a heterogeneous immunoassay for the determination of triazine herbicides in water

2
Fresenius J Anal Chem (1991) 339:468-469 Fresenius' Journal of @ Springer-Verla9 1991 A heterogeneous immunoassay for the determination of triazine herbicides in water L. Weil, R. J. Schneider, O. Schiifer, P. Ulrich, M. Weller, T. Ruppert, and R. Nie6ner Institute of Hydrochemistry, Munich Technical University, Marchioninistrasse 17, W-8000 Munich 70, Federal Republic of Germany Summary. A heterogeneous enzyme-linked immunosorbent assay (ELISA) for the screening of triazine herbicides, e.g. atrazine in ground and surface water is reported, which is able to detect the pesticide at concentrations down to 10 ng/1 without a preconcentration step. Introduction Legislation in the field of drinking water has proved to be stimulating for the development of new analytical methods and procedures. The EC guideline for the examination of drinking water, for example, fixes the maximum limit for pesticide concentrations to 0.1 pg/1 for each single substance and to 0.5 pg/1 for the sum of all pesticides present in the sample. Methods of determination have to cope with these low values, which are in the range of the lower detection limit of the elaborate analytical procedures for most of the applied pesticides [1, 2]. The immunoassay, widespread in clinical chemistry for routine determination of high- and low-molecular sub- stances, has recently been brought to application for small molecules in environmental analysis, too [3, 4]. At present, the top position of water contaminants is occupied by the herbicides, especially those of the s-triazine group. Water resources of West Germany should be submitted to a permanent screening for atrazine (2-chloro-4-ethyl- amino-6-isopropylamino-l,3,5-triazine) concentration be- cause up to about 20% of the water suppliers already have to deal with a contamination of drinking water by this herbicide [5]. A heterogeneous enzyme immunoassay (ELISA) has been developed that is able to detect directly atrazine, simazine and propazine at levels down to 10 ng/1 without clean-up or pre-concentration of the sample. Experimental Antibodies are polyclonal from rabbit and have been made available by our co-workers in the Botanical Institute of Munich Technical University, Freising-Weihenstephan, Prof. Hock. Our synthesis of the enzyme tracer begins with the construction of a derivative of atrazine that is suitable for Offprint requests to." L. Weil 0.8- l ! I L 0.7- t T Four parameters:. = O,~7879EI~4 .~ = 0,1301~7055 0.6 - .L I regressiorl ~oeffident= D.~9987658 0.5- 0.2- 0. I" ~ -~-calibrator s - - . ] l | ~' ~ , ~ zero sample (uater for HPLC) | I I [ ] ! talc. values I (~our-parameter reqress, on) ~ , 0- 0-001 0.01 0.1 iO atrazine concentration l't~g/1] Fig. 1. Calibration graph for atrazine. Antibody dilution 1:20.000 of AK 1/93. Dilution of Tracer (P.U.-Tgc) 1 : 10.000. Each point represents the mean +_ 2 SD for calibrators run in quadruplicate. The limit of detectability is about 10 ng/1 coupling to the enzyme horseradish peroxidase (HRP, POD) [6, 7]. Cyanuric chloride is subsequently reacted with isopropylamine and 6-aminohexanoic acid, the latter serving as a spacer for further conjugation. The derivative is activated by reaction with N-hydroxysuccinimide (NHS) and dicyclohexylcarbodiimide (DEC) in dry 1,2-di- methoxyethane (glyme). The NHS-ester in glyme is finally added to an enzyme solution in 0.1 mol/1 sodium hydrogen carbonate buffer. The tracer has been purified by gel permea- tion chromatography on Fractogel TM TSK HW-40 (F) from Merck. The enzyme tracer still ressembles atrazine and the immunogen, yet does not bear the ethylamino group any more. Before running the test, commercially available poly- styrene plates (GREINER) are coated with an antibody dilution (carbonate buffer, pH 9.6, overnight at room tem- perature). Then, 200 gl of the samples (12 calibrators and 12 unknowns) are pipetted into the wells in quadruplicate and 100 ~tl of peroxidase tracer dilution (in phosphate buffered saline, pH 7.6) are added. While mixing well (on an orbital shaker) incubation lasts one hour. After washing away the excess of unbound pesticide, the enzyme's substrate is pipetted into the wells. The substrate cocktail consists of a mixture of hydrogen peroxide and TMB (3,3',5,5'-tetra- methylbenzidine) in a citrate/acetate buffer (pH 5.5). Due to the optimized procedure of tracer synthesis, development of _ m m ]00

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Page 1: A heterogeneous immunoassay for the determination of triazine herbicides in water

Fresenius J Anal Chem (1991) 339:468-469 Fresenius' Journal of

@ Springer-Verla9 1991

A heterogeneous immunoassay for the determination of triazine herbicides in water

L. Weil, R. J. Schneider, O. Schiifer, P. Ulrich, M. Weller, T. Ruppert, and R. Nie6ner Institute of Hydrochemistry, Munich Technical University, Marchioninistrasse 17, W-8000 Munich 70, Federal Republic of Germany

Summary. A heterogeneous enzyme-linked immunosorbent assay (ELISA) for the screening of triazine herbicides, e.g. atrazine in ground and surface water is reported, which is able to detect the pesticide at concentrations down to 10 ng/1 without a preconcentration step.

Introduction

Legislation in the field of drinking water has proved to be stimulating for the development of new analytical methods and procedures. The EC guideline for the examination of drinking water, for example, fixes the maximum limit for pesticide concentrations to 0.1 pg/1 for each single substance and to 0.5 pg/1 for the sum of all pesticides present in the sample. Methods of determination have to cope with these low values, which are in the range of the lower detection limit of the elaborate analytical procedures for most of the applied pesticides [1, 2].

The immunoassay, widespread in clinical chemistry for routine determination of high- and low-molecular sub- stances, has recently been brought to application for small molecules in environmental analysis, too [3, 4].

At present, the top position of water contaminants is occupied by the herbicides, especially those of the s-triazine group.

Water resources of West Germany should be submitted to a permanent screening for atrazine (2-chloro-4-ethyl- amino-6-isopropylamino-l,3,5-triazine) concentration be- cause up to about 20% of the water suppliers already have to deal with a contamination of drinking water by this herbicide [5].

A heterogeneous enzyme immunoassay (ELISA) has been developed that is able to detect directly atrazine, simazine and propazine at levels down to 10 ng/1 without clean-up or pre-concentration of the sample.

Experimental

Antibodies are polyclonal from rabbit and have been made available by our co-workers in the Botanical Institute of Munich Technical University, Freising-Weihenstephan, Prof. Hock.

Our synthesis of the enzyme tracer begins with the construction of a derivative of atrazine that is suitable for

Offprint requests to." L. Weil

0 . 8 -

l ! I L 0.7- t T Four parameters:. = O, ~7879EI~4

.~ = 0,1301~7055

0.6 - .L I regress ior l ~ o e f f i d e n t = D. ~9987658

0 . 5 -

0 . 2 -

0. I " ~ -~-calibrator s - - . ] l | ~ ' ~ , ~ zero sample ( u a t e r fo r HPLC)

| I I [ ] ! ta lc. values I (~our-parameter reqress, on) ~ , 0-

0-001 0.01 0.1 iO

a t r a z i n e c o n c e n t r a t i o n l ' t~g /1 ]

Fig. 1. Calibration graph for atrazine. Antibody dilution 1:20.000 of AK 1/93. Dilution of Tracer (P.U.-Tgc) 1 : 10.000. Each point represents the mean +_ 2 SD for calibrators run in quadruplicate. The limit of detectability is about 10 ng/1

coupling to the enzyme horseradish peroxidase (HRP, POD) [6, 7]. Cyanuric chloride is subsequently reacted with isopropylamine and 6-aminohexanoic acid, the latter serving as a spacer for further conjugation. The derivative is activated by reaction with N-hydroxysuccinimide (NHS) and dicyclohexylcarbodiimide (DEC) in dry 1,2-di- methoxyethane (glyme). The NHS-ester in glyme is finally added to an enzyme solution in 0.1 mol/1 sodium hydrogen carbonate buffer. The tracer has been purified by gel permea- tion chromatography on Fractogel TM TSK HW-40 (F) from Merck. The enzyme tracer still ressembles atrazine and the immunogen, yet does not bear the ethylamino group any more.

Before running the test, commercially available poly- styrene plates (GREINER) are coated with an antibody dilution (carbonate buffer, pH 9.6, overnight at room tem- perature). Then, 200 gl of the samples (12 calibrators and 12 unknowns) are pipetted into the wells in quadruplicate and 100 ~tl of peroxidase tracer dilution (in phosphate buffered saline, pH 7.6) are added. While mixing well (on an orbital shaker) incubation lasts one hour. After washing away the excess of unbound pesticide, the enzyme's substrate is pipetted into the wells. The substrate cocktail consists of a mixture of hydrogen peroxide and TMB (3,3',5,5'-tetra- methylbenzidine) in a citrate/acetate buffer (pH 5.5). Due to the optimized procedure of tracer synthesis, development of

_ m

m

]00

Page 2: A heterogeneous immunoassay for the determination of triazine herbicides in water

469

blue colour can be stopped by addition of sulphuric acid (2 mol/1) about 20 min later. By this the blue colour turns to yellow. The absorbance of each well is measured in an 8- channel photometer designed for ELISA tests (microplate reader) at a wavelength of 450 nm.

Results and discussion

First the data are transferred to a microcomputer. The reader software MikroTEK TM from SLT-Labinstruments, Austria, permits to create and export an ASCII-file of raw data. Results are obtained by comparing the absorbance of samples to an intra-test calibration graph (Fig. 1) that is set up by standard solutions on the same plate. Spline interpola- tion is possible, but adaptation by four-parameter regression [8] proved to be the best approach to minimize measurement bias. Calculations, display of data and the setup of a calibra- tion graph can be done by the programme MULTIGRAF TM

from midas Micro-Datensysteme GmbH that has been adapted to our specific problems by automated macro routines. Users of MULTIGRAF TM may obtain those routines by request on the authors [9].

Thus, up to 12 samples with a 4-fold determination can be run simultaneously in a procedure that lasts less than two hours permitting up to one hundred samples to be screened per day.

The cross-reactivities of propazine and simazine in the test are about 180% resp. 8%, showing the antibodies' pref- erence for isopropylamino-substituted triazine derivatives, according to the structure of the immunogen and the tracer. A positive result in a screening of samples therefore detects both atrazine and propazine well, while samples that contain only a small concentration of simazine may show false- negative results.

The limit of detection for atrazine reached up to now is at about 10 ng/1, the optimal measuring range where the total maximum error does not exceed 50% extending from about 20 ng/1 to 4 ~tg/1.

A first comparison of gaschromatographic results from water samples with those obtained by ELISA showed a good correlation [8].

Conclusions

The ELISA test described has already in the present state a low detection limit with an acceptable maximum error and shows that immunological test systems are a valuable tool to satisfy legal demands like those that are included in the German Drinking Water Ordinance.

At present the ELISA procedure undergoes further verification of exactness of results in comparison with gas chromatography for artificial and natural water samples and soil extracts. Assays for the herbicides metabenzthiazuron, metazachlor and the triazine metabolites desethyl-atrazine and hydroxy-atrazine are under development.

Acknowledgement. We would like to thank Prof. B. Hock/Weihen- stephan for making available antibodies and the Deutsche Forschungsgemeinschaft (DFG) for financial support.

References

1. Oehmichen U, Karrenbrock F, Haberer K (1987) Fresenius Z Anal Chem 327:715-719

2. Reupert R, P16ger E (1988) Fresenius Z Anal Chem 331:503- 509

3. Jung F, Gee S J, Harrison RO, Goodrow MH, Karu AE, Braun AL, Li QX, Hammock BD (1989) Pestic Sci 26:303- 317

4. Hemingway RJ (1984) Pure Appl Chem 56 : 1131 - 1152 5. Siiddeutsche Zeitung (1989) 30.09/01.10.89, p 34 6. Weller M, Ulrich P, Ruppert T, Schneider R, Weil L, Niel3ner R

(1990) Poster presented at the Jahrestagung der Fachgruppe Wasserchemie in der GDCh, Timmendorfer Strand, May 21- 23, 1990

7. Schneider RJ, Ruppert T, Weller M, Weil L, Niel3ner R (1990) Fresenius J Anal Chem 337:74-75

8. Schneider RJ, Ruppert T, Weller M, Well L, Niel3ner R (1990) Poster presented at the 12th International Conference on Bio- chemical Analysis accompanying ANALYTICA, Munich, May 8-11, 1990

9. Requests to: Olaf Schfifer, Institut ffir Wasserchemie, Marchioninistr. 17, W-8000 M/inchen 70, Federal Republic of Germany

Received August 13, 1990