Determination of pesticides by enzyme immunoassay

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1061-9348/05/6003- 2005 Pleiades Publishing, Inc.0202Journal of Analytical Chemistry, Vol. 60, No. 3, 2005, pp. 202217. Translated from Zhurnal Analiticheskoi Khimii, Vol. 60, No. 3, 2005, pp. 230246.Original Russian Text Copyright 2005 by Morozova, Levashova, Eremin.The increasing use of pesticides, mineral fertilizers,pharmaceuticals, surfactants, and many other biologi-cally active substances results in serious environmentalproblems. Among the pesticides, chlorine-containingpesticides, which can accumulate and contaminate soil,water bodies, and food products, are most widely used;they exert toxic effects on human and animal organ-isms. These compounds are the most important pollut-ants in rivers and ground water [13]. Moreover, someorganochlorine pesticides such as chlorophenoxy acidsand their chlorophenol derivatives contain polychlori-nated dibenzodioxins and dibenzofurans (PCDDs andPCDFs), which are extremely toxic and stable com-pounds, as impurities. Dioxins are formed both in thecourse of the production of the chlorophenols and chlo-rophenoxy acid pesticides and during their metabolismin the environment.To solve environmental problems, contaminatedregions and contamination sources must first be estab-lished. The environmental monitoring of unfavorableregions is performed in all developed countries. How-ever, it is well known that this problem has received lit-tle attention. One of the reasons for this slow develop-ment of environmental monitoring consists in technicalproblems and high costs of analysis. Other reasons arerelated to the low productivity of analysis and thenecessity of performing routine determinations. Theabove problems are also typical of routine food moni-toring: simple and rapid techniques for determining pri-ority pollutants are required [4].DETERMINATION METHODS FOR PESTICIDESChromatographic analytical techniques are com-monly used for determining pesticides and their metab-olites in environmental samples and food products.Gasliquid chromatography (GLC) is used for deter-mining nonpolar pesticides, while liquid chromatogra-phy is used for determining polar and nonvolatile pesti-cides [2, 5, 6]. Thin-layer chromatography [7], flow-injection analysis [8], and capillary electrophoresis [9,10] are also in use. These techniques are indispensablefor the reliable identification of an analyte or for thesimultaneous determination of several pesticides. How-ever, these techniques are not free of disadvantages.Among these are expensive instrumentation and thedemand for highly skilled personnel. Moreover, theanalysis of each particular sample is preceded by time-consuming sample preparation, which takes from sev-eral hours to several days. Because of this, environmen-tal monitoring is difficult to perform; usually, a greatnumber of samples should be analyzed at regular inter-vals. Chromatographic instrumentation is continuallybeing developed to improve sensitivity and accuracy; atthe same time, the costs of instruments continue to rise.In addition, note that chromatographic techniques areunsuitable for rapid monitoring under field conditions.New instrumental techniques are currently underdevelopment. For example, a method based on flow-injection analysis with micellar-enhanced fluorescencedetection for determining 2,4-dichlorophenoxyaceticacid (2,4-D) and mecoprop [11] and a method with theuse of a PVC membrane electrode selective for pen-tachlorophenolate [12] were developed. These methodsprovide an opportunity to determine pollutants morerapidly; however, they are inadequately developed (andhence insufficiently sensitive) and require expensiveinstrumentation.Recent advances in analytical chemistry are associ-ated with the development of bioanalytical techniquesthat make it possible to overcome the above problems.REVIEWSDetermination of Pesticides by Enzyme ImmunoassayV. S. Morozova*, A. I. Levashova**, and S. A. Eremin** Department of Chemistry, Moscow State University, Vorobevy gory 1, Moscow, 119992 Russia** Institute of Physiologically Active Substances, Russian Academy of Sciences, Chernogolovka, Moscow oblast, 142432 RussiaReceived April 9, 2004AbstractImmunochemical methods of analysis, which are based on the binding of an antigen (pesticide)molecule to specific antibodies, are finding increasing use for determining pesticides in various samples (water,soil, food products, and biological fluids). Among these, enzyme-linked immunosorbent assay (ELISA), whichcombines the unique specificity of immunoassay with the high sensitivity of the detection of an enzymaticmarker, is the most widely used method. Moreover, in ELISA, the components of an immunochemical reactionare separated; as a consequence, the effect of interfering components in the sample (a so-called matrix effect)is reduced. In this review, the principles of enzyme immunoassay for pesticides are considered, and the deter-mination of pesticides in environmental samples and food products is exemplified. The main directions of thefurther development of immunoassay techniques for determining pesticides are also discussed. JOURNAL OF ANALYTICAL CHEMISTRYVol. 60No. 32005DETERMINATION OF PESTICIDES BY ENZYME IMMUNOASSAY 203The evaluation of the most commonly used currentlyavailable analytical techniques in terms of productivity,cost, and accuracy demonstrated that, economically,rapid immunochemical methods of analysis are moreconvenient for screening natural samples [1316].Immunochemical methods are based on a highlyspecific and highly sensitive reaction of an antigen withantibodies. Antibodies are proteins from the class ofimmunoglobulins (molecular weight of 150 000 Da)that are produced in the immune system of any verte-brate or human being as a result of a defense reaction(immunity) to a foreign substance (antigen). Antigen isa substance that induces the production of antibodies.However, substances with molecular weights lowerthan 1000 Da (for example, the majority of pesticides)are not immunogenic but acquire immunogenicity uponaddition to larger molecules (such as albumin-like pro-teins). These low-molecular-weight substances arereferred to as haptens; their antibodies are produced bythe immunization of laboratory animals with a proteinconjugate (immunogen).The advantages of immunochemical methods are asfollows: (1) simplicity and rapidity of determinations;(2) the possibility of automation and applicability toroutine analyses under field conditions; and (3) simpleand nondestructive sample preparation, which is mostoften not required for aqueous samples (so-called directassay). Immunochemical methods are also highly reli-able. Moreover, these methods do not require expensiveinstrumentation (the majority of immunochemicalmethods are based on photometric, fluorimetric, lumi-nescence, or electrochemical detection), and semiquan-titative evaluation is performed visually [17]. The nar-row specificity of determination and the effect of matrixcomponents are among the disadvantages of immu-nochemical methods. At the same time, the determina-tion of a group of substances rather than an individualcompound is of the greatest current interest for environ-mental monitoring. However, in recent years, antibod-ies have been produced and immunoassay techniqueshave been developed for substance-class specific anal-ysis, making it possible the determination of the con-centrations of an entire class of compounds with similarstructures and properties (for example, pesticides of thetriazine group [18] and herbicides of the sulfonylurea[18] and benzoylphenylurea groups [19]) in the testsample.Immunochemical methods are widely used in ana-lytical practice, various branches of medicine, andmicrobiological and food industries [2022]. Theyhave been successfully applied to the detection ofviruses, hormones, and pharmaceuticals in medicaldiagnostics for several decades. The advance of immu-nochemical methods can be readily traced using labo-ratory analyses in medicine as an example. A decadeago, chromatographic techniques dominated in thisarea. However, a chromatograph is currently used inmedical diagnostic laboratories only in specific caseswhen the results obtained by immunochemical methodsshould be confirmed. Various biologically active sub-stances are determined in increasing frequency with theuse of immunochemical methods. Immunochemicalmethods are finding increasing use in environmentalmonitoring; however, they are not as commonly used aschromatographic techniques.Enzyme-linked immunosorbent assay (ELISA)occupies a leading position among immunochemicalmethods. The fraction of ELISA in immunochemicalmethods for determining pesticides is about 90% [23].In enzyme immunoassay, the unique specificity ofimmunochemical analysis is combined with the highsensitivity of the detection of an enzymatic marker. Agreat number of publications in both Russian and for-eign journals were devoted to ELISA. Thus, the theoryof ELISA and the principles of its development andoptimization were considered in Russian reviews [2427]; the use of ELISA for determining pesticides wasalso discussed in [2831]. The fundamentals ofELISA and trends in the development of this tech-nique for determining pesticides were surveyed byforeign authors [3235]; procedures for the ELISAdetermination of pesticides in environmental samples[3638] and food products [2, 39, 40] were also con-sidered.The table summarizes the references and the maincharacteristics of ELISA procedures developed fordetermining pesticides and their metabolites. The deter-mination of certain pesticides was considered in ahandful of works, whereas more than hundred publica-tions were devoted to the determination of widely usedpesticides such as 2,4-D and atrazine. These herbicidesare most frequently used as model compounds inresearch studies on the optimization of various immu-nochemical methods for determining pesticides. Thedevelopment and use of ELISA methods for determin-ing atrazine were considered in review [41] and refer-ences [4246]; references [4752] may be consideredamong the most important and interesting publicationson the determination of 2,4-D.In this review, we consider the following items:(1) special features of the determination of pesticidesusing immunochemical analysis, (2) main principlesand stages in the development of ELISA methods,(3) recent advances in the development of ELISAmethods for determining pesticides and the use of thesemethods for analyzing real materials, (4) sample prep-aration procedures for immunoassay, and (5) possibledirections in the development of ELISA for determin-ing pesticides.ANTIBODIESAntibodies are the main reagent in ELISA. Thequality of the antibodies essentially affects the sensitiv-ity and specificity of analysis. The development ofimmunoassay procedures for determining pesticides 204JOURNAL OF ANALYTICAL CHEMISTRYVol. 60No. 32005MOROZOVA et al.Main parameters of the ELISA determination of pesticides and other pollutants in various test materialsAnalyte name Antibody type Test materialDetection limit, g/LIC50***,g/L Analytical range, g/L Refer-encesAlachlor P* Water, soil **** 0.727.9 [53]Alachlor P Water, vegetables 1.76 [54]Atrazine P Water, oil 0.7 110 [55]Acetochlor P Water 0.2 0.265 [56]Acephate P Water, vegetables 2 25 5140 [57]N-Acetyl-glufosinate P Water 17 2010000 [58]Azadirachtin P Coffee, cotton 75 0.51000 [59]Azinphos-methyl M** Water 6 nM [60]Bensulfuron-methyl P Water 0.002 0.09 0.010.60 [61]Bromophos-ethyl P Water, vegetables 0.3 3.9 11000 [62]Glyphosate P Water 0.6 125 [63]HCH P Water 20 [64]Hydroxytriazines M Water 0.01 0.154 0.0062 [65]DealkylatedhydroxytriazinesP Water 0.32 3.75 0.837.8 [66]Diazinon P Water, juice 0.05 [67]2,4-D M Water 0.05 0.55000 [68]2,4-D P Urine 19 [69]2,6-Dichlorobenzamide; dichlobenilM Water 0.05 190 [70]DDT M Water 0.4 [71]DDT P Water 0.2 [72]Deltamethrin P Water 1.1 17.5 [73]Dioxin P Soil 0.004 0.036 [74]Dicamba P Water 195 [75]Inabenfide M Water 0.1 1.3 [76]Rice 5 g/kg Imidacloprid P Water 0.1 0.8 [77]Carbaryl M Cucumbers,strawberries10 g/kg [78]CarbofuranMethiocarbCarbaryl M Vegetables, fruits 10 g/kg 101 g/kg [79]Carbofuran M Vegetables, fruits 10 g/kg 740 g/kg [80]Metolachlor P Water, soil 0.14.2 [81]Metolachlor mercapturate P Urine 0.220 [82]Molinate M Water 1 82 [83]Parathion-methyl P Water 0.05 0.9 [84]Nitrophenol P Water 0.61 9.32 [85]Pentachlorophenol P Water 0.1 0.330.5 [86]Pentachlorophenol P Soil 0.550 mg/kg [87]Polychlorinated biphenyls P Water 2 0.11000 [88]Propanide P Rice 1 g/kg [89] JOURNAL OF ANALYTICAL CHEMISTRYVol. 60No. 32005DETERMINATION OF PESTICIDES BY ENZYME IMMUNOASSAY 205became possible because of an exclusive property ofthe immune system to produce antibodies to moleculesof almost any structure. Antibodies to a great number ofpesticides have now been obtained, and they are readilyavailable (table).The production of high-affinity specific antibodiesis one of the most complicated priority problems in thedevelopment of immunochemical methods when anti-bodies to a particular pesticide are unknown. Antibod-ies produced by the B lymphocytes of animals as aTable. (Contd.)Analyte name Antibody type Test materialDetectionlimit, g/LIC50***,g/L Analytical range, g/L Refer-encesPropanide P Water 0.2 2.2 [90]Rice 20130Propoxur M Water 4.4 [91]Thiabendazole M Juice 1 200 [92]5 20 Thiram P Lettuce [93]Thiobencarb M Rice [94]Thiamethoxam P Water 9 [95]Triazines P Water 0.01 [96]Triazines (total) P Water 0.5 [97]Atrazine 0.1Triclopyr P Drinking 0.037 0.15.2 [98]3,4,6-Trichloropyri-dineand natural water 0.052 0.1362,4,5-T P Water, juice 0.0235000 [99]2,4,5-TP P Water, soil 0.05 [100]2,4,6-Trichlorophenol P Water 0.2 0.484.14 [101]2,4,6-Trichlorophenol P Water 0.2 [102]Urine (SPE purification) 12,4,6-Trichloroanisole M Wine 0.01 35 [103]Fenoxycarb P Leaves 1 g/kg [104]Fenitrothion P Water 6 [105]Fenitrooxon P Water 0.32 4.2 0.7127 [106]Fenthion P Water, rice, vegetables 1 16 1010000 [107]Fenbendazole M Milk 7 120 [108]Flucythrinate M Water 102000 [109]Soil 440 mg/kgapples 770 mg/kgtea 6006000 mg/kgFlutolanil M Water, rice 0.3 mg/kg 0.310 [110]Chlorpyrifos M Water 10 nM 1100 nM [111]Chlorsulfuron P Water 1 1100 [112]Cypermethrin P Water 1.3 13.5 1100 [113]Cyanazine M Water 0.01 [114]* P refers to polyclonal antibodies.** M refers to monoclonal antibodies.*** IC50 refers to the analyte concentration that decreases the analytical signal by 50%.**** Data are not given in the reference. 206JOURNAL OF ANALYTICAL CHEMISTRYVol. 60No. 32005MOROZOVA et al.response to the introduction of foreign substances (anti-gens) into the organism are referred to as polyclonalantibodies. Polyclonal antibodies are isolated from theserum of immunized animals (antiserum), and they areproteins from the class of immunoglobulins, heteroge-neous in physicochemical properties, primarily, speci-ficity and affinity. The success of immunizationdepends significantly on the following conditions: theconjugate immunogenicity, which depends on solubil-ity, molecular weight, and hapten content; immuniza-tion procedure; antigen presentation (with or withoutan adjuvant, a substance that enhances the immuneresponse); adjuvant in use; mode of antigen introduc-tion into the recipient, dose, and time constraints; andimmunized animal species [27]. In recent years, high-quality polyclonal antibodies to a number of pesticideshave been obtained; these have been subsequently usedfor the development of ELISA (table).Polyclonal antisera for experiments are commonlyobtained by the immunization of rabbits; recently,sheep immunization is being used in increasing fre-quency. The production of polyclonal antibodies is notvery labor intensive and does not require considerableappropriated funds, sophisticated equipment, or specialreagents. The advantage of using polyclonal antibodiesis that standardized antibody preparations cannot beproduced in large amounts at regular intervals becausean antiserum is a mixture of antibodies from variousclasses in arbitrary proportions. Antibodies heteroge-neous in specificity and affinity are required for per-forming group-specific analysis.Along with polyclonal antisera, monoclonal anti-bodies are used in immune methods for determiningpesticides [27, 32, 33]. Monoclonal antibodies are pre-pared with the use of the immune system of mice, fol-lowed by the application of hybridoma technologies.Monoclonal antibodies are identical in physicochemi-cal properties and specificity. By thorough screeningand selection of antibody clones, which are affine to aparticular determinant of the desired antigen, the unde-sirable activity of antibody preparations can be mini-mized because clones that give cross reactions can berevealed and rejected even at the early stages of screen-ing. In some cases, monoclonal antibodies providehighly sensitive and specific analysis [115]. The hybri-doma cell secretes antibodies in unlimited amounts thatdepend on only the stability of the cell, that is, its lifetime [116]. This is the most important technologicaladvantage of monoclonal antibodies over polyclonalantibodies. With the use of monoclonal antibodies,ELISA procedures for determining various pesticides,such as 2,4-D [47, 68], DDT [71], and molinate [83],were developed (table).The production of polyclonal antibodies is simpler,more rapid, and less expensive; therefore, polyclonalantibodies are used at the first stage of a study. Thequality of the resulting antibodies proven to depend onthe animal immune system, the immunogen, and theimmunization procedure. The use of polyclonal anti-bodies occasionally provides a higher sensitivity in thedetermination of an analyte than the use of monoclonalantibodies (table). The presence of antibodies in anantiserum is determined by their specific binding to alabeled antigen. The antisera prepared are characterizedby titer, antibody concentration, binding efficiency, andspecificity. The primary screening of both polyclonaland monoclonal antibodies is usually based on the lev-els of their positive responsestheir interaction withan antigen (in the case of haptens, with a labeled anti-gen conjugate or another carrier protein). It is also rea-sonable to take into account negative responsestheabsence of cross reactions with analogs of the test pes-ticide and with the carrier protein of the immunogen.The titer of polyclonal antibodies is determined as afinite dilution of an antiserum in an incubation medium,which allows a certain amount (usually, 50%) of alabeled antigen to be bound to antibodies [27, 35]. Thehigher the titer (it can be as high as 1/10 0001/100 000), the smaller the antiserum amount requiredfor performing ELISA.Along with monoclonal and polyclonal antibodies,imprinting polymers, which are sometimes referred toas synthetic antibodies, have been used in the past fewyears [117, 118]. In foreign publications, they aretermed molecule imprinting polymers (MIPs). They areprepared by the polymerization of special monomers,in a solution of which imprinting molecules (analyte)are present. The resulting imprinting polymers containcavities complementary to the imprinting molecule.These cavities are capable of specifically incorporatingthe analyte. High-stability synthetic imprinting poly-mers are used in ELISA, immunoaffinity chromatogra-phy, and immunosensors [118121].In the past decade, technologies for the productionof recombinant and chimeric antibodies have beendeveloped [38, 52]. In recombinant technologies, anti-body libraries are generated that include a set of possi-ble antibodies to a given compound. These librariesform the basis for the subsequent selection and modifi-cation of a required fraction. Molecular geneticsapproaches provide an opportunity to reproduce vari-ous amino acid sequences of antibodies on the surfaceof a bacteriophage and then to select the requiredsequences using an ELISA-like assay. It is now possi-ble to clone not only antibody molecules but also theirfragments containing the site of binding with an antigen(Fab fragments, the hypervariable regions of antibod-ies) in simple cell systems such as E. Coli [122].Recombinant antibody technologies make it possible toobtain more specific antibodies, to reduce nonspecificprotein interactions, and to vary other properties ofantibodies: to increase affinity, to change antigenicspecificity, and to improve the sensitivity of immunoas-say and the stability of antibodies [123, 124]. For exam-ple, recombinant antibodies have been used in thedetermination of atrazine [123] and chlorpyrifos [116].Yokozeki et al. [125] proposed a homogeneous non- JOURNAL OF ANALYTICAL CHEMISTRYVol. 60No. 32005DETERMINATION OF PESTICIDES BY ENZYME IMMUNOASSAY 207competitive immunoassay with enzymatic complemen-tation. The assay is based on the fact that, for some anti-bodies, the reassociation of separated heavy-chain vari-able-region and the light-chain variable-regionfragments becomes much more favorable in the pres-ence of an immunogen. At the same time, the fusion oftwo inactive -galactosidase subunits occurs to form anactive molecule.An alternative approach to the production of specificantibodies consists in using the receptors and enzymesof living systems, which bind a required antigen or agroup of compounds, and an attempt to simulate theirnatural ability in specific binding with the use of antiid-iotypic antibodies. Initially, antibodies of a startingreceptor are obtained; next, secondary antibodies of theFab fragment or the hypervariable regions of the pri-mary antibodies are produced. This technology pro-vides an opportunity to obtain antibodies not only witha given specificity but also with catalytic activity (withthe use of enzymes). The production of antiidiotypicantibodies has made it possible to develop a multiassayfor pesticide residues [126].Another approach to the production of antibodieswith specified properties consists in the expression ofsingle-chained antibody fragments (scFab), for exam-ple, in transgene plants. Even modified single-chainedvariable fragments (scFv) of known antibodies can beprepared. For example, clones obtained by molecularevolution are used for this purpose. The direct expres-sion of enzyme-labeled scFv prepared with the use of afusion technology in E. coli is also possible. These anti-bodies do not require an additional stage of the synthe-sis of a labeled conjugate [127].In general, the use of antibodies as a natural tool forthe recognition and separation of an analyte has a num-ber of advantages. Antibodies are readily available andcan be produced in any amount at low cost. At the sametime, very small amounts of antibody are required forperforming ELISA, so that, for example, immuneserum from a rabbit suffices for the determination of ananalyte in more than 5 million samples [16]. Antibodiesto most of pesticides have been prepared and success-fully used in ELISA (table).FUNDAMENTALS OF IMMUNOCHEMICAL METHODS FOR DETERMINING PESTICIDESImmunochemical methods, including ELISA, arebased on the reversible binding of an antigen (pesticide)to specific antibodies, which are specially prepared fora given analyte:,where Ab is a specific antibody, Ag is an antigen (pes-ticide), Ag : Ab is an antigenantibody immune com-plex, and Kas is the constant of formation of the com-plex.Ag + Ab Ag : Ab(free components)Kas(bound fraction)ELISA methods are classified in accordance with anumber of parameters: the reagent that is immobilizedon a solid phase, the reagent with an enzymatic markerintroduced, the system component used for determina-tion, the type of the analyte (competitive or noncompet-itive), etc. [27].The majority of pesticides are monovalent antigens;therefore, they are primarily determined with the use ofcompetitive ELISA methods. The principle of thismethod consists in the competitive interaction of alabeled antigen and an analyte with a limited number ofthe binding sites of specific antibodies. Figure 1 sche-matically illustrates the most widespread methods usedfor determining pesticides.In so-called direct ELISA (Fig. 1b), antibodies areimmobilized on a solid phase, whereas an enzymaticmarker is introduced into an antigen. In accordancewith this scheme, an analyte and the labeled antigen areadded to the immobilized antibodies. After incubationand washing the carrier for the removal of unboundcomponents, the formation of the immunocomplex isdetected by enzymatic activity on the solid phase,which is inversely proportional to the concentration ofthe antigen to be determined. The advantage of this pro-cedure consists in the small number of stages, whichprovides the capability of easily automating the analy-sis. The complexity and nonversatility of methods forthe synthesis of enzyme conjugates and the possibleeffect of sample components on enzyme activity areamony the disadvantages of this procedure. Neverthe-less, the majority of commercial kits for determiningpesticides are based on this scheme of assay [34]. Inrecent publications, a genetic engineering method forthe production of enzyme conjugates and the use ofthese conjugates for determining, for example, 2,4-Dwere reported [52].However, simpler methods and immunoreagents aremost frequently used in the development of new ELISAprocedures. So-called indirect ELISA with the use oflabeled secondary antibodies (Fig. 1a) is most com-monly used [128]. An antigen immobilized on a solidphase and enzyme-labeled secondary antibodies (anti-species antibodies against corresponding immunoglob-ulins) are used in indirect ELISA. Because the directadsorption of a low-molecular-weight antigen on thesurface of polystyrene plates is impossible or undesir-able in a number of cases, immobilization is performedthrough a macromolecular carrier protein, which is sub-sequently strongly bound to the solid phase. For thispurpose, the conjugate of a hapten covalently bound tothe carrier protein is initially prepared. In the synthesisof a pesticideprotein conjugate, the modification ofgroups that form the antigen determinant should beavoided. Moreover, in order to decrease nonspecificinteractions, a protein and a coupling agent other thanthose employed in the synthesis of the immunogenshould be used. This analytical procedure is commonlyused for screening the resulting antibodies. The use of 208JOURNAL OF ANALYTICAL CHEMISTRYVol. 60No. 32005MOROZOVA et al.a versatile labeled reagent provides an opportunity todetect antibodies to various antigens. The advantage ofthis procedure also consists in the possibility to elimi-nate the interference of various effectors contained inthe sample with the catalytic properties of the enzy-matic marker because the test sample and the labeledreagent are introduced into the system at differentstages. However, this analytical procedure makes theassay more complicated than direct ELISA becauseadditional stages are introduced.Peroxidase is commonly used as an enzymaticmarker; alkaline phosphatase is used more rarely, andother enzymes such as -galactosidase [35] and glucoseoxidase [112, 129] are used occasionally. Various tech-niques are used in ELISA for assessing enzyme activ-ity: spectrophotometry is the most widespread tech-nique; chemiluminescence [130] and fluorescencetechniques [131, 132], which are more sensitive, arealso in use. To perform ELISA, one of the componentsof an immune reaction is usually immobilized on asolid phase for the separation of bound and free antigenfractions; this is a so-called heterogeneous method ofanalysis. As carriers, 96-well plates of polystyrene aremainly used [18]. The use of polystyrene spheres, mag-netic particles [43], and modified glass tubes has alsobeen reported. These materials increased the surfaceand sorption capacity of a solid phase and therebyincreased the sensitivity of ELISA somewhat [33]. Themain types of solid-phase carriers were characterizedby Dankwardt [35].Heterogeneous ELISA makes it possible to separateeffectively the components of an analytical systembefore the stage of the determination of enzymaticmarker concentrations. Many examples of successfulIndirect ELISA Direct ELISA() (b)SSSSSPPPPesticideprotein conjugatePesticideEnzymepesticide conjugateSpecific antibodiesConjugate of specific antibodieswith enzymeEnzymesecondaryantibody conjugateSubstrateProductSPFig. 1. Schematic diagrams of (a) indirect and (b) direct ELISA. JOURNAL OF ANALYTICAL CHEMISTRYVol. 60No. 32005DETERMINATION OF PESTICIDES BY ENZYME IMMUNOASSAY 209ELISA applications to environmental monitoring, foodcontrol, and biological monitoring for pesticide resi-dues have been published. Moreover, ELISA kits fordetermining pesticides are commercially manufac-tured; these include all of the components required forthe determination in both laboratory and field [34, 133].However, ELISA exhibits some negative attributes.Among them are the possibility of the nonspecific bind-ing of components to a carrier, a long analysis timebecause the antigenantibody reaction on the surfacetakes a long time to complete, and the considerableeffect of the nonuniformity of the sorption properties ofpolymer carriers on the results of analysis.OPTIMIZATIONOF IMMUNOASSAY CONDITIONSThe immunoreagents (antibodies and haptens) pre-pared are tested under noncompetitive (in the absenceof the pesticide to be determined) and competitive con-ditions, and immunoreagent combinations that areappropriate for solving the problems in hand are cho-sen. After choosing the appropriate combinations, ana-lytical conditions are optimized taking into consider-ation the following parameters: assay format, analyticalsignal detection system, antibody and hapten concen-trations to be determined, incubation and/or preincuba-tion times, successive or simultaneous addition of thereagents, effects of blocking agents, effects of the pHand ionic strength of buffer solutions, and temperatureconditions [34]. All of these parameters are selected sothat a maximum sensitivity of assay is ensured with theretention of a high accuracy of the results.Along with the selection of the above conditions ofELISA, additional efforts may be made to improve thesensitivity of assay. The sensitivity of the determinationof a number of pesticides increases when specific anti-bodies are preincubated with the test sample before theintroduction of a competitor (a pesticideprotein conju-gate) into the system [42, 134]. The replacement of thedirect adsorption immobilization of antibodies by ori-ented immobilization onto a solid phase through pro-tein A or secondary antibodies is also favorable for anincrease in the sensitivity of determination with the useof immobilized antibodies. In the determination of var-ious pesticides, the use of protein A increased the sen-sitivity by a factor of 2 to 3 [2, 183]. Approximately thesame increase in sensitivity can be reached with the useof monovalent derivatives (so-called Fab fragments) inplace of native immunoglobulins. These monovalentderivatives are prepared by the reduction of SS bondswith the use of hydrosulfite in the presence of an excessof cysteine. The described effect is based on the preven-tion of the formation of bivalent bonds between theantibody molecule and the pesticideprotein conjugate,which cause a decrease in the sensitivity of determina-tion with the use of competitive immunoassay proce-dures.Another technique, which is also oriented towardrestricting the formation of bivalent antibodyconju-gate complexes, consists in optimizing a pesticidepro-tein molar ratio in conjugates. This technique consider-ably increased sensitivity (by a factor of 10 or higher)in the determination of 2,4-D and 2,4,5-trichlorophe-noxyacetic acid (2,4,5-T) pesticides. These substanceswere converted into conjugated form during preincuba-tion for 15 min due to the presence of reactive carboxylgroups; in this case, additional procedures for the puri-fication or stabilization of conjugates were not required[48].The specificity and sensitivity of any immunochem-ical method significantly depends on the quality of spe-cific antibodies. Nevertheless, the structure of a labeledcompound (tracer) has a noticeable effect on the analyt-ical characteristics of ELISA in many cases. The tracercan be tentatively separated into the following threestructural elements: a hapten, an enzymatic marker, anda spacer bridge between the hapten and the marker.Colbert et al. [135] found that the greater the size of themarker and the closer the marker to the antigenic deter-minant of the hapten, the greater the change in theimmunological properties of the tracer. The chemicalnature of a spacer bridge is often responsible for the sta-bility of the tracer; this may have an indirect effect onthe sensitivity of ELISA. It is likely that a change in thepolarity of the bridge reduces the nonspecific interac-tions and, consequently, increases the sensitivity ofimmunoassay [26]. The effect of the hapten structure inthe tracer on the sensitivity of ELISA was described inmany publications, for example, [43, 130, 136]. Exper-imental data were described in detail, for example, fordetermining 2,4,6-trichlorophenol using a competitiveELISA [137]. Five different conjugates for sorbtion,having different degrees of heterology with respect tothe immunogen, were studied. It was found that theaffinity of antibodies to the conjugate decreases with anincrease in the degree of heterology, whereas the sensi-tivity of determination increases. The most sensitiveELISA procedure was developed using a conjugatewith the highest degree of heterology [32, 43, 138].This was studied in more detail in the determination ofhormones. Thus, Giraudi et al. [136] studied tracerswith different (homologous and heterologous) spacerbridges between antigen and enzyme (horseradish per-oxidase) molecules in developing the ELISA determi-nation of progesterone. They found that heterologoustracers have lower Kas, than homologous tracers, andprovide the most sensitive determination.In studies on the effect of the structure of immu-noreagents on the parameters of determination, theauthors of the publications cited above primarily basedon empirical data and simple conclusions on the physi-cochemical fundamentals of the antigenantibodyinteraction. The most detailed approach to this problemconsists in the use of molecular simulation methods.Molecular simulation, which is oriented to the determi-nation of the molecular mechanisms of antigenanti- 210JOURNAL OF ANALYTICAL CHEMISTRYVol. 60No. 32005MOROZOVA et al.body binding, studies three-dimensional structures;electronic properties; hydrogen bond formation; andvan der Waals, hydrophobic, and electrostatic interac-tions. Structureactivity relationships are found andbinding models are calculated based on considerationof all the above with the use of computer programs.Theoretical approaches to the optimization of ELISAwere considered in a number of review publications[24, 27, 139]. Immunoassay methods, in particular,ELISA, are not labor intensive and do not require spe-cially trained personnel. Moreover, the process of anal-ysis can be easily automated; because of this, the pro-ductivity of determinations is dramatically increased,which is of importance for routine environmental mon-itoring.ELISA PRODUCTIVITY AND COSTSHennion and Barcelo [34] comparatively assessedthe cost effectiveness of ELISA using the determina-tion of pesticides (molinate, metalaxyl, chlorsulfuron,and diflubenzuron) as an example. The specific featuresof the ELISA technology allow an operator to performthe screening of more than 100 samples a day withouta considerable increase in analysis costs with increas-ing throughput. In this case, the operators work time isabout 2 h; a considerable time is taken to perform thestages of incubation.High-throughput automated laboratories, which use384- and 1536-well plates in place of classical 96-wellplates, have been increasingly incorporated into currentpractice. This provides an opportunity to multiplyincrease the productivity of an assay up to tens of thou-sand of samples a day [140]. At the same time, the pro-ductivity of analysis using procedures based on high-performance liquid chromatography (HPLC) is foursamples a day. Moreover, the reagents required for theELISA analysis of a sample are less expensive than thereagents used in GLC and HPLC by a factor of 20100,and instrumentation costs are lower by a factor of 5 to6. In many countries, ELISA is considered as the mosteconomically sound for environmental screening [13].APPLICATIONS OF ELISASince the latter part of the 1980s, the development ofimmunochemical methods for determining pesticideshas been particularly intensive [2, 14]. Hammock (Uni-versity of California, Davis, the United States) [32],Egorov (Moscow State University, Moscow, Russia),and Dzantiev (Bach Institute of Biochemistry, RussianAcademy of Sciences, Moscow, Russia) [141] are rec-ognized leaders in the development of ELISA for pesti-cides. In Russia, early publications on ELISA for pesti-cides have been prepared in the group of Chkanikovand Umnov at the Institute of Phytopathology [142],and the first review on the immunochemical methodsfor determining pesticides and growth regulators waspublished by Zhemchuzhin and Gorobets in 1990[143]. In Russia, the early publications in this area weredevoted to the development of the ELISA determina-tion of the following pesticides: 2,4-D [48, 144]; 2,4,5-T [48]; simazine and atrazine [42]; and chlorsulfuron[112, 142]. An extremely large number of proceduresfor determining various pesticides have been devel-oped. The table summarizes recently developed meth-ods for determining the most commonly used pesti-cides.The ELISA procedures described were adapted tothe development of test systems (ELISA kits) suitablefor the monitoring of pesticides in both the environment[15, 16, 145] and food products [2]. The determinationof pesticides with the use of commercial diagnostic kitsis primarily based on the principles of ELISA. Bakerand Millipore are leaders in this area. Ohmicron, Milli-pore, and Idetek manufacture commercial kits for theclass-specific ELISA determination of chloroacetanil-ides (alachlor, acetochlor, butachlor, metalaxyl, andmetolachlor) [13, 16]. Polyclonal antibodies are used inthe majority of commercial kits because their produc-tion is less expensive. The kits are most often based onmicroplates or tubes. In some cases, the determinationof pesticide degradation products rather than the pesti-cide itself can be effectively performed. This is due tothe fact that some pesticides are unstable in the environ-ment and undergo rapid degradation to form stablemetabolites, which can be no less toxic than the parentpesticides. Thus, 2,6-dichlorobenzamide is a dichlobe-nil degradation product [146]. ELISA procedures havebeen developed for determining the main metabolitesof the herbicides alachlor (2-chloro-2',6'-diethylaceta-nilide and diethylacetanilide [147]) and metolachlor(metolachlor mercapturate, which was detected inhuman urine [148]). An ELISA procedure for determin-ing spinosad and its metabolites in food products andenvironmental samples was described by Young et al.[149].SAMPLE PREPARATION IN ELISAEnzyme immunoassay is a good alternative toinstrumental analysis in the determination of pesticideresidues in various samples. However, the presence ofimpurities in the sample can affect the results of analy-sis; therefore, the determination of a pesticide is oftenpreceded by sample preparation. In some cases, amatrix effect can be removed using simple procedures.Thus, in the ELISA determination of the herbicide flu-tolanil in rice, rice samples were extracted with metha-nol (1 g/10 mL). The extract was filtered, and the filtratewas diluted with methanol by a factor of 5 and with aphosphate buffer solution by a factor of 10; the result-ing solution was analyzed. The linearity range of thecalibration function was 0.310 g/L with consider-ation for dilutions [150]. To determine the thiocarbam-ate herbicide thiobenzcarb in rice, the methanolextracts were filtered and diluted in a borate buffer solu-tion [151]. Rice samples were prepared in the same JOURNAL OF ANALYTICAL CHEMISTRYVol. 60No. 32005DETERMINATION OF PESTICIDES BY ENZYME IMMUNOASSAY 211manner for determining the inabenfide plant growthregulator [152]. However, not only liquid extraction butalso other types of extraction are frequently used for theremoval of impurities from the sample and for the sep-aration of an analyte; among these are solid-phaseextraction, ultrasonic extraction [153], microwaveextraction [154, 155], supercritical fluid extraction[156], etc. [2].Liquid extraction is used for the removal of sub-stances that are insoluble or sparingly soluble in water.The technique does not require special equipment andis easy to perform. The disadvantages of liquid extrac-tion consist in a large consumption of solvents, the pos-sibility of the formation of unavoidable emulsions atthe solventsample interface, a low rate of extraction,analyte losses during sample transfer and evaporation,and difficulty of automation [157]. All of these have ledto the development of alternative techniques that aremore rapid and less labor intensive while using muchsmaller amounts of organic solvents. Soxhlet extractionis an improved technique for the liquid extraction ofsolid matrices [158] with the use of a special extractor,in which a 5- to 50-g sample and about 100 mL of anorganic solvent or an aqueous organic mixture areplaced. The duration of this extraction varies from 4 to48 h.Microwave extraction is a new technique that hasbeen recently developed and applied to pesticideextraction from solid matrices [154, 155]. It is based onthe use of microwave radiation energy and has a num-ber of advantages over other extraction techniques:microwave extraction makes it possible to extract quan-titatively organic compounds from a matrix in 1015 min with the use of minimum amounts of organicsolvents (210 mL of a solvent per gram of a sample).In the past two decades, solid-phase extraction(SPE) has become the main alternative to labor-inten-sive and time-consuming liquid extraction. SPE isbased on the adsorption of a substance on a solid carrierunder the action of various noncovalent (usually hydro-phobic) sorption forces [159]. SPE combines the stagesof preconcentration and extraction of organic com-pounds from water [160, 161]. Special cartridgescoated with an adsorbent (C-18 cartridges, divinylben-zene, etc.) are immersed in the test solution for severalhours; next, the analyte is washed away from the car-tridge with a solvent and analyzed. The miniaturizationof SPE has resulted in the development of solid-phasemicroextraction (SPME) with the use of needles basedon Carbowax, divinylbenzene, and Carboxene for sorp-tion and preconcentration [162].Methods for the extraction and preconcentration ofliquids with the use of liquid membranes, semiperme-able liquid phases that separate two other solutions(donor and acceptor), have also been developed in thelast few years. The use of liquid membranes of anorganic solvent placed in the pores of a polymer sup-port is more convenient [110].Supercritical fluid extraction (SFE) requires the useof small solvent volumes and is highly selective [2].Supercritical fluids have a density close to the densityof ordinary liquids; however, they exhibit a lower vis-cosity and a high diffusion coefficient. Because of this,supercritical fluids extract pollutants more rapidly thanunder conditions of ordinary extraction [163]. As asupercritical fluid, CO2 is frequently used (critical tem-perature of 31C). An automated process makes it pos-sible to perform extraction and purification rapidly in asingle stage. Good results were obtained for the separa-tion of nonpolar and weakly polar pesticides, polychlo-rinated biphenyls (PCBs), and dioxins [156].Immunoaffinity chromatography is a special samplepreparation technique based on biospecific interactions.The operating principle of immunoaffinity chromatog-raphy consists in the use of a complexation reactionbetween an antibody immobilized on a solid carrier andan antigen (pesticide), which is supplied and theneluted with a mobile phase. The fundamentals of thistechnique were considered in review [164]. Immunoaf-finity chromatography makes it possible to extractselectively either a single compound or groups ofrelated substances from samples of complex composi-tion. Immunoaffinity chromatography also solves theproblem of time-consuming multistage sample prepa-ration and, consequently, improves the accuracy ofanalysis. The possibility of adsorbent regeneration andrepeated use for a long time are among the advantagesof immunoadsorbents over the adsorbents used in SPE.As compared with liquid extraction, the consumptionof organic solvents is considerably reduced because thedesorption of substances is performed using a smallvolume (14 mL) of an organic solvent. To extract amaximum number of substances from a group, mixedimmunosorbents are also used; these consist of anti-bodies against different compounds of the given group[165] and provide complete extraction. Immunosor-bents are responsible for high sensitivity and selectivityin the analysis of various materials characterized bycomplex matrix composition and the presence of impu-rities: surface water, wastewater, industrial effluents,biological fluids, and food products [166].CURRENT TRENDS IN THE DEVELOPMENTOF ELISA FOR THE DETERMINATION OF PESTICIDESThe immunochemical analysis of environmentalsamples is now being intensively developed andimproved. The requirements imposed on the sensitivityand selectivity of determination are growing every year,and the variety of environmental pollutants becomeswider. ELISA methods for determining not only classi-cal pesticides (atrazine and 2,4-D) but also their metab-olites, as well as polyaromatic hydrocarbons [167,168], PCBs [88, 163], and dioxins [74, 169, 170], arecurrently under development. Dioxins are particularlycomplex immunogens, which require the use of non- 212JOURNAL OF ANALYTICAL CHEMISTRYVol. 60No. 32005MOROZOVA et al.standard procedures in the production of antibodies ora change in the assay format. Moreover, the maximumpermissible concentrations (MPCs) of new pollutantsare low (at a level of picograms for dioxins) so that clas-sical ELISA procedures become inapplicable. New ver-sions of immunochemical methods, such as a homoge-neous phosphorescent immunoassay for dioxins [171],are under development.To perform continuous monitoring in polluted andsuspect regions, reliable analytical results must be rap-idly obtained by processing a very great number ofsamples. In this context, the main trends in the develop-ment of immunoassay techniques are the following:(1) the reduction of the assay time, (2) the possibility ofperforming analysis under field conditions, (3) thereduction of analysis costs, (4) the miniaturization ofanalysis, (5) the automation of determination, and(6) the simultaneous determination of several analytes(multiassay).To shorten the determination time, homogeneousmethods are used that do not include the stages ofreagent separation (for example, fluorescence polariza-tion immunoassay [172]). Methods based on imprint-ing polymers in place of antibodies result in assays thatare less expensive and more environmentally safe.Rapid tests for the use under field conditions havebeen developed for rapid environmental monitoring.These tests may be exemplified by the determination ofpentachlorophenol in water and soil with visual detec-tion at a level no lower than 1 mg/L. The method isbased on the agglutination of latex microparticles withgrafted pentachlorophenol upon binding to antibodies.The assay time is 5 min [173]. An immunoassay for thedetermination of atrazine and other triazines is based onthe same principle; atrazine can be determined in soil ata level of 50 g/kg or higher and in water at a level of1 g/L or higher [174]. Weetal and Rogers [175]described immunoanalytical test strips for determining2,4-D. In this method, antibodies are adsorbed on col-loidal gold particles; this complex migrates along thestrip with the sample and reaches the 2,4-DBSA con-jugate. The visual determination of 2,4-D at a levelfrom 0.02 mg/L can be performed. Wang et al. [176]described test strips for determining diflubenzuron incereals at a level from 0.75 mg/kg. The developed colorintensity is determined with the use of a portable pho-tometer; the total analysis time with sample preparationis 15 min.Various aspects of immunoassay miniaturizationwere considered in [177179]. The use of disposableglass capillaries coated with carboxymethyldextranwith grafted monoclonal antibodies is an example ofthe miniaturization of ELISA with chemiluminescencedetection. The method decreases the detection limit of2,4-D to 0.02 ng/L [130]. The determination of2,4,6-trichlorophenol in a microdroplet (58 m) is per-formed with the use of a homogeneous immunoassaybased on fluorescence quenching. The detection limitsof 2,4,6-trichlorophenol in water and urine are 0.04 and1.6 g/L, respectively. When this assay is performed ina microplate, the detection limit is 0.36 g/L [180].Microchips, miniature analytical devices, have becomepopular in recent years [178, 181]. Thus, for example,an ELISA method for determining atrazine in a micro-chip flow system was described [182].Some publications [23, 182, 183, 184] were devotedto the group determination of pesticides and otherorganic pollutants. A multiassay is based on either theuse of group-specific antibodies, which provide thedetermination of pesticides from one group, or thecombined use of antibodies for different analytes.The automation of analysis has resulted in the devel-opment of flow-injection immunoassay, which is capa-ble of continuously analyzing a great number of sam-ples [8, 60, 185, 186]. Highly productive assays can beperformed with the use of automated microplateimmunochemical methods based on immunoenzyme[93, 110], fluorescence [131, 187], and other immu-noassay modifications. These systems are the most eco-nomically feasible systems for the purpose of routinescreening.Attempts to improve the performance characteris-tics of analytical procedures and to reduce analysiscosts have resulted in the use of new types of antibodies(recombinant, antiidiotypic, and synthetic antibodies)or only individual antibody fragments (Fab fragmentsand single-chained antibodies) (see Antibodies sectionabove). The development of the above lines in the elab-oration of immunoassay techniques has also resulted inthe appearance of new more practically feasible typesof immunochemical analysis. Capillary electrophoreticimmunoassay with electrochemical detection reachesdetection limits at a level of tens of ng/L [188]. Biosen-sors [189] and immunosensors, miniature instrumentsbased on specific immune recognition and electro-chemical detection [190], have been intensively devel-oped in the last few years.Many immunosensors are based on fluorescencedetection [191, 192]. For example, a fiber-optic immu-nosensor for determining 2,4-D uses a fluorophore andconsists of a compact solid-phase laser source and aphotodetector. The detection limit of 2,4-D is 60 pM[191]. Other examples are the following: an electro-chemiluminescence immunosensor for determining2,4-D with a detection limit of 0.2 g/L [193], a biosen-sor for determining 2,4-D (0.2288.4 g/L) with theuse of imprinting polymers [194], and a biosensor for2,4,6-trichloroanisole with the use of so-called screen-printed electrodes [195]. Piezoelectric immunosensorsbased on a gold electrode were developed for determin-ing 2,4-D [192] and atrazine [196]. An immunosensorbased on competitive immunoassay with the use of sur-face plasmon resonance (working range) was devel-oped for determining 2,4-D (3100 g/L) [197].Systems based on photoinduced electron transferare quite new. For example, a solgel luminescence JOURNAL OF ANALYTICAL CHEMISTRYVol. 60No. 32005DETERMINATION OF PESTICIDES BY ENZYME IMMUNOASSAY 213sensor was developed with the use of imprinting poly-mers specific to 2,4-D [198]. An electrochemical deter-mination method with amperometric transducers wasalso proposed using a portable device for measure-ments under field conditions; it exhibits a wide linearityrange and a low detection limit [199].Currently available approaches provide an opportu-nity of performing time-resolved observations of bind-ing both with the use of labeled reagents and withoutthe use of labels (for example, reflectometric interfer-ence spectroscopy). Two methods for the fluorescenceimmunoanalysis of estrogens have been described: aheterogeneous method with the use of internal reflectedfluorescence and a homogeneous method with fluores-cence energy transfer. These approaches are used formultiassay because the reading of signals from differ-ent sites in a waveguide makes it possible to detect sev-eral signals from a sample in a short time, especiallywhen an optical transducer is connected to a CCDchamber [200].* * *Note that in the 1960s1970s, hormones and drugsin human serum were determined by chromatographictechniques, whereas only immunochemical methods ofanalysis (classical ELISA methods and test-strip sys-tems) are in current use in medical diagnostics. As aresult of the elaboration of various immunoanalysistechniques, effective methods have been developed inthe last few years for monitoring pesticides and otherorganic pollutants in the environment [16, 3638,133]. 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