Development of an Enzyme Immunoassay for the Determination of Metazachlor

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This article was downloaded by: [The University of Manchester Library]On: 06 December 2014, At: 16:11Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number:1072954 Registered office: Mortimer House, 37-41 Mortimer Street,London W1T 3JH, UKAnalytical LettersPublication details, including instructions forauthors and subscription information: of anEnzyme Immunoassayfor the Determination ofMetazachlorHeike Maria Scholz a & Bertold Hock aa Technische Universitt Mnchen atWeihenstephan, Department of Botany , D-8050,Freising 12, FRGPublished online: 23 Oct 2006.To cite this article: Heike Maria Scholz & Bertold Hock (1991) Development of anEnzyme Immunoassay for the Determination of Metazachlor, Analytical Letters,24:3, 413-427, DOI: 10.1080/00032719108052915To link to this article: SCROLL DOWN FOR ARTICLETaylor & Francis makes every effort to ensure the accuracy of allthe information (the Content) contained in the publications on ourplatform. 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Terms & Conditions of accessand use can be found at by [The University of Manchester Library] at 16:11 06 December 2014 LETTERS, 24(3), 413-427 (1991) Development of an Enzyme Immunoassay for the Determination of Metazachlor Key words: Metazachlor, EIA, ELISA, enzyme immunoassay, solid phase, herbicide, polyclonal antibodies Heike Maria Scholz and Bertold Hock Technische Universitlt Miinchen at Weihenstephan Department of Botany D-8050 Freising 12, FRG Abstract A sensitive, solid phase microtitre plate enzyme immunoassay (ELISA) was developed for the determination of the herbicide metazachlor, which belongs to the class of a-chloroacetamides. The antisera were raised in rabbits .by immunization with a metazachlor-BSA conjugate containing 27 metazachlor residues per molecule. A competitive ELISA with a peroxidase-metazachlor tracer was performed. Metazachlor could be detected within the range of 0.01 and 1 pgh. The test showed no cross-reactivity with other structurally similar chloroacetamide herbicides and little cross-reactivity with derivatives of metazachlor. Introduct ion An increased activity in the development of immunoassays for the detection of pesticides has been noticed during the last ten years'-4. This is due ta the fact that immunological methods have several advantages over the classical procedures, like GC and HPLC. because of their simplicity, high sample throughput, and lower reagent costs. Furthermore. there is need to expand options available for the analysis of pesticide residues because the EC guideline for drinking water states a limit for pesticide concentration of 0.1 ,ug/l for a single substance. and 413 Copyright 0 1991 by Marcel I k k k e r . Inc Downloaded by [The University of Manchester Library] at 16:11 06 December 2014 414 MARIA SCHOLZ AND HOCK Figure 1: Structure of metazachlor N-(2,6-dimethyl-phenyl)-N-( 1 - pyrazoly I-methy1)chloroacetamide 0.5 pg/l for the sum of pesticides. The demand for an efficient, rapid, and dependable screening method may be met by the enzyme-linked immunoassays. The purpose of this study was to develop an ELISA for the identification and quantification of metazachlor, the active ingredient of Butisan S@ (Fig. I ) . Metazachlor is a pre-emergence, a-chloroacetamide herbicide used for weed control in rape, potatoes, tobacco, soybean. and many other crops . Currently, the acetamide herbicides are analyzed by GC with a nitrogen-selective detector6-8. Feng et al. reported an enzyme immunoassay with polyclonal antibodies to alachlor, one representative of this class of herbicides. Even a commercial test for alachlor. based on monoclonal antibodies, is now available from ImmunoSystems. Inc., Maine. USA. However, there have not yet been any reports on ELISAs for the determination of metazachlor. 5 Materials and Methods Chemicals Metazachlor, the derivatives metazachlor-OH and the oxanilic acid derivative, its methylated analogue and OfuraceTM were a gift from BASF. Limburgerhof. The pesticides acetochlor. butachlor, propachlor. and alachlor were kindly provided by Monsanto Company, St. Louis, USA. Pretilachlor and dimethachlor were obtained from Ciba Geigy, Switzerland. Diethyl-Ethyl was supplied by Schering, Agrochemicals LTD. Cambridge, United Kingdom. Metolachlor was purchased from Pestanal Riedel-de Haen. Additional materials used were: bovine serum albumin, carbonyldiimidazole, dithiothreitol, Freunds adjuvant and Downloaded by [The University of Manchester Library] at 16:11 06 December 2014 415 ELISA FOR DETERMINATION OF METAZACHLOR polyoxyethylenesorbitan monolaurate (Tween 20) from Sigma Chemie, Deisenhofen; horseradish peroxidase (I350 U/mg) from Serva; tetramethylbenzidine from Riedel-de Haen; ethanol, p.a., and peroxide (30%) from Merck. All other chemicals used were of analytical grade or chemically pure. Buflers and solutions Carbonate buffer for coating; 50 mmol/l, pH 9.6 phosphate buffered saline (PBS): 1.37 moln NaCI, 0.02 mol/l KCI. 0.015 mol/l KH2P04, 0.08 moljn Na2P04 washing buffer (PBST): phosphate-buffered saline containing 0.1 % Tween 20 substrate buffer for peroxidase: 0.1 mol/l NaAc buffer; the pH was adjusted to 5.5 by adding citric acid substrate solution for peroxidase: 400 pl TMB (6 mg TMB/ml dimethyl sulfoxide and 100 p1 1 % H202 in 25 ml substrate buffer; stopping reagent: 4 normal H2SO4. Preparation of standards A stock solution of metazachlor was prepared by dissolving 1 mg/ml in ethanol abs.. followed by sonication. Appropriate dilutions were made with PBS to yield standards containing 0.01. 0.1. 1, and 10 pgjn metazachlor for the immunoassay. Instrumentation EIA photometer: SLT Easy Reader EAR 400 (SLT, Groding/Salzburg, Austria); microtitre plate washer: SLT EASY Washer EAW 81 12 (SLT, Austria); Ultrasonic bath (Sonorex, Bandelin, Berlin). Further Mat eria Is Microtitre plates (Greiner Labortechnik, D-7443 Frickenhausen) Itnmunogen preparation Metazachlor-BSA conjugate I Ten mg BSA were dissolved in 6 mol/l guanidine/HCl. 0.1 mol/l Tris-HCI (pH 8 . 5 ) . DTT was added to a final concentration of 15 mmol/l and the solution was allowed to react for one hour while nitrogen was flushed throughlo. The alkylation was performed with 50 mmolll metazachlor, dissolved in DMF. Then the protein conjugate was dialyzed against 6 mol/l guanidinelHC1, 0.1 mol/l Tris-HCI with several changes of buffer. Downloaded by [The University of Manchester Library] at 16:11 06 December 2014 416 MARIA SCHOLZ AND HOCK I- 0 II B S A - S - C H r C K - R I I R2 Figure 2: Preparation of rnetazachlor irnrnunogen by using DTT to reduce BSA M e Metazachlor-BSA conjugate 11 Metazachlor was covalently linked to BSA after cleavage of the disulfide bonds with dithiothreitolll (Fig.2). For this purpose, 10 rng of BSA were dissolved in deoxygenated 0.1 rnol/l KCI. The pH was adjusted to 8.1 and maintained during the reduction. 4.8 pmoM DTT were added and the reaction was carried out under nitrogen for 1 h at RT. For alkylation. the pH was raised to 8.4, and 13.2 rng rnetazachlor, dissolved i n 100 pl DMF, were added. The reaction was allowed to proceed for Ih. The resulting product was extensively dialyzed against 0.01 rnol/I NH4HC03 and lyophilized. Downloaded by [The University of Manchester Library] at 16:11 06 December 2014 ELISA FOR DETERMINATION OF METAZACHLOR 41 7 Determination of the epitope density The molar ratio of metazachlor coupled to BSA was determined by amino acid analysis on an Alpha Plus Amino-Acid Analyzer (Pharmacia- LKB. Freiburg, FRG). Each cysteine which had been alkylated by metazachlor to the S-carbamidomethyl derivative could be detected as S-carboxymethylcysteine after acid hydrolysis. Production of antisera Three rabbits (German land breed) were intradermally injected into the back with 600 pg metazachlor-BSA-conjugate, dissolved in 1 mi isotonic saline and mixed with 1 ml Freunds complete adjuvant. The second and third immunizations were similarly performed on days 7 and 14, except for the use of incomplete Freunds adjuvant. After one booster immunization on day 28, blood was collected two weeks later and clotted. Then, the serum was separated, centrifuged, and lyophilized. Further identical immunizations were performed at intervals of 1 month. For seven months, conjugate I was applied for immunizations. then all booster injections were performed with conjugate 11. Enzyme conjugate preparation Metazachlor was linked to horseradish peroxidase to produce a covalently bound metazachlor-HRP complex. The enzyme conjugate was synthesized according to Dreher and Podratzky2 as shown in Fig.3. Ten mg metazachlor, dissolved in 100 pl DMF. were incubated with carbonyldiimidazole (5 mg in 100 y l DMF) at 37OC for 1 h. 200 yI ethylenediamine were added and incubated at RT for 1 h and then lyophilized. The oxidation of the carbohydrate moiety of the HRP was performed according to the simplified method of Tjissen and Kurstak13. Two mg of peroxidase were oxidized with sodium-m-periodate, then diakyzed against NaAc buffer, pH 5.5. 0.4 mg of the previously synthesized metazachlor-NH2 intermediate, dissolved in PBS, were added and incubated for 4 h at RT. For the reduction of the resulting Schiff base linkages, 1 mg of sodium borohydride was added and incubated for 4 h at 40C 14. The resulting enzyme conjugate was extensively dialyzed against PBS. Enzyme Immunoassay Protocol The ELISA procedure employed was the direct competitive method, which was carried out in the following manner: Step 1: Coating. 96 well microtitre plates were filled with 300 yl of Downloaded by [The University of Manchester Library] at 16:11 06 December 2014 0 MARIA SCHOLZ AND HOCK carbonyldiimidazole I NHZ-C,Hd-NH, ethylenediamine I HRPO Nal0,wxidized Figure 3: Synthesis of the metazachlor-HRP conjugate. The OH-derivative of metazachlor was activated with carbonyldiimidazole and allowed to react with ethylenediamine. The amine intermediate was coupled to NaIOq-oxidized HRP (after Dreher and Podratzky; ref.12). 0 Downloaded by [The University of Manchester Library] at 16:11 06 December 2014 419 ELISA FOR DETERMINATION OF METAZACHLOR antiserum [No. I 1 (date of blood collection: 4/10/89)] in a dilution of 1:16 000 and incubated for at least 12 hours at 40C. Step 2: Washing. 3 times with PBST. Step 3: Immune reaction. 200 of metazachlor standards or samples were added and incubated with 50 p i of HRP tracer (29/1/90; diluted 1:4000 in PBST) for 1 h at RT. Enzyme reaction. 200 pl of substrate solution were added and incubated for 20-30 minutes in the dark. Colour detection. The HRP reaction was stopped with 4 normal sulfuric acid and the absorption read at 450 nm. Step 4: Washing as above Step 5: Step 6: Calculations Each sample was assayed in quadruplicate and the absorbance values were transformed using the formula of percentage calculation of in ax i ni um binding : x 100 %BIB,) = Sigmoidal curves were linearized using the formula for logit-log t ransformation: A - Aexcess Ac0 , tro l - Aex,,,, After reference to a calibration curve, mean values and standard deviations of the ELISA were calculated. Specificity of the antibodies In cross-reactivity studies. the antibodies were checked with a series of metazachlor derivatives, structural analogues, and compounds of other herbicide classes. For this purpose. the ELlSA protocol was carried out as described above. Instead of using metazachlor as the standard, various compounds were tested at different concentrations and the cross-reactivitiy was determined as % cross-reactivity = m o l e s o f m e t a z a c h l o r a t SO % BIB, ~~~ m o l e s of s t r u c t u r a l l y r e l a t e d c o m p o u n d at 50% BIB, x 100 Results Determination of the epitope density An appropriate immune response depends upon the epitope density of the immunoconjugate. For this reason. an antigen was prepared with Downloaded by [The University of Manchester Library] at 16:11 06 December 2014 420 MARIA SCHOLZ AND HOCK A O.* 1 0.6 0.4 0.2 0 a concent rat ion [pg/l] 0 0.01 0.1 1 10 E Figure 4: Competitive ELISA for the determination of metazachlor a) absorption values, measured 25 minutes after addition of the substrate solution (means of 4 determinations and standard deviations) b) % B/B,-plot of the standard assay c) logit-log transformation two different amounts of metazachlor residues. According to the amino acid sequence, reduced BSA contains 35 cysteine residues's. Seven residues of cysteine were detected as S-carboxymethylcysteine for conjugate I and 27 residues for conjugate 11. thus 7 or 27 molecules of metazachlor, respectively, had been coupled to one molecule of BSA. Antibody response Antibodies to metazachlor were produced in 3 rabbits of the land breed after pre-immunization with conjugate I for a period of seven months. As the titre did not increase, conjugate I1 with a higher epitope density was administered. A two-dimensional titre determination for the optimal dilution of the antiserum and the enzymatic tracer was carried out. Serum 11 (date of blood collection: 4/10/89) proved to be superior. and all the following data refer to this serum and the HRP-tracer (No. 29/1/90). German Downloaded by [The University of Manchester Library] at 16:11 06 December 2014 E L I S A FOR DETERMINATION OF HETAZACHLOR 4 logit (YoBIBo) ' 2- 0- -2 - -4 - -6 421 I I 1 I 100 O/OB/BO 80 60 40 20 0 b 0 0.01 0.1 1 10 concentration in [~g l l ] (Figure 4 Continued) Downloaded by [The University of Manchester Library] at 16:11 06 December 2014 422 MARIA SCHOLZ AND HOCK Table 1 : Cross-reactivity with the degradation products of metazachlor in relation to metazachlor ( = I 0 0 %) R Derivative cross-reactivity in % cH2a metazachlor 100 mflH metazachlor-OH 1 3 C0a-I metazachlor-COOH 2.8 -3 methylated metazachlor-COOH 4.5 The titre (i.e. the antiserum dilution used in the assay to obtain absorbance values between 0.5 and 0.8) turned out to be 1:16,000; the dilution of the HRP tracer (No. 29/1/90) was 1:4,000. Assay parameter s EIAs are defined by their sensitivity in terms of both limit of detection '6 and mid-point (= 50% B/B,-value) of the calibration curvel7. The detection limit of the assay was found to be at 0.01 pgA metazachlor and the logit/log plot to be linear between 0.01, and 1 pgA. The 50% B/B,-value was 0.09 pg/l. The standard deviations are very low, as shown in Fig. 4a, with percentage coefficients of variability ranging from 2 to 6%. Figure 4b shows the absorption values transformed according to the formula of maximum binding, and Fig. 4c presents the logitllog transformation. S p e cifi ty The cross-reactivity of several metazachlor-related compounds was determined. The derivaties metazachlor-OH, metazachlor-COOH and metazachlor-COOCH3 showed cross-reactivities of 13, 2.8, and 4.5 %, respectively (Table 1). The cross-reactivity of metazachlor analogues (Table 2) was below 1%. None of the assayed structurally unrelated Downloaded by [The University of Manchester Library] at 16:11 06 December 2014 E L I S A FOR DETERMINATION OF METAZACHLOR 423 Table 2: Cross-reactivity with metazachlor analogues in relation to metazachlor (= 100%) name of compound R1 R2 R3 cross- reactivity in % CP-5747 Ofurace dimethachlor acetochlor pretilachlor al achl or metolachlor butachlor propachlor diethyl-ethyl Me Me Me Et Et Et M e Et Et M e M e M e Et Et Et Et Et Et CH3 I (H-CH3 0 .34 0 . 0 7 0 . 0 3 0.19 0.05 0 . 0 4 0.1 0.5 n.d. n.d. Downloaded by [The University of Manchester Library] at 16:11 06 December 2014 424 M A R I A SCHOLZ A N D HOCK compounds. such as atrazine. simazine, propazine. terbuthylazine. ametryne. aziprotryne. simetryne, prometryne. terbutryne. and methabenzthiazuron, were cross-reactive. Discussion The most critical part during the immunoassay development for the chloroacetamide herbicide metazachlor was the coupling procedure for the conjugate, since none of the common functional groups like -COOH, - NH2. -OH. and -SH are available for the covalent bond to a protein. As it is known that chloroacetamides react with sulfhydryl groups, this reaction mechanism was used in hapten synthesis. Further indications of this mechanism were found in studies of the metabolism of this family of herbicides, e.g. by Lamoureux et al.18. Hussain et al.19. and Ezra et al. 20. These compounds are initially metabolized by conjugation with the SH-group of glutathione. A coupling procedure using the chloroacetamide moiety, which is common to all pesticides of this class, is thought to be advantageous because the pyrazolyl group characteristic only for metazachlor is preserved as a possible determinant group for antibody recognition. By attaching the hapten to the protein carrier in this manner, cross-reactions with other representatives of this family should be theoretically excluded and the specifity of the antiserum guaranteed. The obtained cross-reactivity data justify this strategy. Feng et al.9 also coupled the chloroacetamide-moiety to a protein, to which the sulfhydryl groups had been introduced by AMSA or AHT, and reported similar results. In our work, metazachlor was coupled at first to guanidineMC1 treated BSA after the disulfide bridges had been cleaved by DTT. This approach led to insoluble conjugates which were not suitable for repeated immunizations. The reduction of disulfide bonds in the absence of denaturants and with a lower concentration of D I T , as described by Bewley and Lill, resulted in better soluble conjugates and a higher coupling efficiency. The obtained antiserum is highly specific for metazachlor, since cross- reactivity with structural analogues is below 1 % . The elimination of the chloro-moiety causes an approximately 7-fold reduction in the antibody binding. For instance, the binding to metazachlor-OH is much weaker than to metazachlor. Although the antiserum was prepared with an Downloaded by [The University of Manchester Library] at 16:11 06 December 2014 ELISA FOR DETERMINATION OF METAZACHLOR 425 immunoconjugate which had been coupled by the reaction of the chloro- moiety with a SH-group. i t could obviously discriminate between the OH- and the Cl-group. A comparison of the cross-reactivities of the antibody with metazachlor-OH and metazachlor-COOH or its rnethylated analogue suggests that the CH2-moiety, which is absent in the oxanilic derivative and its methylated analogue. is an important factor in structural recognition. Thus, a serum could be obtained which enables measurements in the range of 0.01 and 1 pgh and therefore fulfills the requirements of the EC guideline. Abbreviat ions A AHT AMSA BSA DMF DMSO m EIA ELISA Et Q7 HPLC H RP Me NaAc-buffer n.d. PBS PBST RT TMB Tris Acknowledgements absorpt ion N-acetylhomocysteine thiolactone S-acetylmercaptosuccinic anhydride bovine serum albumin N,N-dimethylformamide dimethyl sulfoxide dithiothrei to1 enzyme immunoassay enzyme-linked immunosorbent assay e thyl gas chromatography high perfomance liquid chromatography horseradish peroxidase methyl Sodium acetate buffer not detectable phosphate buffered saline phosphate buffered saline containing 0. I % Tween 20 room temperature 3.3 ' ,5 ,5 ' - te tramethylbenzidine Tris(hydroxymethy1)aminomethane We thank the Deutsche Forschungsgemeinschaft for a grant (Ho 383/26- I ) . We are indebted to BASF AG, Landwirtschaftliche Versuchsstation Downloaded by [The University of Manchester Library] at 16:11 06 December 2014 426 MARIA SCHOLZ AND HOCK Limburgerhof, for financial support and to Drs. R. T. Hamm and W. Turk for providing pure metazachlor and its derivatives. We are grateful to Mr. I. Krause and to Mrs 1. Sperrer of the Department of Dairy Science at Weihenstephan (Technical University Munich) for the determination of carboxymethylcysteine by amino acid analysis. We also wish to thank Monsanto Company, St. Louis, USA, Ciba Geigy. Switzerland, and Schering, Agrochemicals LTD, Cambridge. United Kingdom, for the gifts of compounds used in the cross-reactivity study. References 1 . B. D. Hammock. and R. 0. Mumma: Potential of immunochemical technology for pesticide analysis. In Pesticide Analytical Methodology;: J. Harvey, G . Zweig. G.. Eds.; ACS Symposium Series 36; American Chemical Society: Washington, DC, pp. 321-352 (1980). 2 . J . M. van Emon, J. N. Seiber, and B. D. Hammock: Applications of immunoassay to paraquat and other pesticides. In Bioregulators for Pest control; ACS Symposium Series 276; American Chemical Society: Washington. pp. 307-3 16 (1985). 3 . M. Schwalbe-Fehl, Inter. J . Environ. Anal. Chem. 26, 295-304 ( 1 9 8 6 ) . 4 . F. Jung, S. J. Gee, R. 0. Harrison, M. H. Goodrow, A. E. Karu, A. L. Braun, Q. X. Li, and B. D. Hammock, Pestic. Sci.. 303-317 ( 1 9 8 9 ) . 5. Technisches Merkblatt. BASF AG, Landwirtschaftliche Versuchsstation Limburgerhof, 1984. 6. J. W. Worley, M. L. Rueppel. and F. L. Rupel, Anal. Chem.. 1849 (1980). 1845- 7. K.-D. Wenzel. M. Mohnke, and J. N. Seiber, Fresenius Z. Anal. Chem. - 322. 423-425 (1985). 8 . A. Balinowa: J. Chromat.. 455, 391-395 (1988). Downloaded by [The University of Manchester Library] at 16:11 06 December 2014 ELISA FOR DETERMINATION OF METAZACHLOR 427 9 . P. C. C. Feng, S. J. Wratten. S. R. Horron. C. R. Sharp . and E.W.Logusch, J . Agric. Food Chem. 18. 159-163 (1990). 10. D. Holowka, Anal. Biochem. lJ. 390-397 (1981). 1 I . T. A. Bewley and C. H. Li. Int. J. Protein Res. 1; 117-124 (1969). 12. R. M. Dreher and B. Podratzki, J. Agric. Food Chem. & 1072- 1075 (1988). 13. P. Tijssen, and E. Kurstak, Anal. Biochem. 136. 451-457 (1984). 14. B. F. Erlanger and S. M. Beiser, Natl. Acad. Sci. U.S.A.. 2. 68-74 ( 1 9 6 4 ) . 1 5 . J . R. Brown and P. Shockley: In Lipid-protein interactions. Eds. P. Jost. 0. H. Griffith. Vol. 1. pp. 25-68. Wiley, New York (1982) . 16. G. E. Abraham. J . Clin. Endocr. Metab. 29. 866-870 (1971). 17. H. Feldnian, and D. Rodbard: Mathematical theory of radioimmunoassay. In Principles of competitive protein- binding assays, Eds. W. D. ODell & W. H. Daughaday, Lippincott Co.. Philadelphia. pp. 158-203 (1971). 18. G. L. Lamoureux, L. E. Stafford. and F. S . Tanaka, J. Agric. Food Chem. 19: 346 (1971). 19. M. Hussain. I . P. Kapoor. C. C. Ku, and S. Stout. J. Agric. Food Chem. 31. 232 (1983). 20. G. ELra. G. R. Stephenson, and G. L. Lamoureux: Metabolism of 14C metaiachlor and action of safener 145 138 i n corn. In nte National Meeting of the Weed Science Society of America, Houston. TX. (1986). Received December 20 , 1990 Accepted January 4 , 1991 Downloaded by [The University of Manchester Library] at 16:11 06 December 2014


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