An Immunochromatographic Assay of 2,4-Dichlorophenoxyacetic Acid ...

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A method of qualitative express analy- sis is necessary to monitor the content of pesticides in environment; it should enable one-step reliable detec-.
Russian Journal of Bioorganic Chemistry, Vol. 30, No. 2, 2004, pp. 178–183. Translated from Bioorganicheskaya Khimiya, Vol. 30, No. 2, 2004, pp. 201–207. Original Russian Text Copyright © 2004 by Lyubavina, Zinchenko, Salomatina, Zherdev, Dzantiev.

An Immunochromatographic Assay of 2,4-Dichlorophenoxyacetic Acid and Simazine Using Monoclonal Antibodies Labeled with Colloidal Gold I. A. Lyubavina*, 1, A. A. Zinchenko*, I. S. Salomatina*, A. V. Zherdev**, and B. B. Dzantiev** *Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, GSP Moscow, 117997 Russia **Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia Received October 16, 2002; in final form, January 29, 2003

Abstract—A method of the competitive immunochromatographic assay of the pesticides 2,4-D (2,4-dichlorophenoxyacetic acid) and simazine (2-chloro-4,6-bis(N-ethylamino)-1,3,5-triazine) in aqueous samples was developed. Monoclonal antibodies to these pesticides labeled with colloidal gold were used to visualize the results. The sensitivity of the 2,4-D and simazine assay is 12 ng/ml, and the time of analysis is 3–7 min. The method does not differ in sensitivity from the competitive EIA using conjugates of monoclonal antibodies to the pesticides with horseradish peroxidase; however, the time of the EIA is 1.5 h. The immunochromatographic method of the pesticide detection is available and simple and may be recommended for the development of assays of any other low-molecular compounds. Key words: competitive EIA, complex of monoclonal antibodies with colloidal gold, 1,4-dichlorophenoxyacetic acid, immunochromatography, simazine 1

INTRODUCTION

The contamination of environment by the products of human industrial activities and agriculture makes urgent the development of express methods for detecting such substances, in particular pesticides, in food, water, soil, etc.2 A method of qualitative express analysis is necessary to monitor the content of pesticides in environment; it should enable one-step reliable detection of pesticides in water solutions without any special equpment. The method of competitive immunochromatographic assay meets these requirements. In recent years, it is widely used for the express detection of various haptenes, such as steroid hormones, narcotics, medicinal preparations, etc. [1, 2]. The testing by competitive immunochromatography is based on the distribution of molecules in a biphasic system. A polyvalent conjugate of a test compound with OVA is fixed on the surface of a nitrocellulose membrane in the test zone and remains stationary during the analysis. The mobile phase, which contains a 1 Corresponding author; e-mail: [email protected] 2 Abbreviations: CG, colloidal gold dispersion;

2-CPA and 4-CPA, 2- and 4-chlorophenoxyacetic acids; 2,4-D, 2,4-dichlorophenoxyacetic acid; HRP, horseradish peroxidase; MAb, monoclonal antibodies; OVA, ovalbumin; PBS, phosphate-buffered saline, pH 7.4; PBST, PBS containing 0.05% Tween-20; PBS/OVA, PBS containing ovalbumin (2 mg/ml); PBST/OVA, PBST containing ovalbumin (2 mg/ml); and Sm, simazine (2-chloro-4,6-bis(N-ethylamino)-1,3,5-triazine.

test sample and a limited amount of labeled antibodies to the test compound, enters the test zone and migrates in it with a constant rate. The binding of labeled antibodies to the test compound sorbed on the solid phase leads to the formation of a complex, which can be evaluated visually. If the an assay sample contains the test compound, free and bound antigens begin to compete for the antibody binding sites. Unbound reaction components are removed from the system. The rate of an antigen binding to its antibody in the immunochromatographic method is by one order of magnitude higher than in the conventional dot assay, and the kinetics of the process is close to that in solution [3]. The time of assay is 5–10 min. Previously we reported the use of immunofiltration [4] and a comb-format dot immunoassay [5] for the noninstrumental detection of the 2,4-D (class of aryloxyalkylcarboxylic acids) and Sm (class of sim triazines) pesticides. The goal of this study was the development of a method for the assay of 2,4-D and Sm by immunochromatography [6] using the CG-labeled MAb to the pesticide as a detecting agent. RESULTS AND DISCUSSION The competitive immunochromatographic detection of 2,4-D and Sm in water samples is based on the following principle. During the assay, the pesticide in a water sample competes with the pesticide immobilized

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in the test zone of the test strip of nitrocellulose membrane (Fig. 1) for the binding sites of a limited amount of labeled antibodies. For the one-step qualitative immunochromatography, antibodies are labeled with enzymes and various corpuscular markers such as CG [7], latexes [8], colloidal dyes [8], and others. The use of an enzyme as a label for immunochromatography requires an additional step for the detection of the enzyme itself using an unstable substrate, chromatogen, which considerably complicates the preparation of test strips and the assay procedure. In most cases, the immunochromatography is carried out using a complex of antibodies with CG as a detecting agent [7–9]. The advantages of CG as a marker are: (1) the ease of preparation of particles of required size with an optimum molecular-mass distribution; (2) the possibility of drying antibody–CG complexes; and (3) a high sensitivity of assays using such complexes. A considerable drawback of complexes with CG is their insufficient stability.

Absorbing part (cellulose membrane) Control band

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We first obtained a gold sol with an average diameter of particles of 20 ± 5 nm using a standard method [9] (Fig. 2). We previously found that the particle size of 20 nm is optimal for protein sorption and subsequent migration of the resulting complex through the membrane pores of the immunochromatographic test strip. The antibodies to 2,4-D or Sm were then sorbed on the surface of gold particles [10]. The visualization capacity of the resulting complexes was compared by the dot analysis in a model system by detecting the polyvalent conjugates of the pesticides with OVA (Sm–OVA and 2,4-D–OVA) at a series of twofold dilutions and applied in dots to the strips of nitrocellulose membrane. It was found that, in the range of the sol loads with antibodies of 0.1–1.6 mg/ml of sol, the optimal load was 0.8 mg/ml. The antibody load less than 0.8 mg/ml leads to a drop in the assay sensitivity of the Sm–OVA and 2,4-D–OVA, whereas an increase in the antibody load up to 1.6 mg/ml did not decrease the detection limit of the polyvalent conjugates. An antibody load above 1.6 mg/ml resulted in a flocculation of CG. The conjugates of HRP with antibodies to 2,4-D (MAb to 2,4-D) and to Sm (MAb to Sm) synthesized by the standard method [11] served as a control in the dot analysis. One can see in Fig. 3 that the detection limit for both 2,4-D–OVA and Sm–OVA using the MAb to 2,4-D and MAb to Sm complexes with CG is 12.5 ng/ml. The detection limit for the pesticides using conjugates with HRP is 50 ng/ml. The temperature stability of the resulting complexes was characterized in a model system by comparing the sensitivity of the pesticide assay using these complexes during the storage under various conditions. The sensitivity of assay by the complexes stored at 4°ë remained unchanged during 12 months; the storage at 22°ë did not change the sensitivity for 6 months; it was then deteriorated. RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

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Fig. 1. A schematic representation of a test strip for qualitative one-step assay of pesticides in a test sample by competitive immunochromatography.

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Fig. 3. a, Detection of Sm–OVA and b, 2,4-D–OVA by the dot immunoassay using 1a, the (MAb to Sm)–CG complex; 1b, the (MAb to 2,4-D)–CG complex, 2a, the (MAb to Sm)–HRP conjugate, and (2b) the (MAb to 2,4-D)–HRP conjugate.

The competitive immunochromatographic analysis of pesticides in water samples was carried out using the prefabricated test strips provided by the Fisher Scientific company (UK). A schematic representation of a strip is given in Fig. 1. A nitrocellulose membrane, which serves as a test zone, is mounted in the central part of a polystyrene base. Above and below the membrane, large-pore cellulose membranes are fastened, which act as taking up (lower membrane) and absorbing (upper membrane) regions. Preliminarily, we created a detecting band in the test zone of the nitrocellulose membrane by applying a line of polyvalent antigen, an OVA–pesticide conjugate containing 5–10 mol of pesticide per mol of OVA. In addition, antimouse antibodies were applied onto nitrocellulose in the form of a line (control band) above the detectable band; they served for the control of the correctness of analysis and the quality of test system. Preliminarily, the complexes of MAb to 2,4-D and CG or MAb to Sm and CG were applied onto the upper part of the taking up region of the test strip. After applying the components, the strip was thoroughly dried. The assay was started from the immersion of the lower end of the test strip into the aqueous solution of sample. The solution was sucked in by the taking up part of the strip, rises up the strip due to capillary

forces, and interacts with the CG-labeled specific MAb applied above. Then, all the reaction components move up to the test zone of the strip. The higher the content of the pesticide in the sample, the less the number of labeled antibodies bound to the polyvalent antigen sorbed in the solid phase. At a certain concentration of pesticide in a sample, a complete inhibition of binding of labeled antibodies to the polyvalent antigen occurs. As a result, only one (control) band of a bright rose color is observed in the test strip. If a sample contains no pesticide, or if its content is less than the level leading to the complete inhibition of the reaction, two brightly colored bands were observed in the test zone of the test strip. After 3–7 min, the assay process is completed. The results of a typical assay of 2,4-D and Sm by the method of competitive immunochromatography with the use of the conjugates of CG-labeled MAb to 2,4-D and Sm are given in Fig. 4. A complete inhibition of the reaction occurs at the concentrations of 2,4-D and Sm in the sample of 12 ng/ml. Our preliminary studies showed that the sensitivity of assay and the character of the reaction inhibition— the response of the + or – type (i.e., the band is present or absent) or of the +, ±, or – type (bright band, pale band, or no band) depends on the ratio of concentrations of the polyvalent antigen and the CG-labeled antibodies. The inhibition of the reaction by the + or – type takes place at the concentration of polyvalent antigen of 4 µg/ml and the concentration of antibody–CG complex of 1 OU520 unit [12]. We compared the results of the noninstrumental analysis and the competitive EIA. We also chose optimal concentrations of reagents to provide a maximum sensitivity of EIA: 1 µg/ml of polyvalent antigen (for 2,4-D and Sm); 2 µg/ml of the (MAb to 2,4-D)–HRP conjugate; and 2 µg/ml of the (MAb to Sm)–HRP conjugate. The sensitivity of EIA for Sm and 2,4-D was 12.5 ng/ml (Fig. 5). To evaluate specificity of the methods, the crossreactions between Sm, 2,4-D, and some compounds structurally related to 2,4-D were studied. The results given in the table indicate a high specificity of the developed methods. Thus, the method of qualitative one-step assay of the 2,4-D and Sm pesticides using competitive immunochromatography enables the detection of the pesticides in aqueous samples with a high specificity for 3–7 min. According to our scheme, the detection of any lowmolecular compound is possible in an aqueous sample provided MAb to this compound exist. The immunochromatographic assay procedure for the pesticides takes 3–7 min (detection of pesticides by the comb-format dot assay takes 45 min and by immunofiltration, 5– 15 min). No special devices are necessary for the immunochromatography. The testing procedure is very simple, one-step, and can be carried out in field conditions using easily accessible aqueous samples. The final result is assessed visually and is easily interpreted. Test

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Fig. 4. Detection of (a) simazine and (b) 2,4-D by the competitive immunochromatography assay. The concentrations of pesticides in samples (ng/ml): 1, 18; 2, 12; 3, 9; and 4, 6.

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Fig. 5. The dependence of optical absorption on the concentration of (a) simazine and (b) 2,4-D in competitive EIA.

strips for immunochromatography can be stored at room temperature for long periods of time. EXPERIMENTAL The following preparations were used: 2,4-D, simazine, and sodium citrate (Sigma, United States); horseradish peroxidase (Center of Agricultural Technology, Lvov; Rz 3.0); and gold chloride (Fluka, SwitRUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

zerland). Monoclonal antibodies to simazine were kindly provided by Dr. D. Knorr (Technical University of Munich, Germany). The preparation of biospecific reagents for detecting simazine and 2,4-D (the polyvalent conjugates Sm–OVA and 2,4-D–OVA [13], and monoclonal antibodies to 2,4-D [15]) was described previously. Complexes of antibodies with colloidal gold. A 1% solution of sodium citrate (1.2 ml) was added to a

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Assay of simazine, 2,4-D, and structurally related compounds by competitive immunochromatography and competitive EIA Sensitivity of assay, ng/ml, of Compound

Simazine 2,4-D 2-CPA 4-CPA

simazine immunochromatography 12 ** – –

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*Background level. **Test band is observed.

0.02% hydrogen tetrachloroaurate(III) (50 ml) in twice-distilled water upon boiling, and the mixture was further boiled for 3 min [9]. Immediately after the addition of sodium nitrate, the colorless solution of HAuCl4 turned to gray, then to violet, and finally to claret-colored. The resulting sol was cooled, and the mean diameter of sol particles was measured using a Coulter N4MD automated analyzer of submicron particles (Coultronics, France). The content of major fraction with a particle diameter of 20 nm was 95%. The concentration of the sol was 2.0 ± 0.05 OU520 unit/ml, as determined in absolute arbitrary units [12] from the maximum absorption in the visible spectrum region (400–600 nm) at a wavelength of maximal absorption of 520 nm and optical path length of 1 cm. The antibody–CG complexes were obtained by adding the antibodies to 2,4-D or Sm in PBS to a stirred gold sol (1 ml, 2.0 OU520 unit/ml, pH 6.5) to a concentration of 100–1600 µg/ml [10]. The mixture was incubated for 15 min at room temperature, an OVA solution in PBS was added to a concentration of 0.1%, the mixture was further incubated for 1 h at room temperature and then washed off from unreacted components of the PBS mixture by centrifugation (4°ë, 12500g). The complexes were stored at 4°ë. The working concentration of the MAb–CG complex was 2 OU520/ml for the dot assay and 1 OU520/ml for immunochromatography. Conjugates of antibodies with HRP were obtained by the method [11] with minor modifications. HRP was dissolved in water to a concentration of 10 mg/ml, and one tenth of its volume of freshly prepared 0.25 M sodium periodate was added. The mixture was incubated for 20 min at room temperature in the dark and dialyzed at 4°ë overnight against 0.1 M carbonate– bicarbonate buffer, pH 9.0. A solution of antibodies to 2,4-D or Sm preliminarily dialyzed against the same buffer was added and incubated in the dark for 3 h at room temperature. The peroxidase–antibody ratio was 2 : 1 w/w. Then, one tenth of the volume of freshly prepared sodium borohydride solution (4.0 ng/ml) in

10 mM NaOH was added, and the mixture was incubated for 2 h at 4°ë and dialyzed against PBS overnight at 4°ë. Determination of the sensitivity of the method for the pesticide detection using the complexes of antibodies to pesticides with colloidal gold. Aqueous solutions of the polyvalent antigen 2,4-D–OVA or Sm– OVA (2 µl) in a series of twofold dilutions (from 200 to 0.1 µg/ml) were applied in dots onto the surface of nitrocellulose strips (Schleicher & Schuell, Germany). The strips were air-dried, blocked with PBS/OVA for 30 min at room temperature, air-dried again, and stored in a hermetically sealed container at 4°ë. For the assay, strips were placed into a solution of the antibodies–CG complex (2 OU520/ml in PBS/OVA) or into a solution of the antibodies–HRP conjugate (4 µg/ml in PBS/OVA), incubated for 30 min at room temperature under continuous stirring, and washed three times with PBST. In the analysis of 2,4-D–OVA and Sm–OVA using (MAb to 2,4-D)–HRP and (MAb to Sm)–HRP conjugates for a visual assessment of the analytical signal, strips were additionally incubated in 50 mM imidazole buffer, pH 7.5, containing 2.0 mg/ml of 4-chloro-1-naphthol and 0.02% ç2é2 with the addition of 0.2 mg/ml of o-phenylenediamine and 0.1 mg/ml of sodium bisulfate [16]. Each assay was repeated three times. The detection of pesticides by competitive immunochromatography. A polyvalent antigen 2,4-D–OVA or Sm–OVA (1 µl, 4 mg/ml in PBS) was applied using a Hamilton syringe as a thin cross line of 3-mm width in the middle of a nitrocellulose membrane of a prefabricated immunochromatographic test strip (Fisher Scientifics, UK). Three microliters of either a (MAb to 2,4D)–CG or (MAb to Sm)–CG complex (concentration of 1 OU520/ml) was applied onto the upper part of the taking up part of the test strip. The chromatographic strip was dried for 12 h at room temperature and stored in a hermetically sealed container at room temperature. The storage time of the strips is up to 12 months. Serial 1.5-fold dilutions of the test sample (2,4-D or Sm) solution in PBST (50 µl) were prepared in the wells of immunological or culture microplates. For the assay, the taking up parts of immunochromatographic test-strips were immersed into the wells. Driven by the capillary forces, the sample solutions rose up the taking up parts of the test-strips and dissolved the antibodies– CG complex applied. Then the mixture of the sample and CG-labeled antibodies migrated to the test zone of the strip and passed through it with a rate of about 1.5 cm/min. After 3–4 min, the results of the assay can be visually assessed from the presence (negative test for the presence of a pesticide in the sample) or absence (positive test) of a bright rose band at the place of application of the polyvalent antigen. The work of the test strip was controlled by the presence of a violet control band. The solution containing no test compounds served as a negative control. Each assay was repeated three times.

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Detection of pesticides by competitive EIA. Either 2,4-D–OVA or Sm–OVA (100 µl, 4 µg/ml in 0.1 M NaHCO3, pH 9.2) was placed into the wells of a microplate for EIA (Costar) and incubated overnight at 4°ë. The plates were three times washed with PBST and blocked with PBST/OVA for 30 min at 37°ë. Serial twofold dilutions of test samples of Sm or 2,4-D (from 0.2 to 0.003 µg/ml) in PBST/OVA were prepared in the wells of immunological plates. Equal volumes of the (MAb to 2,4-D)–HRP or (MAb to Sm)–HRP conjugates (2 µl/ml) in PBST/OVA were added into the wells, and the plates were incubated for 1 h at 37°ë. The plates were three times washed with 250 µl of PBST. To detect the bound conjugate, 100 µl of 50 mM phosphate-citrate buffer, pH 5.0, containing 0.05% ç2é2 and 0.4 mg/ml of o-phenylenediamine (Serva) were added to each well and incubated for 5–10 min in the dark. The enzymatic reaction was quenched by adding 50 µl of 1.7 N ç2SO4. Each assay was repeated three times. Optical absorption at 492 nm was measured on a Multiscan multichannel spectrophotometer (Titertek). REFERENCES 1. Principles and Practice of Immunoassay, Price, C.P. and Newman, D.J., Eds., London: Macmillian Reference, 1999. 2. Birnbaum, S. and Uden, C., Anal. Biochem., 1992, vol. 206, pp. 168–171.

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