Immunochemical Technology for Environmental Applications

Chapter 7

Development of Various Enzyme Immunotechniques for Pesticide Detection

Downloaded by NORTH CAROLINA STATE UNIV on October 6, 2012 | http://pubs.acs.org Publication Date: May 5, 1997 | doi: 10.1021/bk-1997-0657.ch007

B. B. Dzantiev, A. V. Zherdev, O. G. Romanenko, and J. N. Trubaceva A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninskiy prospect 33, 117071 Moscow, Russia

Different enzyme immunotechniques were developed for the following pesticides: 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), simazine, atrazine, permethrin, phenothrin and their derivatives. The sensitivities obtained using solid-phase tech niques such as ELISA varied from 0.02 to 8 ng/mL, and the assay duration ranged from 1 to 2 hrs. A new homogeneous assay technique based on the inhibition of the catalytic activity of amylase-pesticide conjugate by anti-pesticide antibodies was examined. Two types of immunosensors that could measure 2,4-D and 2,4,5-T for 12 min with sensitivities close to ELISA were also developed. Furthermore, a new visual membrane immunoassay based on polycation-polyanion interaction is proposed. The latter allows detection of 2,4-D up to 10 ng/mL and is useful for on-site analysis.

Determination of pesticides in soil, water, and food is an actual problem nowadays. Enzyme immunoassay methods are very effective solutions to analytical problems because they combine the specificity of antigen-antibody interactions and the high sensitivity characteristic of enzyme detection. To date, there are enzyme immunoassay systems developed for more than 70 pesticides, as mentioned in a number of reviews Applications of a new analytical technique depend substantially on its quickness and simplicity. If a technique can reduce analysis time without significant loss of assay sensitivity, it becomes appropriate for a variety of tasks. Therefore, simple and fast assays, including the ones with instrument-free detection, are of great interest for pesticide residue monitoring and environmental quality studies. This paper will describe different enzyme immunotechniques developed in our laboratories for some of the widely used pesticides. Solid-Phase Immunoassays Enzyme-linked immunosorbent assay (ELISA) is the most traditional and extensively developed type of enzyme immunotechniques. We have developed ELISAs for four herbicides, namely 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), simazine and atrazine (Figure 1).

© 1997 American Chemical Society In Immunochemical Technology for Environmental Applications; Aga, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.


IMMUNOCHEMICAL TECHNOLOGY FOR ENVIRONMENTAL APPLICATIONS

3-Phenoxybenzoic acid Figure 1. Chemical structures of selected pesticides.

In Immunochemical Technology for Environmental Applications; Aga, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.


7. DZANTIEV ET AL.

Enzyme Immunotechniques for Pesticide Detection 89

Direct vs. Indirect ELISA. Monovalent antigens can be detected only by competitive ELISA formats, where a pesticide-protein conjugate and a free pesticide in the sample compete for the binding sites of the antibodies. Competitive ELISAs can either have labeled antibodies and immobilized pesticide-protein conjugate (indirect ELISA) or labeled antigens and immobilized antibodies (direct ELISA). Both formats were examined (7). For this purpose we have produced specific anti-pesticide polyclonal antibodies and have synthesized pesticide-protein conjugates. Then the ELISAs have been optimized, i.e. reagent concentrations and stages duration have been chosen in order to reach maximal assay sensitivities. In the cases of 2,4-D and 2,4,5-T, higher sensitivities were achieved using the scheme with labeled antigen (i.e. with pesticide-peroxidase conjugates), whereas for simazine and atrazine the scheme with labeled antibodies had 12-30 times better sensitivities (Table I). Table I. Detection Limits of Two Standard ELISA Formats Pesticide Detection Limit ELISA Formats 2,4-D 5 ng/mL The scheme with labeled antigen 20 ng/mL The scheme with labeled antibodies 2,4,5-T

The scheme with labeled antigen The scheme with labeled antibodies

8 ng/mL 50 ng/mL

Simazine


10 ng/mL 0.8 ng/mL

Atrazine


30 ng/mL 1 ng/mL

In addition to the traditional assay optimization methods, some additional approaches for improving ELISA sensitivity have been developed (8-9). Effect of Pre-incubation. First, the anti-pesticide antibodies were incubated with pesticide-containing sample before addition of the competitor (pesticide-protein conjugate). This approach proved to be useful for simazine and atrazine. The pre incubation of simazine with the antibodies increased ELISA sensitivity 16 fold (from 0.8 to 0.05 ng/mL), and in the case of atrazine the gain was 10 fold (from 1 to 0.1 ng/mL). On die other hand, the sensitivities of 2,4-D and 2,4,5-T assays did not change after the pre-incubation. Use of Protein A . Assay sensitivities using labeled antigen may be increased (2-5 fold for different pesticides) if antibodies are immobilized on polystyrene by means of staphylococcal protein A . The same effect was observed in earlier studies on testosterone ELISA (10). This effect may be attributed to the fact that protein A binds to the Fc region of the antibody molecule, resulting in a favorable orientation of the antibody for antigen binding in solution. Effect of Conjugation Ratios. Composition of the pesticide-protein conjugates significantly influences the assay sensitivity. Conjugates with minimal loading of antigenic groups have low rate of antibody-binding which prolongs the assay duration. On the other hand, increase in pesticideiprotein molar ratio leads to an increased conjugate-antibody equilibrium binding constant due to bivalent interactions. In contrast, free pesticide is not capable of bivalent binding, therefore, the sensitivity of the competitive assay falls.


90



We have synthesized 2,4-D-peroxidase and 2,4,5-T-peroxidase conjugates via hydroxysuccinimide and carbodiimide technique with different pesticide:peroxidase ratios. The compositions of the resulting conjugates were determined by comparing the ultraviolet spectra and the number of surface amino groups of the free protein and the hapten-protein conjugates. Amino groups were detected using 2,4,6-trinitrobenzensulfonic acid. Figure 2 shows that the conjugates with pesticide:peroxidase molar ratios of 2:1 and 5:1 are optimal for competitive ELISAs. To obtain such conjugates the initial molar ratio of the pesticide and peroxidase should be within the range of 4:1 to 16:1. Use of Monovalent Antibodies. The bivalent complex formation may be eliminated by using antibody monovalent derivatives, which can be prepared by reducing the IgG (antibody) S-S-bonds using hydrosulfite in the presence of cystein. The reduced IgG (half-antibody) may be purified by gel-filtration on Sephadex G-100. When the halfantibodies are immobilized on the solid support using protein A , the sensitivity of the 2,4-D ELISA with labeled antigen becomes 3 times greater than the sensitivity of the ELISA using native IgG (it changes from 5 to 1.5-2 ng/mL). Analyte Activation. The affinity of an antibody to a pesticide-conjugate may be significantly higher than its affinity towards free pesticide. For example, the affinities of the anti-2,4-D antibodies towards 2,4-D-BSA, 2,4-D-ovalbumin or 2,4-D-soybean trypsin inhibitor conjugates were about 10 times higher than the affinity towards free 2,4-D. However, this negative effect may be minimized if the free 2,4-D in the sample is conjugated with an amino acid or protein (8). Preliminary incubation of water samples containing 2,4-D with activation agents (N-hydroxysuccinimide and 1cyclohexyl-3(2-morpholinoethyl)-carbodiimide) and carrier (amino acid or protein: glycine, lysine or BSA) results in 10 to 30 times increase in sensitivity of the direct ELISA technique (using 2,4-D-peroxidase conjugate as competitor). The same gain in the ELISA sensitivity has been demonstrated for 2,4,5-T. Conjugation reaction was allowed to proceed for 15 min at 37°C to reach optimum sensitivity. The proposed technique does not require additional steps of separation because excess reagents do not interfere in the assay. The ratio of the sample:activation reagent:carrier was 4:1:1.

A 490 2,4-D/HRP molar ratio:

0.8

0.6

0.4

0.2

r

_l

0.01

0.1

I I I I Mil

I

I I I I III!

10

I

I I I I Mil

100

1 I I I I Mil

1000

[2,4-D], ng/mL Figure 2. Competitive curves for 2,4-D ELISA with 2,4-D-peroxidase conju gates of different composition.



7. DZANTIEV ET AL.

Enzyme Immunotechniques for Pesticide Detection

Addition of the activation step slightly increases the total duration of the assay from 60 to 80 min. The resulting limits of detection for the modified immunoassay techniques are presented in Table II. The coefficients of variation and recoveries were determined using 10 to 25 samples for each format. The pesticide concentrations varied from 0.5 to 5 ng/mL and each sample was analyzed in four repetitions. Measured C.V. in seria are 7-12%, and between series - 12-20%. Addition of pesticides in known concentrations (0.5-5 ng/mL) to water or milk, results in recoveries ranging from 80% to 115%. The modified enzyme immunoassays described above have considerable advantages. Whereas traditional ELISA optimization has permitted to reach only a few ng/mL as a limit of detection, the above modifications improved the detection to levels that correspond with the requirements for environmental monitoring and agricultural production control. The principles described here are universal and may be used in immunoassay development for other pesticides and other low-molecular weight compounds. Ta ble II. Detection Limits of Optimum f XISA Formats Detection Limit Pesticide ELISA Format The scheme with labeled antigen 2,4-D using immobilization of IgG monovalent fragments through 0.1 ng/mL protein A and the sample activation 2,4,5-T

Simazine Atrazine

The scheme with labeled antigen using immobilization of IgG monovalent fragments through protein A and the sample activation

0.02 ng/mL

The scheme with labeled antibodies using the sample pre-incubation

0.05 ng/mL

The scheme with labeled antibodies using the sample pre-incubation

0.1 ng/mL

Homogeneous Immunoassays Homogeneous immunoassays are based on modulation of the catalytic activity of enzyme-antigen conjugates by the formation of complexes with antibodies. Although homogeneous immunoassays are known to be fast and simple, its use has been hindered by the limited availability of suitable enzyme labels and conjugation chemistry that will cause significant changes in the catalytic activity of the enzyme. Therefore, it is important to find new labels that are appropriate for homogeneous assays. We have shown the suitability of alpha-amylase from Bacillus subtilis on the examples of homogeneous immunoassays for 3-phenoxybenzoic acid (derivative of some pyrethroid pesticides, see Figure 1) and 2,4-D (7). The necessary amylasepesticide conjugates were prepared via succinimide reaction, and purified from unreacted compounds of low molecular weight by gel-filtration using Sephadex G-25 and dialysis. They keep 60-70% of the initial enzymatic activity and contain about 5 to 10 pesticide groups (for different preparations) on one amylase molecule. The main advantage of a homogeneous assay is its methodical simplicity. In contrast to ELISA, the homogeneous enzyme immunoassay does not demand preliminary immobilization or long incubations. Thus, the homogeneous assay


91


92


developed in our laboratory includes three simple steps: 1) incubation of the pesticidecontaining sample and pesticide-amylase conjugate with the anti-pesticide antibodies, 2) addition of the enzyme substrate (starch hydrolysis), and 3) detection of the extent of hydrolysis. Specific anti-pesticide antibodies at high concentrations inhibit the catalytic activity of the pesticide-enzyme conjugate. On the other hand, if the pesticide is present in the sample, the pesticide reacts with the antibodies and displaces the pesticideenzyme conjugate, restoring the initial activity of the enzyme conjugate (Figure 3). Therefore, the higher the pesticide concentration in the sample, the greater the starch hydrolysis. The amount of starch hydrolysis may be quantified using either the starchiodine test, or the test for the presence of free aldhydes. In the former test, iodine is added to the test solution to indicate the presence of unhydrolyzed starch. If there is pesticide present in the sample, the enzyme amylase will be free and active, resulting in starch hydrolysis. This will be indicated by the discoloration of the iodine solution or low absorbance at 630 nm. On the other hand, a high absorbance at 630 nm means high amount of unhydrolyzed starch, and thus, low pesticide concentration in the sample.

ABSENCE

OF TESTED

' Ab

PESTICIDE

INHIBITED AMYLASE

Pest Starch

I PRESENCE

I OF TESTED

PESTICIDE

Starch

Figure 3. Principle of the proposed homogeneous immunoassay technique. Ab anti-pesticide antibodies, Pest - pesticide in the tested sample, Pest-E - conjugate of bacillary alpha-amylase with the pesticide.



7. DZANTIEV ET AL.

Enzyme Immunotechniques for Pesticide Detection

The occurrence of starch hydrolysis may also be monitored by detecting the presence of free aldehydes released during the breakage of glucose links in starch. The aldehyde test uses 3,5-dinitrosalicylic acid for color development. The color intensity is measured at 570 nm; a high absorbance corresponds to a high concentration of free aldehydes (greater starch hydrolysis), thus higher pesticide concentration. In both tests, the colored products are formed quickly after stopping of the starch hydrolysis; within 1 min for iodine probe and 5 min for interaction with 3,5-dinitrosalicylic acid. Concentrations of the colored products obtained were measured using standard photometers for ELISA. The amylolytic activity modulations prove to be sufficient for reliable detection of 3-phenoxybenzoic acid and 2,4-D. The assay time is 40 min, which is half the time required for the improved ELISA described above. However, the sensitivity of the homogeneous assay for 2,4-D (about 50-100 ng/mL) is poorer compared to ELISA, thus, its use is limited only to samples with high pesticide concentration. Nonetheless, such system is appropriate for testing solid and heterogeneous samples particularly when the pesticide had been extracted before assay. Determination of 3-phenoxybenzoic acid by the homogeneous assay results in better sensitivity as compared with the 2,4-D homogeneous technique. 3-phenoxyben zoic acid can be detected at concentrations as low as 5 ng/mL (Figure 4). As indicated in the figure, native pesticides permethrin and phenothrin, both containing 3-phenoxy benzoic group, also react with the antibodies and thus influence the conjugate catalytic activity. So the proposed technique may be used both for the native pyrethroid pesticides and products of their degradation. Electrochemical Immunosensors Immunosensors have become popular as immunoanalytical techniques. Their principal advantage is possibility to provide many assays in a short time with minimal handling procedures. We have developed two different types of immunosensors for 2,4-D and 2,4,5-T detection. The first type uses graphite electrodes with antibodies adsorbed on their surface (77). This assay is based on the competitive binding of pesticide and pesticideperoxidase conjugate with the antibodies, followed by the detection of activity of the bound peroxidase. An automatic potentiometric device measures the change in redox potential during the peroxidase reaction using 5-aminosalicylic acid and hydrogen peroxide as enzyme substrates. The optimum conditions for this assay have been determined, such as conditions for antibody immobilization and concentrations of reagents. The developed immunosensor allows detection of 2,4-D at concentrations as low as 40 ng/mL (both in water and biological fluids). The total time of the assay including electrode regeneration is 12 min. One electrode can be used for 60 sequential analyses. The measured signal retains no less than 85% of its amplitude after 20 analyses or after storage under the operating conditions at room temperature. The same parameters have been reached by the sensor for 2,4,5-T. The principle of the second immunosensor is based on potentiometric detection of the enzyme label by field-effect transistor (FET). The FET-sensor preparation includes the following stages (72). Aldehyde groups are introduced onto membranes (80-100 nm pore diameter) from regenerated cellulose on nylon net by photo-activation. Then, specific antibodies react with active groups at the membrane surface. After washing, the membrane is attached to the electrode. In this assay the quantity of formed complexes between the immobilized antibodies and 2,4-D-peroxidase conjugate reflects the amount of 2,4-D in the sample. Enzymatic activity of label on the solid phase is detected potentiometrically from pH changes during catalytic reaction. Peroxidase substrate solution used for this purpose contains 5-aminosalicylic acid, ascorbic acid, and hydrogen peroxide.


93

94



A 630

• Permethrin 0 I

i

i i i i 1111

0.1

i

1

i

i i i i in

i

i i i i nii

10

i—1_

100

[Pesticide], ng/mL Figure 4. Competitive curves for the homogeneous enzyme immunoassay of pyrethroid pesticides. Enzymatic activity of the 3-phenoxybenzoic acid-amylase conjugate is detected by starch-iodine reaction.

Sample (Pesticide)

Antipesticide antibodies

! •

Protein A polyanion

I

conjugate

Peroxidase pesticide conjugate Reaction mixture Holder 1 Membrane

(with polycation adsorbed)

Filter papers Holder 2 Substrate

o o • o o o • • • • o o o • o 0 •o o o • • • o • 0 • Developing

of spots'

color

Figure 5. Principle of the proposed visual membrane immunoassay for pesticides detection.


7. DZANTIEV ET AL.

95 Enzyme Immunotechniques for Pesticide Detection

At optimum conditions, this system permits detection of 2,4-D at concentrations as low as 1 ng/mL. The total time of testing is 45 min. The use of changeable membranes reduces the FET preparation and eliminates the electrode regeneration. The potentiometric measurement demands no more than 5 min.


Visual Membrane Enzyme Immunoassays The instrument-free assay with visual detection is a very promising direction in pesti cide immunoanalysis. We have proposed a new visual method based on "dot"-assay principle employing water-soluble linear polyelectrolytes (13-14). The polyelectrolyte interaction is used for quick reagent separation after immunochemical reaction. A pair of such polyelectrolytes was selected: salts of poly-N-ethyl-4-vinylpyridine (polycation) and polymethacrylate (polyanion). These ions interact with each other with extremely high affinity due to cooperative binding between their chains' links. Insoluble interpolymeric complexes are formed instantly. This reaction and its applications for enzyme immunoassays have been described (75). The scheme of the visual immunoassay for pesticides is presented in Figure 5. First, four solutions are mixed: 1) sample containing the pesticide to be determined, 2) pesticide-peroxidase conjugate, 3) anti-pesticide antibodies, and 4) staphylococcal protein A covalently bound to the polyanion. During the incubation, complexes are formed, such as polyanion-protein A : antibodies : pesticide and polyanion-protein A : antibodies : pesticide-peroxidase. Then, the reaction mixture is applied onto the nitrocellulose porous membrane where the polycation is already adsorbed (the membrane has been placed in special holder with microwells. The polyanion (and the immune complex bound to it) reacts with the immobilized polycation almost instantaneously. The other components are removed through the membrane into layers of filter paper by washing. Finally the membrane is placed in peroxidase substrate solution, giving insoluble colored product. In the absence of pesticide in the test sample, only polyanion-protein A : antibodies : pesticide-peroxidase complexes bind with the membrane; the quantity of the peroxidase immobilized is high, and therefore, the resulting spots are darker. In contrast, a decrease in the spot intensity (up to pale) corresponds to the presence of pesticide in the sample. This principle was realized for 2,4-D detection in water. The optimum assay conditions are presented at Table HI. The total duration of the assay is no more than 20 min. The proposed qualitative technique allows detection of 2,4-D in water samples at concentrations as low as 10 ng/mL. Table III. Optimum Conditions of 2,4-D Detection by the Proposed Visual Membrane Enzyme Immunoassay 5 pg/mL Concentration of protein A-polyanion conjugate 1 pg/mL Concentration of specific antibodies against 2,4-D 0.5 pg/mL Concentration of 2,4-D-peroxidase conjugate 10 min 450 nm

Duration of the reaction mixture incubation Membrane pores diameter Duration of the filtration and washing Peroxidase substrate solution

3 min N,N'diaminobenzidene + CoCl + H 0 2

Duration of the spot color developing

2

2

3 min


96



Conclusion The developed enzyme immunotechniques allowed the determination of 2,4-D, 2,4,5-T, simazine, atrazine, permethrin, phenothrin and 3-phenoxybenzoic acid. Solidphase techniques (ELISA) proved to be the best format for quantitative measurements; their proposed modifications improved the assay sensitivities to 0.02-0.1 ng/mL. Homogeneous techniques and immunosensors reduce the assay duration significantly. Measurements for the homogeneous technique using alpha-amylase can be realized by means of standard photometers for ELISA. The main advantages of immunosensors include reduction of handling procedures and possible automation of the analytical procedure. The proposed visual membrane immunoassay is useful for on-site qualitative determination of pesticide. Nowadays, we apply the developed rapid enzyme immunoassays (immunosensors, visual membrane assay) for the detection of j-triazines and pyrethroids in environmental samples. Literature Cited 1. 2. 3. 4. 5. 6. 7.

8. 9. 1995, 10. 11. 12. 1994, 13. 14. 15.

Mumma, R.O.; Brady, J.F. In: Pesticide Science and Biotechnology; Greenhalg, R., Roberts, T.R., Eds.; Blackwell Scientific, Oxford, 1987, 341-348. Jung, F.; Gee, S.J.; Harrison, R.O.; Goodrow, M.N.; Karu, A.E.; Braun, A.E.; Li, Q.X.; Hammock, B.D. Pestic Sci. 1989, 26, 303-317. Van Emon, J.M.; Mumma, R.O. ACS Symp. Ser. 1990, 442, 112-139. Gazzaz, S.S.; Rasco, B.A.; Dong, F.M. Crit. Rev. Food Sci. Nutr. 1992, 32, 197-229. Hock, B. Acta Hydrochim. Hydrobiol. 1993, 21, 71-83. Knopp, D. Anal. Chim. Acta 1995, 311, 383-392. Dzantiev, B.B.; Zherdev, A.V.; Moreva, I.Yu.; Eremin, S.A.; Romanenko, O.G.; Sapegova, L.A. In: Modern Enzymology: Problems and Trends; Kurganov, B.I., Kochetkov, S.N., Tishkov, V.I., Eds.; Nova Sci. Publ., Commack, 1995, 803-807. Dzantiev, B.B.; Zherdev, A.V.; Moreva, I.Yu.; Romanenko, O.G.; Sapegova, L.A.; Eremin, S.A. Appl. Biochem. Microbiol. (Moscow) 1994, 30, 752-759. Dzantiev, B.B.; Zherdev, A.V.; Romanenko, O.G.; Titova, N.A.; Trubacheva, J.N.; Cherednikova, T.V.; Eremin, S.A. Appl. Biochem. Microbiol. (Moscow) 31, 120-125. Rukavishnikova, G.E.; Dzantiev, B.B.; Liozner, A.L.; Eremin, S.A.; Sigal, E.R. In: Advances in Steroid Analysis '90; Gorog, S., Ed.; Akademiai Kiado, Budapest, 1991, 103-108. Dzantiev, B.B.; Zherdev, A.V.; Yulaev, M.F.; Sitdikov, R.A.; Dmitrieva, N.M.; Moreva, I.Yu. Biosensors & Bioelectronics 1996, 11, 179-185. Khomutov, S.M.; Zherdev, A.V.; Dzantiev, B.B.; Reshetilov, A . N . Anal. Lett. 27, 2983-2995. Dzantiev, B.B.; Zherdev, A.V.; Romanenko, O.G.; Izumrudov, V.A.; Zezin, A.B. In: Advances in Steroid Analysis '93; Gorog, S., Ed.; Akademiai Kiado, Budapest, 1994, 119-125. Dzantiev, B.B.; Choi, M.J.; Park, J.; Choi, J.; Romanenko, O.G.; Zherdev, A.V.; Eremin, S.A.; Izumrudov, V.A. Immunol. Lett. 1994, 41, 205-211. Dzantiev, B.B.; Blintsov, A.N.; Bobkova, A.F.; Izumrudov, V.A.; Zezin, A.B. Doklady Biochemistry (Moscow) 1995, 342, 77-80.


Immunochemical Technology for Environmental Applications

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