Table Of Content

1 IMMUNOCHEMICALASSAYSINPESTICIDEANALYSIS Immunochemical Assays in per trillion to lower parts per billion range.A lot of samples can be analyzed within a short time, while only low Pesticide Analysis samplevolumesarenecessary.Inmanycases(water,some liquid food samples) no extraction step and no cleanup arenecessary.Notallassaysarecompletelyspecifictoone AndreaDankwardt single compound. Cross-reactivities of the Abs with hap- SensionGmbH,Augsburg,Germany tenssimilartotheanalytecanbeobserved.Insomecases, matrix effects may occur, especially with soil or colored food extracts. Therefore, validation of the assays for the matrixofinterestshouldbecarriedout.AsIAsareusually 1 Introduction 1 targeted at a single analyte or a group of analytes, multi- 2 Antibodies 2 analyte approaches using Ab arrays or a combination of 2.1 Antibody Structure 2 immunochemical techniques with liquid chromatography 2.2 Antibody Production 3 (LC)arepursued. 2.3 Immunogens 3 3 Immunoassay Types 5 3.1 Assay Formats 5 1 INTRODUCTION 3.2 Labels 5 3.3 Solid Phases 6 Interestinimmunochemicalassaysforthedetermination 4 Properties of Competitive Immunoassays 6 of pesticides has been steadily increasing. IAs are now 4.1 Dose–Response Curve 6 commonly applied for the analysis of contaminants 4.2 Quality Control 7 in water, soil, food and body fluids..1–9/ The first 4.3 Cross-reactivity 8 immunologicalexperimentshadalreadybeencarriedout 4.4 Sample Preparation and Matrix as early as the late eighteenth century when Edward Effects 8 Jenner, an English physician, used cowpox to prevent 5 Application to Environmental Samples 9 infection with smallpox. Based on these studies Louis 5.1 Water 9 Pasteur developed the use of attenuated strains of 5.2 Soil 13 microorganisms for successful vaccinations. Emile Roux 5.3 Food 13 andAlexandreYersinthenfoundthatimmunityiscaused 5.4 Biomonitoring 14 by soluble compounds of microorganisms, which they 6 New Developments 14 calledtoxins.Thesetoxins inducespecificcompounds in Acknowledgments 16 theimmunizedanimal,whichwerenamed‘‘antitoxins’’by Emil von Behring and Shibasaburo Kitasato (1890) and Abbreviations and Acronyms 16 arenowcalledAbs.TheAb‘‘generating’’compoundsare Related Articles 16 knownasAgs. References 16 AroundtheturnofthecenturyitwasshownthatAbs arenot only produced against microorganisms and their toxins,butalsobyothersubstancessuchasmilk,protein or plant-derived toxins. Paul Ehrlich was the first to Immunochemical assays (immunoassays, IAs) are bio- carry out quantitative studies on Ag–Ab interactions. chemicalassayswhichworkaccordingtothelawofmass The great interest in this field led to the first book action. They are based on the recognition of an antigen on immunochemistry, published by Svante Arrhenius in (Ag) or a hapten by antibodies (Abs). Abs are serum 1907..10/KarlLandsteineralsobelongstothepioneersin glycoproteins of the immunoglobulin (Ig) class and are immunochemistry.Hesystematicallyusedsmallartificial producedbythevertebrateimmunesystemagainstforeign molecules, which he called haptens, coupled to a carrier materialofhighmolecularmass.Theresultofthebinding molecule for immunization. In 1923 Heidelberger and reaction between the Ab and an analyte is usually made co-workersfoundpolysaccharidestobeantigenicaswell. visiblebymeansofenzymatic,chemiluminescent,fluores- StudiesbyRodneyPorter(1959)andGeraldEdelman cent or radioactive markers. According to the label used (1961) have provided the chemical structure of the Ab IAs can be classified into enzyme immunoassays (EIAs), molecule.TheenormousvarietyofAbswasexplainedby radioimmunoassays (RIAs), fluorescence immunoassays FrankMcFarlaneBurnetin1957, basedonahypothesis (FIAs)orchemiluminescentimmunoassays(CLIAs).The ofNielsJernedatingfrom1955,thenowwidelyaccepted measuring range of most IAsfor pesticides is in the parts clonal-selection theory. It describes each Ab-producing EncyclopediaofAnalyticalChemistry EditedbyRobertA.Meyers.(cid:211) JohnWiley&SonsLtd,Chichester.ISBN0471976709 2 PESTICIDES cell as carrying on its surface only one type of Ab as a receptor. The binding of a respective Ag to this Occupancy of antibody binding sites by analyte receptorleadstoaclonalexpansionofthiscellandtothe maturationofAb-producingcells. Immunochemical methods have their origin in the medicalfield.ThefirstIA,aRIAforthequantificationof Occupied Unoccupied insulininserum,wasdescribedbyYalowandBerson..11/ binding sites Later, radiolabels were replaced by enzymes in EIAs by Engvall and Perlmann.12/ and Van Weeman and Competitive Measurement of Schuurs..13/ Since then radiolabels have obtained broad assays unoccupied application in medical diagnostics and environmental binding sites analysis. IAs belong to the most common methodology in the (a) Immobilized antibody (b) Immobilized coating field of immunoanalysis. Even though Abs are (still) conjugate produced by a biological process, IAs are nevertheless chemical analytical procedures. The basic principle applyingtoallimmunoreactionsisbaseduponthelawof mass action. In the equilibrium reaction between a free Agorahapten,suchasapesticide,andtheAbformingthe hapten–AbcomplexHAb(Dboundhapten),represented Inverse relation between Inverse relation between byEquation(1), antibody-bound tracer and coating conjugate-bound analyte concentration labelled antibody and HCAbDHAb .1/ analyte concentration theaffinityconstantKdeterminestheconcentrationratio betweentheboundhaptenandthefreereactionpartners, Antibody Iamntmiboobdiylized Lanatbibeoleddy Equation(2): Analyte Immobilized Labeled [HAb] K D mol(cid:0)1 .2/ (hapten) analyte analyte [H][Ab] A low detection limit (DL) in an IA therefore requires Figure1 Principle of the competitive IA. In the first format a high affinity of the Ab toward the analyte, which is withimmobilizedAb(a)theplatesarecoatedwithAb.Analyte expressed by a high affinity constant. For further details andenzyme-labeledanalytecompetefortheAbbindingsites.In refertoHocketal..14/ thesecondformat ahapten–proteinconjugateisimmobilized in the solid phase(b). This protein conjugate and the free IAs are based upon the measurement of Ab binding- analyte compete for the binding sites of the Ab in solution. siteoccupancybytheanalyte(Figure1).Thisreflectsthe (Reproduced from Dankwardt and Hock.7/ with permission analyte concentration in the sample. Since the binding fromFoodTechnologyandBiotechnology.) reactiondoesnotproduceasignalwhichcanbedetected by simple means, various markers, e.g. radioactivity, enzymes, or fluorescence, are employed for the detec- of the Ig class produced by the immune system against tion of the immunoreaction (see section3.2). However, foreign material such as pathogens or xenobiotics, and moresophisticatedtechniqueslikesomeimmunosensing bindthetargetsubstancewithhighselectivityandaffinity. methodsdonotrelyonalabel(seesection6). Although there are five distinct classes of Ab in most higher mammals (IgA, IgD, IgE, IgG, IgM) IgG makes up approximately 80% of the total Ig in human serum. MostIAsrelyuponIgGasthemajorIg. 2 ANTIBODIES The basic structure of an Ab molecule is shown in Figure2. It consists of two identical heavy (H) chains 2.1 AntibodyStructure and two identical light (L) chains stabilized and linked Immunochemical analysis is based upon the specific by inter- and intrachain disulfide bonds. The H- and reaction between an Ab and its corresponding Ag or L-chainsareorganizedintovariableandconstantregions. hapten.Absarepartofthevertebratedefensesystem(for The Ag binding site (combining site) is formed by the moredetailsrefertoimmunologytextbooks,forexample association of parts of the variable regions of the H- Golub.15/ and Roitt.16/). They are serum glycoproteins and L-chains, located at the amino terminal end. The 3 IMMUNOCHEMICALASSAYSINPESTICIDEANALYSIS Heavy chain FR1 Antigen binding site of interactions such as hydrophobic, ionic, H-bonding, Light chain FFRRF 4 R32 CDR1 bpi-npdeinlegcetrnoenrgiynt(errealacttiivoen,afafinnditvyaonfdtheerAWba)ailnscfroeracseess.wTihthe VH CDR2 VL CDR3 the number of specific chemical interactions between the analyte and the amino acid residues in the Ab combining site. Therefore, the selectivity and sensitivity CH1 ofanIAiscontrolledbythenatureoftheAg–Abbinding CL VL VH process. 2.2 AntibodyProduction VL VH CH2 Fv Ab production is conveniently carried out in warm Carbohydrate bloodedanimals,e.g.rabbits,sheep,miceorchickens..17/ Polyclonal antibodies (pAbs) are obtained from the CL CH1 CH3 CL VH serum and comprise a mixture of different Ab popu- lations.Monoclonalantibodies(mAbs)consistofasingle monospecific Ab population. These Abs are produced Fab Antibody scFv in cell culture by a single hybridoma cell derived from the fusion of B-lymphocytes with myeloma cells..18/ The Figure2 StructureofIgGAbsandtheirfragments(modified hybridoma cells can then be propagated almost indefi- afterHocketal..17/).scFvistherecombinantantibodyfragment, nitely in culture and will continue to produce the Ab of asinglechainfragmentcontainingonlythevariableregion,FR the lymphocyte parent. Since an individual lymphocyte istheframeregion,V isthevariableregionoflightchain,V L H isthevariableregionofheavychain,C istheconstantregion produces only a single Ab type, all of the Ab molecules L onthelightchain,CH ,CH ,CH isaconstantregiononthe produced by a hybridoma cell line derived from a sin- 1 2 3 heavychain. gle hybrid cell are identical and have the same binding properties. Therefore, the hybridoma technology guar- variable regions of both chains are organized into three antees the unlimited production of mAbs with constant hypervariable or complementarity determining regions characteristics..19/ Owing to the great effort involved in (CDRs) separated by four framework regions. The mAb production many IAs still employ pAb. A third greatest amino acid sequence variation occurs within possibility for creating Abs has emerged, recombinant the CDRs whereas the framework regions are more antibody(rAb)techniques.Here,Iggenescanbecloned, conserved. It is assumed that the association of the introduced and expressed in inexpensive and relatively CDRregionsformsthecombiningsite.Thelowerpartof simple host systems..20;21/ Although several nonmam- the molecule, the Fc (antibody fragment containing the malian host systems (yeast, plant and insect cells) have crystallizablefragment)isresponsibleforsomeimportant been used to produce rAbs, the most common vehicle biologicaleffectorfunctionssuchascomplementfixation is Escherichia coli..22;23/ The main properties of pAbs, andisnotnecessaryforAgorhaptenbinding.Itcontains mAbsandrAbsarelistedinTable1. the last heavy chain domains. The whole of the Ig molecule or Ab fragments, F(ab) and Fab (antibody 2 2.3 Immunogens fragments containing the antigen binding site(s)) can be usedinIAs. Mostpesticidesareoflowmolecularmassandtherefore A substance that after injection into the body of a arenot ordinarily antigenic. Theyhave to be coupled to vertebrateinducesaspecificAbsynthesis,iscalledanAg. a carrier molecule, usually a protein, in order to induce Agsareprincipallymacromolecules,forinstanceproteins, anAbresponseinthevertebrateimmunesystem..14/The polysaccharidesornucleicacids.Syntheticpolymersalso site of coupling to the carrier, the coupling procedure belongtotheantigens,i.e.theycanbeusedas,oractas, as well as the number of haptens bound to one Ags. Small molecules (haptens) such as pesticides have carrier molecule can be of major importance for the tobecoupledtoamacromolecularcarriertoelicitanAb sensitivity and the selectivity of the resulting Ab (for response(seesection2.3).TheabilityofanAbmolecule reviews refer to Erlanger,.24;25/ Goodrow etal..26/ and to bind an Ag or a hapten specifically is controlled Szurdokietal..27/). by structural and chemical interactions between the The protein carriers used in various laboratories ligand and the Ab at the combining site. The Ag–Ab include globulin fractions, serum albumins of different interaction is reversible and does not involve formation species, hemocyanin, ovalbumin, thyroglobulin, and fib- ofcovalentbonds..16/ Thebindingisaresultofavariety rinogen.Alsononproteinaceouscarriershavebeenused 4 PESTICIDES Table1 Propertiesofpolyclonal,monoclonalandrecombinantAbs Properties pAb mAb rAb (Abfromserum) (Abfromhybridomacells) (Abproducedbygenetechnology) Supply Limitedandvariable Unlimitedproductionpossible Unlimitedproductionpossible, immunizationnotmandatory Uniformity Changingpropertieswith ConstantpropertiesofamAb ConstantpropertiesofarAb,canbe differentseraandbleedings changedbygeneticmanipulations Affinity MixtureofAbwithdifferent Uniformlyhighorlow,canbe Uniformlyhighorlow,canbe affinities,affinityoftenhigher selectedbytesting selectedbytestingandcanbe withpAb modified Cross-reactivity Resultsfromdifferent Different,dependentuponthe Different,dependentuponthe selectivitiesandlowaffinity individualAb individualAb,canbemodified interactions Classesand Typicalspectrum Onedefinedisotype Different,dependingonmolecular subclasses design DemandsonAg Highpurityrequiredforspecific ImpureAgsormixtureofAgs ImpureAgsormixtureofAgscan antisera canbeusedforimmunization, beusedforimmunization,pure pureAgsnecessaryfor Agsnecessaryforscreening, screening immunizationnotmandatory Costs Low High Onceestablished,low suchasliposomesordextran..28;29/Keyholelimpethemo- determinant structures..26/ Usually C –C spacers are 3 6 cyanin (KLH), a protein from mollusks, is often viewed used; if the spacer is too long, it may bend back to the asasuperiorcarrierbecauseitisforeigntothevertebrate carrier and the hapten will not be properly exposed. immunesystem..30/ StrategiesforIAhaptendesignforthetriazine,arylurea Anotherimportantissueconcernstheoptimalnumber and chloroacetanilide herbicides have been summarized of haptens bound to the carrier protein (i.e. optimal byGoodrowetal..26/ epitopedensity).Highlysubstitutedcarriersusuallylead The functional groups of the hapten govern the to the best results. For bovine serum albumin (BSA) selection of the method to be used to conjugate the molar ratios of 10:1 to 20:1 (hapten:carrier) are hapten to the functional groups of the carrier. The desirable;forlargermoleculessuchashemocyanin,ratios functional groups of the protein carrier available for of800:1to1000:1shouldbeobtained..17/However,very attachment of thehaptens arethecarboxyl groupof the highratiosmayreduceimmunogenicitybecauseofeither Cterminalandoftheasparticandglutamicacidresidues, the changes in tertiary structure of the protein caused theaminogroupoftheNterminalandthelysineresidues, by masking of the essential free amino groups or the the imidazol and phenolic functions of the histidine and removal of critical determinant sites on the carrier by tyrosineresidues,respectively,andthesulfhydrylgroupof haptenicblocking. cysteineresidues.Generalproceduresforthepreparation For the production of pAbs the purity of the hapten ofconjugatescanbefoundinErlanger..24;25/ used to prepare an immunoconjugate should be as After coupling, characterization of the conjugates can high as possible. After synthesis of haptenic substances, be carried out (see Erlanger.25/). Generally, the hap- closely related substances may be present in small tenic groups have an absorbance spectrum that can be quantities, leading to the production of nonspecific Abs differentiated from the protein carrier. Elemental anal- and, consequently, to unwanted cross-reactivities of the ysis for the chlorine content can be carried out for antisera.ThisisnotaproblemwithmAb,becauseasingle some triazine conjugates. A more direct procedure is celllineproducingonlyonekindofAbwiththedesired theincorporationofsomeradioactivehapteninthecon- propertiescanbeselected. jugation procedure. Another approach is quantitating Another important consideration is the point of the change in free amino groups as a result of conjuga- attachment on the hapten. Ab specificity is directed tion.Arecentlyappliedtechniqueisthedeterminationof primarily at the part of the hapten molecule farthest haptendensitybymatrix-assistedultravioletlaserdesorp- away from the functional group that is linked to the tion/ionization mass spectrometry (MALDIMS).31/ and proteincarrier..25/Evenbetterspecificitycanbeobtained electrospray ionization mass spectrometry (ESIMS)..3/ with conjugates in which the hapten is coupled to the Application of energy-minimized molecular modeling carrierviaaspacer,therebygivingmuchbetterexposure methods to hapten design will help to choose the best of the hapten on the surface of the carrier. The spacer derivatives and conjugation methods for successful Ab should be attached as far as possible from the unique production..32/ 5 IMMUNOCHEMICALASSAYSINPESTICIDEANALYSIS 3 IMMUNOASSAYTYPES withthetracerforbinding..37/Whiletheseassaysareeasy tocarryoutandverysuitableforautomation,theyusually 3.1 AssayFormats show a lower sensitivity than EIAs, e.g. for simazine a DLof5m gL(cid:0)1wasobserved..37/ For low-molecular-mass analytes (haptens) such as Noncompetitive assays can only be applied for high- pesticides in solution, competitive tests have to be molecular-mass analytes with more than one antigenic employed,usinglimitingAbconcentrations.Thetestscan determinant (i.e. Ag) or low-molecular-mass analytes beperformedashomogeneousassayswithoutseparation (haptens)boundtoasolidphase,exposingtheantigenic ofthereactants,.33/butmorecommonareheterogeneous determinant. They work with an Ab excess. Noncom- tests where unreacted reagents are removed before petitive IAs have been employed for the detection of evaluation. Twodifferent formats areavailable, (1)with soil-bound pesticides..38;39/ In this case the soil particles, immobilized Ab (Figure1a) and (2)with immobilized towhichthepesticideresidueshavebound,formthesolid coatingconjugate(Figure1b).Invariant(1)analyteand phase,andtheresiduescanbedetectedbyalabeledAb alabeledanalyte(tracer)competeforthefreeAbbinding specifictotheanalyte. sites. After removal of unbound reactants the bound tracer yields a signal that is inversely proportional to the analyte concentration. The variant (2)employs an 3.2 Labels immobilizedhapten-carrierconjugateonthesolidphase to which analyte and Ab are added. The Ab binds to Depending on the label, IAs are classified in different the free analyte or to the immobilized hapten according groups. Radioisotopes are used in RIAs, enzymes in to the concentration of the reactants. If a labeled Ab is enzyme-linkedimmunosorbentassays(ELISAs)orEIAs, used,theamountofAbboundtothesolidphasecanbe fluorophores in FIAs or PFIAs and chemiluminescent directly determined after a washing step. Alternatively, compounds in CLIAs. Additional types of IA exist, a secondary labeled Ab may be used to detect the Ab but are not very common in pesticide analysis. A whichhasboundtothesolidphase.Thesignalisinversely more detailed description of these IAs can be found proportionaltotheamountoffreeanalyteinthesample. inGosling..40/ VerysensitivecompetitiveIAshavebeendevelopedwith EIAs are most commonly used in pesticide analysis DLsbetween1and50ngL(cid:0)1,forexampleforthetriazines as they avoid the necessity of working with radioactive andureaherbicides..34–36/ material and low DLs can be reached. Simple and An example for a homogeneous assay system is cheap photometers which give an extremely rapid thepolarizationfluoroimmunoassay(PFIA).PFIAmea- measurement capability and long-lasting stability of the sures the increased polarization of fluorescence when a coloredproductafterthereactionhasstoppedmakeEIA fluorophore-labeledhapten(tracer)isboundbyaspecific superiortofluorimetryorluminometry,eventhoughwith Ab,andthedecreasedsignalwhenfreeanalytecompetes these methods lower DLs may be reached..33/ Enzymes Table2 EnzymesystemscommonlyusedforEIAs Enzyme Source Molecular pH Colorimetric Fluorometric Luminometric weight optimum substrates substrates substrates Alkaline Calfintestine 100000 9–10 p-Nitrophenyl- 4-Methylumbelliferyl- Adamantyl-1,2- phospha- phosphate phosphate dioxyethane tase Phenylphosphate- substituted dioxyethane b-Galacto- Escherichiacoli 540000 6–8 o-Nitrophenyl-b-D- 4-Methylumbelliferyl- – sidase galactopyranoside b-D-galacto- Chlorophenolic pyranoside red-b-D-galactopyranoside Peroxidase Horseradish 40000 5–7 2,20-Azino-di(3-ethyl- p-Hydroxyphenyl- Luminol benzthiazolinesulfonic aceticacid acid-6)(ABTS)/H O p-Hydroxyphenyl- 2 2 3,30-5,50-Tetramethyl- propionicacid benzidine(TMB)/H O 2 2 o-Phenylendiamine (OPD)/H O 2 2 6 PESTICIDES Table3 SolidphasesusedforEIAs Material Form Binding Capacity Polystyrene Microtiterplates,tubes,pins, Noncovalent 250–500ngcm2 beads Polyethylene Tubes Noncovalent ca.300ngcm(cid:0)2 Polypropylene Microtiterplates Noncovalent ca.300ngcm(cid:0)2 Polyvinylchlorideand Microtiterplates,membranes Noncovalent ca.300ngcm(cid:0)2(plates) similar Polycarbonate Beads,membranes Noncovalent ca.300ngcm(cid:0)2(beads) Nitrocellulose Microtiterplates,membranes Noncovalent ca.100m gcm(cid:0)2 ProteinAcoated Microtiterplates,beads Noncovalent 20mgmL(cid:0)1(forAbonly) Activatedpolymer,with Microtiterplates,beads Covalent,using 2(cid:2)1013–1(cid:2)1014reactive aminoorcarboxylgroups bifunctionalreagents sites/cm2 Magnetic Beads Dependsonthe Varies surfaceofthebeads commonlyusedaslabelsinheterogeneousEIAarelisted covalentbinding. Thosesolidsupportscontainaminoor inTable2. carboxygroupsonamodifiedsurfacethroughwhichthe The following requirements are necessary for the use immunoreagentscanbeboundbywater-solublecarbodi- ofanenzymeasamarker: imidesorbifunctionalreagentssuchasglutaraldehyde. Other solid-phase supports for IAs are membranes. (1) high specific activity (turnover number) of free They can be used for dip sticks, which are incubated enzymeandafterlabeling, for a short time in the solution.36/ or for dot blots and (2) availability of soluble, purified enzyme at low cost immunofiltration tests. Here the reactants are filtered andreproduciblequality, through the membrane..43;44/ The test principle is the (3) high stability in free and conjugated form under same as for the microtiter plate tests but the reaction storageandassayconditions, time is much shorter owing to the high surface area of (4) presence of reactive groups for covalent linkage to the membrane and the short distance between reaction hapten, partners. Application of remission measurements yields (5) simpleandgentleconjugationmethods, aproportionalrelationshipbetweenanalyteandremitted (6) inexpensive and stable nontoxic substrates with light.Byusingapocketreflectometer,thisset-upisideally formationofstablechromogenic,fluorogenicand/or suitedforfield-monitoringpurposes..45;46/ chemiluminogenicproducts. 3.3 SolidPhases 4 PROPERTIESOFCOMPETITIVE IMMUNOASSAYS IAs are mainly carried out in 96-well polystyrene, polyethylene, polypropylene or polyvinyl microtiter 4.1 Dose–ResponseCurve plates, owing to the easy separation of the reactants in a washing step, but polystyrene tubes, beads or pins In IAs the signal produced is inversely correlated to are also available (Table3). The plastic plates are of the analyte concentration in the sample (Figure3a). comparativelylowbindingcapacityandlowsurfacearea The typical dose–response curve is of sigmoidal shape to volume ratio. High-binding supports include agarose when the signal is plotted versus the logarithm of and cellulose. Particulate solid phases are very efficient, the analyte concentration. A linear range is obtained because they become scattered throughout the reaction around the middle of the test (IC , middle of assay, 50 mixture and have a much higher surface area to vol- concentration of analyte that causes 50% inhibition), ume ratio..41/ For example, many chemically different which should be used for determinations. Within this beads are available (e.g. polystyrene, latex, polycarbon- working range, the change in absorbance is linearly ate and copolymer beads). Immunological reagents are correlated to the analyte concentration. The linear part bound to the beads in a similar manner as they are to of the curve is confined by the upper and lower limits microtiterplates.Separation ofboundandfreereagents of quantification. These are the cut-off values above occurs by washing and centrifuging. IAs using magnetic or below which quantitative results can be obtained beads employ a magnet for the separation step..42/ Abs with a stated relative precision, or specified degree of and Ags may be immobilized to some solid phases via confidence in real samples..47/ The experimental errors 7 IMMUNOCHEMICALASSAYSINPESTICIDEANALYSIS increase toward these limits. Consequently, the most precise measurements are obtained in the region close tothemiddleofthetest. The DL (or least detectable dose) is the smallest concentrationoftheanalytethatproducesasignalwhich can be significantly distinguished from zero for a given sample matrix with a stated degree of confidence. Very oftenadoseisselectedwhichinhibits10–20%ofenzyme tracer from binding with the Ab or the dose calculated after subtraction of two or three times the standard deviationfromthemeanmeasurementsofthezerodose signal..3/ Linearizationofthecalibrationcurveisusefulformany purposes,forinstance,forthedirectcomparisonofcurves if matrix effects are evaluated. Absorbance curves can be normalized by converting the absorptions to %B=B 0 values.Thesecanbeexpressedastheratioofboundtracer inthepresenceofhaptentoboundtracerintheabsenceof haptenandliesbetween100%(DA ,theupperasymptote 0 of the curve) and 0% (DA , the lower asymptote) Excess (Figure3b).TheyarecalculatedbyEquation(3): %B A(cid:0)A D Excess (cid:2)100 .3/ B A (cid:0)A 0 0 Excess Linearization can be obtained by various mathematical transformations..47/ Usually, IAs are evaluated with commercialIAprograms,oftenbasedonlogisticmodels (cf.Rodgers.48/andDudleyetal..49/),e.g.four-parameter modelsorthemoresimplelogit-logtransformation(two- parameter model, Figure3c) which can also be carried outwithacalculator(s),Equation(4): %B %B=B logit Dln 0 .4/ B 100(cid:0)%B=B 0 0 Figure3 EIA for the determination of atrazine using pAb. 4.2 QualityControl (a)Absorption curve (means of three determinations(cid:6)stan- darddeviations),(b)B=B curve,and(c)logit/logtransforma- Precision and accuracy of IA are important properties 0 tionbythetwo-parameterfit. whichdeservespecialattention.Thequalityandstability of the employed material (microtiter plates, pipettes) andreagents(e.g.Abs,enzymetracerorbuffers),playa Thereproducibilityistheabilitytoyieldthesameresults crucialrole..50/ Thelong-termstabilityofreagentshasto within analyses, between analyses, and between opera- beensured,e.g.byfreeze-dryingofAbsand,ifnecessary, tors. The investigation of the variability of an IA gives addition of stabilizing components to the test reagents valuable information about the consistency of the test. suchastheenzymetracer..51/ Coefficients of variation (CV) of IA measurements are In spite of the simple handling of the assays, expert usuallybetween10and20%foranoptimizedassay,.52;53/ knowledgeisrequired,especiallytorecognizeandremove although more precise results can be obtained..54;55/ incident errors. Therefore, IAs should be performed by Same-dayandday-to-dayCVofsampleshavebeendeter- trainedpersonnel.Thedevelopmentofsimpleandrapid minedindifferentmatrices..53;56/ Interlaboratorytestsof assays, such as dip-stick assays or immunofiltration tests the same IA as that carried out by Hock and the IA reducestherequirementfortrainedusers,butonehasstill StudyGroup.57/ andHayesetal..58/ fortheinvestigation to be aware of potential problems such as interferences of triazines help to evaluate the general applicability of fromthesamplematrix. atest.However,severalconditionslikeexactdescription TheprecisionofanIAisdefinedastheextenttowhich of the assay including calibration curves, DLs, cross- replicate analyses of a sample agree with each other. reactivities, a working range close to the middle of the 8 PESTICIDES test,enoughparallelmeasurements,etc.mustbemet(see 100 alsoAOAC(AssociationofOfficialAnalyticalChemists) ) % criteria).Meanwhile,standardizedproceduresforIAsin ( 80 y waterareadoptedbyAOACInternationalandhavebeen t vi establishedinGermanyasaprenorm..50;58/ ti 60 c A validation of the results obtained by IA should a e be carried out. To a limited extent this can be done r 40 - s by IA itself. Dilution of the samples as well as spiking s o of the authentic sample with known amounts of the r 20 C contaminant can be used to check whether the matrix interferes with the IA..59/ However, spiked samples do 0 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 not completely mimic real unknown samples. They do Selective Intermediate Group-specific not contain potential metabolites of the contaminant nor residues from other compounds which may be Figure4 Selective,intermediateandgroupspecificAbs.This present in real samples. Furthermore, spiked samples exampleusesAbswhichhavebeenproducedagainsthapten1. Substances 2–5 are assumed to be cross-reacting haptens cannot be a model for aged residues which are more (modifiedafterHocketal..14/). difficult to extract and detect because, for example, they may have bound to soil constituents. Therefore, an IA should also be validated by a different established Strong cross-reactivities of an Ab to unexpected method like high-performance liquid chromatography metabolites, for example, can produce false positive (HPLC), gas chromatography (GC) or GC/MS (gas values. An Ab for alachlor was found to react very chromatography/mass spectrometry).Manygroupshave stronglytothesulfonicacidmetaboliteusinganalachlor usedthisapproachandhaveusuallyobtainedcorrelation screeningkit..69/ Thisproblemcouldbesolved,however, coefficients of >0.9..60–63/ Often a slight overestimation by using solid-phase extraction (SPE) prior to IA and of the IA in comparison with HPLC or GC is observed sequential elution of the two compounds with different owingtocross-reactivitiesoftheAbormatrixeffects. organicsolvents. 4.3 Cross-reactivity 4.4 SamplePreparationandMatrixEffects Depending on the conjugate used for immunization Samplescancontaincompoundsinadditiontothetarget and the class of chemicals under investigation, cross- analyte,whichmayinterferewiththetest.Severalgroups reactivities of the Ab with haptens similar to the investigatedtheinfluenceofionsonEIAs..70–72/Ruppert analyte are frequently observed (see e.g. Hock.14/ and etal..70/ observed an inhibition by several anions like Harrisonetal..64/).Therefore,itshouldbecheckedwhich azide, which inhibits the peroxidase by binding to the compoundscross-reacttowhatdegreewiththeAb.This heme group of the enzyme. Most cations did not have is usually done by comparing the standard curves of aneffectexceptforCa2C,whichleadstoanactivationof the analyte under investigation with similar haptens, theperoxidase.Nointerferencebydifferentionssuchas using analyte concentrations at 50% of the inhibition nitrate,copper,magnesiumetc.uptoaconcentration of curve as the reference. However, cross-reactivity with a 250ppmwasdetectedinanEIAforpentachlorphenolin certain analyte is not the same over the whole range water..71/ While ions may inhibit the enzyme used as a of a standard curve. Often higher cross-reactivities can label or lead to precipitates by reacting with the buffer be observed at low concentrations of the cross-reacting components, humic substances present in water or soil analyte..3/ Therefore, it has been recommended that extractsmay bind nonspecifically to the Ab and thereby cross-reactivitiesbemeasuredatdifferentconcentrations interferewiththespecificbindingoftheanalyte..73/These overtherangewheretheassayissuitable..65/ reactionsmayleadtofalsepositiveresults.Watersamples IfanAbisselectiveforasinglecompound,itisregarded from forest stands or soil extracts particularly contain as monospecific.66/ (Figure4). An Ab that recognizes a high content of organic compounds such as humic several compounds to the same extent (e.g. a group of acids(HAs). s-triazines), can be used for the screening of a class Matrixeffectsinfoodsamplesfrequentlyoccurowing of herbicides.67/ (group-specific Ab, Figure4). If cross- to colored extracts or to the content of lipids, proteins reacting compounds are not expected in the samples, or polyphenols that may be coextracted during sample becausethecompoundsarenotlicensed(e.g.propazinein preparation..74/ As food samples usually have to be mostEuropeancountries),agroup-specificAbcanalsobe extractedpriortoimmunochemicalanalysis,themethod usedforquantitativemeasurementsofonecompound..68/ of analyte extraction is of great importance. Analytes 9 IMMUNOCHEMICALASSAYSINPESTICIDEANALYSIS that are water soluble and can be efficiently extracted methods in residue analysis. Not all of them are in aqueous buffer will have the most direct extraction commercially available. Available commercial IAs have method and eliminate the need for organic solvents. been listed in e.g. Dankwardt etal.,.2/ Hennion and However, many pesticides are not readily water soluble Barcelo,.3/ Knopp,.8/ but a lot of movement has been and must be extracted with an organic solvent..5/ For observed in environmental IA markets, leading to the the extraction of pesticides from solid foods a variety disappearance of IA companies. At the moment IAs of solvents have been tested, such as acetone, ether, for environmental contaminants can be obtained for petroleum ether, methanol, acetonitrile or hexane..75/ example from Strategic Diagnostics Inc. (Newark, DE, Direct analysis of extracts by IA requires the use USA, sells former Ensys, Millipore and Ohmicron kits) of solvents that are miscible with water and (at low andEnviroLogix(Westbrook,MA,USA). concentrations) are nondenaturing to proteins such as Ab. IAs are to a certain degree tolerant to a variety of 5.1 Water solvents, but each system must be tested to determine which solvent can be accepted and to what extent (for EIA have been used intensively for the determina- example Hill etal.,.75/ Nugent.76/ and Schneider and tion of pesticides in surface and rainwater.56;60;69;211–217/ Hammock.77/). Usually the extracts are further diluted and groundwater..60;69;214;218;219/ A substantial num- with water prior to the EIA, but an EIA for parathion ber of these studies were carried out for triazine wasdeveloped, inwhichtheanalytedissolvedinhexane herbicides..56;60;211;212;214;216;218;219/ This illustrates the could be directly measured in the EIA without prior widespreadoccurrenceoftheseherbicidesintheaquatic removal of the hexane. This was achieved by using Ab environment. Many groups have used commercial test encapsulatedinreversemicellescomposedofAerosolT kits, which allow the investigation of samples without withaqueouscenters..78/However,a104-folddecreasein time-consuming Ab production. Thurman etal.,.60/ for sensitivitywasobserved. example,usedaRes-I-Munekit(ImmunoSystems)forthe Insomecasesacleanupstepisintroduced,inwhichthe investigationoftriazinesinsurfaceandgroundwater.The analyte of interested is separated from the matrix. This EIAwascomparedtoGC/MSresultsobtainedfromsam- can be carried out by C -columns or immunoaffinity plesthatwereextractedbySPE.Correlationcoefficients 18 columns..69;79;80/ A very interesting approach is the between 0.91 and 0.95 were obtained after introducing applicationofsupercriticalfluidextraction(SFE)priorto cross-reactivity factors for each of the triazines in order immunoanalysis. These methods generally employ CO to calculate a sum parameter for the GC. The majority 2 orCO containingvariousmodifiers..5;81/ of the samples contained only atrazine (up to 3m gL(cid:0)1). 2 Some problems with interfering ions can be solved Therefore, the EIA results corresponded well with the by changing the buffer of the assay system so that no atrazineconcentrationsobtainedbyGC/MS. precipitates may be formed..70/ Addition of BSA to the Mouvet etal..220/ compared four commercially avail- plates prior to the addition of the standard and sample able test kits and one in-house developed assay for the solutions.82/ or to the enzyme tracer.73/ greatly reduces determination of triazines in surface and groundwater. theinfluenceofhumicandfulvicacidsontheEIA.Itmay Operational characteristics, cross-reactivity, sensitivity, alsobehelpfultoswitchtoadifferentbatchofAbsora CV and agreement with GC/LC (gas chromatogra- different assay kit, as different Abs may show different phy/liquid chromatography) measurements were inves- sensitivities to interfering substances. The buffering tigated. DLs were determined between 0.003 and capacity of the assay buffer should also be checked, as 0.07m gL(cid:0)1.Intra-assayCVswerebelow7%foralltests, somewaterorfoodsamplesmayshowrelativelylowpH interassayCVsbelow 20%.Correlation studiesbetween values.Noeffects,however,wereobservedbetweenpH3 the EIA kits and GC/LC were carried out for samples and10bydifferentinvestigators..56;67;83/ from different water matrices. Depending on the water source,differentlevelsofsignificancewereobservedwith differenttests.Thebestresultswereobtainedforsurface water, while not all kits showed a good agreement for 5 APPLICATIONTOENVIRONMENTAL lysimetersamples. SAMPLES Apart from the triazines some other pesticides were investigatedinwatersamples,alsousingcommercialtest IAs have been developed for many environmental kits. Alachlor was determined in ground and surface contaminants during the 1990s. A list of several IAs water using commercial tests..213/ SPE was carried out described in the literature can be found in Table4. priortoEIAtoremoveinterferingsubstancesandtocon- Most of them have been developed in laboratories, centrate the analyte. Concentrations of up to 0.8m gL(cid:0)1 showing the increasing importance of immunochemical wereobserved,andacomparisonwithGC/MSshoweda 10 PESTICIDES Table4 PesticideIAsdescribedintheliterature Pesticides Test Ab Range,DL, Ref. format ormiddleoftest(IC ) 50 Herbicides Alachlor EIA p 0.2–8m gL(cid:0)1 84 EIA p 0.1–10m gL(cid:0)1 85 EIA p,m 0.2–8m gL(cid:0)1 86 Amitrole EIA p 1.7–4200m gL(cid:0)1 87 Atrazine CLIA p 25–500ngL(cid:0)1 88 EIA p 0.5–10m gL(cid:0)1 89 EIA p 0.01m gL(cid:0)1(DL) 90 EIA m 0.03–1m gL(cid:0)1 35 EIA p 0.2–100m gL(cid:0)1 64 EIA p 0.011–33m gL(cid:0)1 91 EIA m 0.05–3m gL(cid:0)1 92 EIA p,m 0.1–100m gL(cid:0)1 93 EIA p 0.5–10m gL(cid:0)1 94 EIA m 0.05m gL(cid:0)1(DL) 95 EIA m 0.01–10m gL(cid:0)1 77 EIA p 0.03–3m gL(cid:0)1 96 EIA p 1–1000ngL(cid:0)1 34 Bentazon EIA p 2–24m gL(cid:0)1 97 Bromacil EIA p 0.1–160m gL(cid:0)1 98 EIA p 0.01–1m gL(cid:0)1 99 Chlorodiamino-s-triazine EIA p 160–480m gL(cid:0)1 100 Chlorsulfuron EIA p 0.1m gL(cid:0)1(DL) 101 Clomazone EIA p 2–250m gL(cid:0)1 102 EIA p 0.5–500m gL(cid:0)1 103 Cyanazine EIA p 0.035–3m gL(cid:0)1 104 EIA p 0.5m gL(cid:0)1(DL) 105 EIA p 0.5m gL(cid:0)1(DL) 106 Diethylatrazine EIA p 0.01–100m gL(cid:0)1 107 Diclofop-methyl EIA p 10–75m gL(cid:0)1 108 2,4-D EIA p 50–5000m gL(cid:0)1 109 EIA m 2–20m gL(cid:0)1 110 RIA p 0.1–10mgL(cid:0)1 111 EIA p 0.05–10mgL(cid:0)1 111 RIA p 5–250m gL(cid:0)1 112 PFIA m 0.6m gL(cid:0)1(DL) 113 RIA p 1–1000m gL(cid:0)1 114 EIA m 0.096m gL(cid:0)1(DL) 115 Dichlorprop PFIA p 0.01–100m gmL(cid:0)1 116 Diuron EIA m 2m gL(cid:0)1(IC ) 117 50 EIA p 0.05–1m gL(cid:0)1 118 Hexazinone EIA p 0.22–17.6m gL(cid:0)1 119 Hydroxyatrazine EIA m 0.03–1m gL(cid:0)1 120 EIA m 0.05m gL(cid:0)1(DL) 95 EIA p 0.01–10m gL(cid:0)1 66 EIA p 3–300m gL(cid:0)1 121 Imazamethabenz EIA p 0.5–32m gL(cid:0)1 122 Imazaquin EIA p 0.45–25m gL(cid:0)1 123 Isoproturon EIA p 0.01–10m gL(cid:0)1 124 EIA m 20–250m gL(cid:0)1 125 EIA NA 0.02–1m gL(cid:0)1 126 Maleichydrazide EIA m 0.01–11m gmL(cid:0)1 127 MCPB EIA p 0.03–0.9m gL(cid:0)1 128 Metazachlor EIA p 10–1000ngL(cid:0)1 129 (continuedoverleaf)