for mercury.

A new non-enzymatic tracer for time-resolved fluoroimmunoassay of triazine herbicides

SummaryThe synthesis, characterization and application of a new kind of conjugate as tracer for use in time-resolved fluoroimmunoassay of triazine herbicides is described. Bovine serum albumin (BSA) served as carrier molecule, to which a triazine herbicide derivative and the Eu(III)-chelate W8044-Eu were subsequently coupled. The conjugate was characterized after each synthesis step by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The total composition was 1 BSA : 5.2 (±0.7) haptens : 5.1 (±0.4) fluorophores as calculated from the MALDI-MS data. The conjugate was successfully applied in competitive fluoroimmunoassays with sequential or simultaneous incubation of herbicide and tracer. With the sequential assay, the lowest detection limit was 0.1 μg/l for terbutryn. Results of assays performed with microtiter strips were compared with those obtained with commercial micro affinity columns.

A procedure for the immunoanalysis of samples containing one or more members of a group of cross-reacting analytes

The information obtained from a single-antibody immunoassay can be ambiguous when the identity of the analyte is unknown and could be one of a group of compounds all having different affinities for the antibody. If we allow the possibility of mixtures of analytes the difficulty of the situation is aggravated. However, additional information can sometimes be obtained by assaying with a number of different antibodies. We demonstrate a rationale for identifying and quantifying analytes from a group of candidates, and illustrate the application of our method with an example from the class of s-triazine herbicides. Using a four-antibody array, correct identification and accurate quantification were usually achieved for singleanalytes samples in the low ppb range. Mixtures of analytes were recognized as such but were more difficult to classify correctly, with some confusion arising between members of subgroups, particularly within the methoxy/methylthio substituted triazines. The mathematical perspective is used to suggest directions for improving the experimental performance of multianalyte immunoassay.

Sources of experimental variation in calibration curves for enzyme-linked immunosorbent assay

Enzyme-linked immunosorbent assays are usually performed by running standard and unknown concentrations together on the same microtiter plate, because the standard curve is known to vary considerably from one assay to the next. Here we examine experimentally the sources and nature of this variation, and discuss the possibility of reducing the cost of the assay by using a batch of plates, only one of which is used to generate the calibration curve. We present a method for doing this, and test it empirically.

FIA Immunoassays with Electrochemical and Optical Detection

Different approaches have been made for developing novel immuno FIA (FIIA) systems for herbicide detection: antigens have been labeled with ferrocene and enzymes, anti-herbicide antibodies were labeled with fluorophores (Europium chelates)

Development of a Flow Injection Immuno-Analysis for Pesticide Determination - First Studies-

In this paper the use of the special Eu 3+ -chelator 4,7-bis(chlorosulphophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) as a fluorophore in immunoasssays rsp. immunosensors is demonstrated. The purpose is the development of a flow injection immunoanalysis for commonly used pesticides in water.