multi-analyte immunoassay for pesticides: a review

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Multi-Analyte Immunoassay forPesticides: A ReviewHe Jiang a & Ming-Tao Fan ba College of Life Science, Hunan University of Arts and Science,Changde, Hunan, P. R. of Chinab College of Food Science and Engineering, Northwest A and FUniversity, Yangling, Shaanxi, P. R. of ChinaAccepted author version posted online: 10 Apr 2012.Publishedonline: 02 Aug 2012.

To cite this article: He Jiang & Ming-Tao Fan (2012) Multi-Analyte Immunoassay for Pesticides: AReview, Analytical Letters, 45:11, 1347-1364, DOI: 10.1080/00032719.2012.675493

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Immunoassay

MULTI-ANALYTE IMMUNOASSAY FORPESTICIDES: A REVIEW

He Jiang1 and Ming-Tao Fan21College of Life Science, Hunan University of Arts and Science, Changde,Hunan, P. R. of China2College of Food Science and Engineering, Northwest A and F University,Yangling, Shaanxi, P. R. of China

The current trend of pesticide immunoassay is developing multi-analyte immunoassays, that

is, more than one target can be detected per test. In this mini-review, the strategies to achieve

multi-analyte immunoassays, which include multi-antibody strategy, broad-specificity anti-

body strategy, and others, are briefly introduced. In addition, the recent developments of

multi-analyte immunoassays for pesticides were summarized. At last, we give a future out-

look of this area, which includes rational design generic hapten with the assist of com-

puter-assisted molecular modeling (CAMM), further improving the properties of

broad-specificity antibody by recombinant antibody (rAb) technology and developing a non-

competitive immunoassay format.

Keywords: Immunoassay; Multi-analyte immunoassay; Pesticide

INTRODUCTION

The issue of pesticide residues is one of the widely concerned food and environ-mental safety problems all over the world, and effective pesticide residue detectionsystems are the first requirement for survey and control pesticide residue. A desireddetection system should including rapid pesticide residue detection methods as localscreening tools and standard pesticide residue detection methods as final qualitativeand quantitative confirmation tools. Recently, standard methods, such as AOACmethods, are typically developed based on gas chromatography (GC) or high perfor-mance liquid chromatography (HPLC) and have been well established. As for therapid pesticide detection methods, immunoassay (IA) is the most popular one.

Immunoassay is a detection method which relies on antibody (Ab) as the keyanalytical reagent, and its principles have been introduced elsewhere in detail. Theapplication of immunoassays to pesticide residue analysis dates back to 1971 whenErcegovich et al. described their early work on the detection of pesticides by

Received 22 December 2011; accepted 28 January 2011.

Address correspondence to He Jiang, College of Life Science, Hunan University of Arts and

Science, Changde, Hunan 415000, People’s Republic of China. E-mail: [email protected]

Analytical Letters, 45: 1347–1364, 2012

Copyright # Taylor & Francis Group, LLC

ISSN: 0003-2719 print=1532-236X online

DOI: 10.1080/00032719.2012.675493

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immunochemical means and indicated the potential usefulness of this method forroutine analyses. Following Ercegovich’s work, an increasing number of researchersfocused their attention on pesticide immunoassay. Initially, the works of pesticideimmunoassay were typically targeted to a single specific pesticide, that is, single ana-lyte immunoassay, and the recent developments of this assay type have beenreviewed in other papers (Fan and He 2011; Morozova, Levashova, and Eremin2005; Bushway and Fan 2001; Kaufman and Clower 1995.). However, the single tar-get immunoassay is difficult to deal with in the diversified situation of pesticidedetection samples, and the current trend of pesticide immunoassay is to developmulti-analyte immunoassays, that is, more than one target can be detected per test(Fan and He 2011; He et al. 2011; He et al. 2010; Spinks 2000).

In this mini-review we focus on the topic of multi-analyte immunoassay forpesticide, and the strategies that can achieve this type of assay will be introducedfirst, then the recent developments of multi-analyte immunoassay for pesticide willbe summarized from the pesticides-type perspective.

STRATEGIES FOR MULTI-ANALYTE IMMUNOASSAY

In order to achieve multi-analyte immunoassay, several strategies can beapplied. These strategies can be classified as multi-antibody strategies, broad-specificity antibody strategies, as well as others. In the following section, the generalprinciple of these strategies will be briefly introduced.

Multi-Antibody Strategy

The first strategy to achieve multi-analyte immunoassay is raising numerousantibodies that recognize individual targets and then incorporating these antibodiesinto a single test. In this strategy, either identical or separate label systems can beapplied. In the former, the multi-analyte can be detected but cannot divide eachother; while in the latter, the content of each target can be confirmed. As to thedetecting model, traditional enzyme-linked immunosorbent assay (ELISA) (Sunet al. 2010; Zhu et al. 2011), convenient immunity-chromatography test strip (Guoet al. 2009; Gabaldon et al. 2003), and advanced immunosensor can all be used(Mauriz et al. 2007; Mauriz et al. 2006). Typical application of this strategy is a workcarried out by Mauriz’s group (2006, 2007). In order to develop a multi-analyte (sur-face plasmon resonance) SPR immunoassay for 1,1,1-trichloro-2,2--bis(4-chlorophenyl)ethane (DDT), chlorpyrifos, and carbaryl, several analyterecognition elements (antibodies) were multiply immobilized on the sensing surfaceof one individual flow cell (Mauriz et al. 2007).

Broad-Specificity Antibody Strategy

Although the multi-antibody strategy has been successfully applied to achievemulti-analyte immunoassay, a more economical alternative is to raise a single anti-body that is able to recognize several analytes, which is called a broad-specificityantibody or class-specificity antibody.

1348 H. JIANG AND M.-T. FAN

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Three methods can be applied to preparing broad-specificity antibody. The firstone is ‘‘generic hapten method,’’ which is based on the similar structure of a groupmolecule (Figure 1 a). In this method, newly synthesized generic haptens or currentlyavailable compounds with the common structure of a group molecule can be used forbroad-specificity antibody preparation. As in a work of our group, in order to pre-paring broad-specificity antibody target to O,O-dimethyl organophosphorus pesti-cides, synthesized generic hapten with the common structure of this kind ofpesticides was conjugated to bovine serum albumin (BSA) and then used for rabbitsimmunization. The result indicated that, the obtained best polyclonal antiserum canrecognize a group of O,O-dimethyl organophosphorus pesticides with acceptableaffinities (Liang, X. J. Liu, Y. Liu, et al. 2008; Liang, Y. Liu, Zhu, et al. 2008). Whilein a work carried out by Alcocer et al. (2000), phosphonic acid (currently availablecompound) was used as a generic hapten to generate broad specificity antibodiesagainst a group of organophosphorus pesticides.

The second approach for broad-specificity antibody production is the‘‘multi-hapten antigen method,’’ which conjugates more than one hapten to the car-rier protein for immunogen production (Figure 1 b). Typical work of this aspect wascarried out by S. T. Wang et al. (2007). In this work, a multi-determinant artificialantigen was prepared by haptens of four pesticides (chlorpyrifos, triazophos, carbo-furan, and parathion methyl) conjugating to the carrier protein BSA in turn. Then,male New Zealand white rabbits were immunized with this multi-determinant immu-nogen to produce polyclonal antibody. Characterization studies indicated that thepolyclonal antibody show high affinity and specificity to the four relative pesticides(S. T. Wang et al. 2007).

Figure 1. Strategies for broad-specificity antibody preparation. (a) generic hapten with the common

structure of a group molecule was conjugated with carrier protein for antibody preparation; (b) more

than one kind of hapten were conjugated to the carrier protein for immunogen production; and (c)

b-type anti-idiotype antibody was used to simulate those naturally binding site of receptors or enzymes.

MULTI-ANALYTE IMMUNOASSAY FOR PESTICIDES 1349

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The third way for broad-specificity antibody production relies on the b-typeanti-idiotype antibody, which can simulate those naturally binding sites of receptorsor enzymes (Figure 1 c). For example, based on the broad binding activity ofcutinase to different kinds of carbamates and organophosphate pesticides, abroad-specificity antibody of organophosphate pesticides was prepared by Wardet al. (1999). After preparation of a monoclonal Ab against cutinase, this antibodywas used as the antigen, and an anti-idiotype antibody (monoclonal antibody) thatmimics the active site of cutinase was acquired. Results indicated that thisanti-idiotype antibody was able to bind the organophosphate pesticides, chlorfenvin-phos, ethyl paraoxon, tetrachlorfenvinphos, and demeton-s-methyl (Ward et al.1999).

Other Strategies

Other strategies for multi-analyte immunoassay mainly rely on a specificimmunoassay model. For example, in a work published by Anfossi et al. (2004), anovel immunoassay format that can non-competitively analyze low-molecular-masscompounds was developed and successfully applied to 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) determination in aqueous samples. This methodis based on the separation of the analyte-bound antibody from the excess of the freeantibody by a chromatographic step, followed by the dissociation of the complexand the capture of the previously bound antibody on a solid phase. The measuredsignal is linearly correlated to the concentration of the complex and, consequently,to the analyte concentration. The authors indicated that by applying this new for-mat, even if a very selective antibody was used, a broad selectivity was observed,which allowed DDTþDDDþDDE to be determined instead of only p,p0-DDTas in the traditional ELISA format performed with the same antibody (Anfossiet al. 2004).

Figure 2. Schematic for the enzyme multiplied immunoassay technique (EMIT) developed by Zherdev et

al. (1997): (a) the hapten-enzyme conjugate was bind to antibody and the enzyme activity site was masked,

hence the enzyme activity was lower; and (b) the analyte competitively bind to antibody and the enzyme

activity site was released, so the enzyme activity was higher.


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Another specific strategy for multi-analyte immunoassay is based on enzymemultiplied immunoassay technique (EMIT). For example, Zherdev, Dzantiev, andTrubaceva (1997)have developed a new enzyme immunoassay for detection of somepyrethroid pesticides (permethrin, phenothrin) and their derivatives containing3-phenoxybenzoic group (3-PBAc) by this method. The general principle of thismethod is outlined in Figure 2. Briefly, 3-PBAc-amylase was conjugated and anti-3-PBAc antibodies were incubated together in plate wells with the pesticide sample tobe analyzed, then starch solution was added and hydrolysis was carried out. Theenzyme activity site was masked when the conjugate was bound to the antibodies;as a result, the hydrolysis activity was proportional to the concentration of targetpesticides. Actually, it also needs the antibody pose broad-specificity; however,homogeneous assay, which is more convenient than traditional ELISA, can beachieved by this strategy.

RECENT DEVELOPMENTS OF MULTI-ANALYTE IMMUNOASSAYFOR PESTICIDES

The general principles of strategies that can achieve multi-analyte immunoassayhave been briefly introduced in the previous section. Among these, the broad-specificity antibody strategy, especially the ‘‘generic hapten method,’’ is the mostpopular one. In the following section, the recent developments of multi-analyte immu-noassay for pesticide will be summarized from the pesticides type perspective.

Multi-Analyte Immunoassay for Organochlorine Pesticides (OCPs)

Although OCPs have been prohibited in most countries for a long time, theystill generate public health concerns because of their unresolved health impact andtheir persistence in living beings. Works of multi-analyte immunoassays for OCPshave been carried out by several groups, and those works are summarized in Table 1.

Obviously, the work of Anfossi et al. (2004) outlined perviously was targetedon OCPs. Apart from this work, an immunoassay kit for the detection of cyclodienepesticide was evaluated by Wigfield and Grant in 1992, and their results indicatedthat this kit can used to simultaneously to detect three OCPs (endrin, endosulfan,and dieldrin) in fruit (apple) and vegetables (tomato and lettuce) at acceptable limitsof detection (LODs). And, in a work of Manclus et al. (2004), an ELISA based onmonoclonal antibodies (MAbs) for the detection of cyclodiene group OCPs wasdeveloped. A generic hapten, characterized by exposure of the common hexachlori-nated bicyclic (norbornene) moiety was prepared and conjugated to the carrier pro-tein. From mice immunized with this conjugate, a MAb displaying the broadestrecognition to cyclodiene compounds (endosulfan, dieldrin, endrin, chlordane, hep-tachlor, aldrin, and toxaphene) was selected for the assay. Results indicated that theperformance of the developed immunoassay was acceptable, with IC50 values in the6–25 nM range. Similar work also include Compagnone and coworkers’ develop-ment of an electrochemical ELISA for the screening of DDT related compoundsin waste waters (Valentini et al. 2003), and Abad et al. (1997) produced a class-specificity antibody target to DDT and related compounds.


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Multi-Analyte Immunoassay for Organophosphorus Pesticides(OPPs)

Currently, OPPs are the most widely applied pesticides in agriculture. Hence,the residues of this type of pesticide have raised great concern for food and environ-mental safety. Multi-analyte immunoassay has been widely used for OPP detection,and the research regarding this aspect are summarized in Table 2.

Table 2 shows that, except for the work published by Ward et al. (1999), whichis based on anti-idiotype antibody, most of these works were based on broad-specificity antibody prepared via a generic hapten method. As previously stated,the generic hapten method was based on the common structure of a group molecule.For example, a class-specific immunochromatographic strip test for the rapid detec-tion of OPPs with a thiophosphate group was developed by Xue et al. (Su et al. 2010).In this work, O,O-diethyl O-(4-carboxy-3-methylphenyl) phosphorothioate wasutilized as the generic hapten for broad-specificity monoclonal antibody (MAb)production. The result indicated that a MAb with desirable properties (IC50 valueis 93 ng=mL) was obtained.

Generally, according to the feature of molecular structure, OPPs can be classi-fied into O,O-dimethyl OPPs and O,O-diethyl OPPs. Multi-analyte immunoassaysfor these two types of OPPs have been widely studied. In addition to the aforemen-tioned work of our group (Liang, X. J. Liu, Y. Liu, et al. 2008; Liang, Y. Liu, Zhu,et al. 2008), multi-analyte immunoassay for O,O-dimethyl OPPs also includes worksof Sun and coworkers (2010), Liu et al. (2009), and Bankset al. (1998). In Sun et al.’sresearch, a series of generic hapten were initially prepared and then the best one wasselected by an extremely laborious evaluation and comparison, while in Liu’s andBanks’s groups, a definite generic hapten was rationally designed. Desirable resultswere obtained by these works, and some differences in their selectivity and specificity

Table 1. Examples of multi-analyte immunoassay for organochlorine pesticides (OCPs)

Targets

Format and

performance Strategy Reference

DDD, DDD, and DDE Specific format

LOD: 8 ng=L

Specific

immunoassay

model

Zherdev et al. 1997

Endrin, endosulfan, and

dielrin

ELISA

LOD: 0.01–0.03mg=kg

Broad specificity

antibody

Wigfield and Grant 1992

Cyclodiene group OCPs

(endosulfan, dieldrin,

endrin, chlordane,

heptachlor, aldrin,

and toxaphene)

ELISA

IC50: 6–25 nM

Generic hapten

method

Manclus et al. 2004

DDT related

compounds

(p,p0-DTT, p,p0-DDE,

p,p0-DDD, and

o,p0-DDT)

ELISA

LOD: 40 pg=mL;

Broad specificity

antibody

Valentini et al. 2003

DDT isomers and

metabolites

ELISA

IC50: 2–11 nM

Generic hapten

method

Abad et al. 1997


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were displayed due to different generic haptens that were applied. Works regardingmulti-analyte immunoassay for O,O-diethyl OPPs have been carried out by Pan andcoworkers (L. J. Zhang and Pan 2010), Lee and coworkers (2009), and Xie and co-workers (Xu et al. 2009; Xu et al. 2010). In Pan’s work (L. J. Zhang and Pan 2010),diethylphosphono acetic acid was directly used as a hapten and conjugated with bov-ine sera albumin (BSA) to prepare immunogen for broad-specificity MAb prep-aration, and the obtained best MAb can cross-react to chlorpyifos, parathion,

Table 2. Examples of multi-analyte immunoassay for organophosphorus pesticides (OPPs)

Targets Format and performance Strategy Reference

O,O-dimethyl OPPs (malathion,

dimethoate, phenthoate, phosmet,

methidathion, fenitrothion,

methyl parathion, and fenthion)

ELISA

IC50:28.9–788.9mg=LGeneric

hapten

method

Liang, X. J. Liu, Y.

Liu, et al. 2008;

Liang, Y. Liu, Zhu,

et al. 2008

Chlorfenvinphos, ethyl paraoxon,

tetrachlorfenvinphos, and

demeton-s-methyl

ELISA

Undefined

Anti-idiotype

antibody

Ward et al. 1999

OPPs with a thiophosphate group Immunochromatogr-aphic

strip test

IC50: 93 ng=mL

Generic

hapten

method

Su et al. 2010

O,O-dimethyl OPPs (cyanophos,

fenthion, parathion-methyl,

dicapthon, fenitrothion, famphur,

lodofenphos and

chlorpyrifos-methyl)

ELISA

LOD: 2.6–104mg=kgGeneric

hapten

method

Li et al. 2010

O,O-dimethyl OPPs

(parathionmethyl,

chlorpyrifos-methyl,

tolclofos-methyl, fenthion,

malathion, and fenitrothion)

ELISA

IC50: 0.58–10.47mg=mL

Generic

hapten

method

Liu et al. 2009

O,O-dimethyl OPPs (fenitrothion,

methacrifos, propetamphos, and

dichlorvos)

ELISA

IC50: 4.8–91.1mg=mL

Generic

hapten

method

Banks et al. 1998

O,O-diethyl OPPs (chlorpyifos,

parathion, profenofos, omethoate,

dichlofenthion, diazinon,

bromophos, and phoxim)

ELISA

Undefined

Generic

hapten

method

Zhang and Pan 2010

O,O-diethyl phosphorothioate and

phosphorodithioate OPPs

ELISA

Average IC50: 89 ng=mL

Generic

hapten

method

Piao et al. 2009

O,O-diethyl OPPs (coumaphos,

parathion, quinalphos,

triazophos, phorate,

dichlofenthion, and phoxim)

ELISA

IC50: 0.013–1.301mg=L

Generic

hapten

method

Xie et al. 2009

O,O-diethyl OPPs (parathion,

coumaphos, quinalphos,

triazophos, phorate,

dichlofenthion, and phoxim.)

ELISA

IC50: 13–1301ng=mL

Generic

hapten

method

Xu et al. 2009; Xu et al.

2010

Parathion, methyl-parathion,

fenitrothion, and isocarbophos

ELISA

IC50: 20.32–58.85ng=mL

Generic

hapten

method

C. M. Wang et al. 2010


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profenofos, omethoate, dichlofenthion, diazinon, bromophos, and phoxim. In Leeand coworkers’ work, four generic haptens with different spacer arm structures weresued to prepare antibodies, while eight haptens were tested for use as coating anti-gen. Their best ELISA showed class- selective response to O,O-diethyl phosphoro-thioate and phosphorodithioate OPPs and with negligible cross-reactivity to othertypes of pesticides. In Xie and coworkers’ research (2009), 4-(diethoxyphosphor-othioyloxy) benzoic acid was directly used as the generic hapten for the productionof broad-specificity antibody for O,O-diethyl OPPs, and the obtained PAb showedhigh sensitivity to seven commonly used O,O-diethyl OPPs in a competitive indirectELISA.

In addition to the aforementioned research groups, C. M. Wang et al. (2010)carried out a work about multi-analyte immunoassay for OPPs. In this work, abroad-selective MAb OPPs was raised using heterologous indirect ELISA to screenhybridomas. On the basis of this MAb, five coating antigens were used to develophomologous and heterologous indirect competitive ELISAs. With the most suitablecompetitor, a sensitive and broad-selective ELISA was developed, and its IC50 valueswere estimated to be 20.32 ng=mL for parathion, 21.44 ng=mL for methyl-parathion,42.15 ng=mL for fenitrothion, and 58.85 ng=mL for isocarbophos. Result of furtherresearch indicated that spiked recoveries were between 70.52 and 103.27% for thedetection of single pesticide residues of the four OPPs in purple-clayed paddy soiland the average recovery and coefficient of variation were 80.91 and 4.82%, respecti-vely, for the detection of mixtures of parathion and methyl-parathion in soil samples.

Multi-Analyte Immunoassay for Pyrethroids

Pyrethroids are synthetic chemical compounds similar to the natural chemicalpyrethrins produced by the flowers of pyrethrums (Chrysanthemum cinerariaefoliumand C. coccineum) and constitute a major proportion of the synthetic insecticidemarket. It is important to develop sensitive and rapid analytical techniques forenvironmental monitoring and assessment of human exposure to these compounds.Multi-analyte immunoassay, as an effectively analytical tool, also has widely usedfor pyrethroids detection, and associated works are summarized in Table 3. Exceptfor the EMIT method previously mentioned (Zherdev et al. 1997), all the otherworks outlined in Table 3 were developed by a generic hapten method. For example,an ELISA of pyrethroid insecticides with the common chrysanthemic acid (CAA)moiety was developed by Miyake et al. (1998).

The synthetic pyrethroids and natural pyrethrins can be divided into twogroups, type I are simple cyclic alcohol esters of 2,2-dimethyl-3-(2-methyl-1-prope-nyl)cyclopropanecarboxylic acid, while type II are esters of an aryl cyanohydrin.The type I and II pyrethroids have different toxicological effects, thus the ability toselectively monitor these two groups’ compounds would be an advantage. Hammockand coworkers (Watanabe et al. 2001;Mak et al. 2005) have carried out lots of work inthis area. In their research, polyclonal antibody selectively target to type I pyrethroidswere generated by immunizing with a permethrin derivative, 2,2-dimethyl-3-(50-carboxy-pent-10-en-yl)cyclopropanecarboxylic acide-(3-phenoxybenzyl) ester conju-gated with carrier protein, and polyclonal antibody selectively targeted to type IIpyrethroids were generated by immunizing with (RS)-a -cyano-3-phenoxybenzyl


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(RS)-cis,trans-2,2-dimethyl-3-carboxyl-cyclopropanecarboxylate conjugated withthyroglobulin. Based on these obtained broad-specificity antibodies, a multi-analyteimmunoassay was developed for type I and type II pyrethroid insecticides, respecti-vely. Q. Zhang et al. (2010) also carried out a systematic work for the class-specificassay to type I and type II pyrethroid insecticides. In this work, multi-analyte immu-noassays for type I pyrethroids without a cyano group, type II pyrethroids with acyano group, and both types of pyrethroids with loss of the ester group, were success-fully developed, respectively.

In addition, works of multi-analyte immunoassay for pyrethroids have alsorecently been carried out by J. P. Wang and coworkers (2011; Lu et al. 2010). Forexample, a new approach was recently used by this group for the preparation ofbroad-specificity antibody targeted to pyrethroids in which a pyrethroids metaboliteanalogue, 3-phenoxybenzoic acid, was used as the generic hapten. After optimization,the IC50 of the developed ELISA were calculated to be 20 mg=L for deltamethrin,

Table 3. Examples of multi-analyte immunoassay for pyrethroids

Targets

Format and

performance Strategy Reference

Some pyrethroid pesticides

(permethrin and phenothrin)

and their derivatives

containing 3-phenoxybenzoic

group

EMIT

LOD: 2–5ng=mL

EMIT Zherdev et al. 1997

Pyrethroid insecticides with the

common chrysanthemic acid

(CAA) moiety

ELISA

Linear range:

1–10mg=L

Generic hapten

method

Miyake et al. 1998

Type I pyrethroid insecticides

(permethrin, phenothrin,

resmethrin, and

bioresmethrin)

ELISA

IC50: 20–30mg=LGeneric hapten

method

Watanabe et al. 2001

Type II pyrethroid insecticides

(cypermethrin, deltamethrin,

cyhalothrin, cyfluthrin,

fenvalerate, esfenvalerate,

and fluvalinate)

ELISA


method

Mak et al. 2005

Type I pyrethroids without a

cyano group; type II

pyrethroids with a cyano

group; both types of

pyrethroids with loss of the

ester group

ELISA

IC50: around

0.02mg=mL

Generic hapten

method

O. Zhang et al. 2010

Deltamethrin, cypermethrin,

fluvalinate, fenvalerate, and

fenpropathrin

ELISA


method

Lu et al. 2010

Pyrethroids with

phenoxybenzene group

(deltamethrin, cypermethrin,

fluvalinate, fenvalerate,

phenothrin, flucythrinate,

fenpropathrin, and

permethrin)

ELISA

IC50: 1.5–5.0mg=LGeneric hapten

method

J. P. Wang et al.

2011


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16 mg=L for cypermethrin, 11 mg=L for fluvalinate, 15 mg=L for fenvalerate, and20 mg=L for fenpropathrin, respectively. Further research indicated that high pre-cision and good correlations between this immunoassay and gas chromatography=electron capture detector data were obtained (Lu et al. 2010). Another relevant workcarried out by this group is develop an ELISA based a MAb for the detection of pyr-ethroids with phenoxybenzene group. The IC50 values of the optimized immunoassaywere 1.8 mg=L for deltamethrin, 1.5 mg=L for cypermethrin, 2.0 mg=L for fluvalinateand fenvalerate, 2.2 mg=L for phenothrin, 2.4 mg=L for flucythrinate, 3.0 mg=L for fen-propathrin, and 5.0 mg=L for permethrin. And, the recoveries of pyrethroids in spikedwater samples ranged from 74 to 108% (J. P. Wang et al. 2011).

Multi-Analyte Immunoassay for Herbicides

Herbicides are widely applied in agricultural production for weed control, andthe s-triazine, sulfonylurea, and imidazolinone herbicides are the most commontypes that cause the greatest concern regarding groundwater and soil contamination.Therefore, development of efficient detection methods for these compounds is anarea of great importance. Multi-analyte immunoassay technology has been widelyapplied in this area, and typical works are summarized in Table 4.

Works regarding multi-analyte immunoassay for s-triazine herbicides were car-ried out extensively by various strategies. Using triazine herbicides as targets, animmunochemical method for simultaneous analysis of cross-reacting analytes, whichis based on a combination of individual enzyme immunoassays for triazine herbicidesusing antibodies with different cross-reactivity patterns toward the selected analytes,was developed by Wortberg et al. (1995). Multi-analyte immunoassays for s-triazineherbicides were also developed by Bruun et al. (2000), Kolar, Deng, and Franek(2002), and Herranz et al. (2008) using generic hapten strategy. In these works, asdifferent haptens were synthesized and used for broad-specificity antibody prep-aration, the developed detection method possessed different group-specificity;whereas, Samsonova et al. (1999) and Franek, Deng, and Kolar (2000) used multi-antibody strategy to achieve multi-analyte immunoassay for s-triazine herbicides.In the former, a competitive chemiluminescent immunoassay (CIA) based on a com-bination of five antibodies was used in a combination with neural network to identifyand estimate amounts of three cross-reacting s-triazines (atrazine, terbythylazine, andametryn). And, in the latter, a flow injection immunoassay (FIIA) based on a sequen-tial injection instrument with a photometric and fluorometric detection unit, wheremonoclonal antibodies against atrzine, simazine, and 2,4-D were immobilized. Inaddition, a biosensor system based on total internal reflectance fluorescence (TIRF)was used to achieve multi-analyte assay for atrazine and simazine, where polyclonalantibodies with cross-reactivity to the two targets were utilized.

Works regarding multi-analyte immunoassay for sulfonylurea and imidazoli-none herbicides were also carried out, and all of these used generic hapten strategy.Kolar et al. (2002) used a single ring hapten strategy to produce antibodies withdominant selectivity toward triazine moieties of metsulfuron-methyl. Resultsindicated that a superior antibody with cross-reactivity values estimated to be100, 142, 95, 60, 40, 167, 83, and 21% for metsulfuronmethyl, cinosulfuron, triasul-furon, primisulfuron-methyl, thifensulfuron-methyl, chlorsulfuron, prosulfuron,


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and tribenuron-methyl, respectively, was obtained. In a work published by Degel-mann et al. (2004), a polyclonal antibody with satisfying broad specificity to arange of sulfonylurea herbicides was obtained by immunizing a mesosulfuronben-zylamine derivative that was coupled via a succinic acid spacer to a keyhole limpethemocyanine. Their result indicated that the direct competitive ELISA developedbased on this polyclonal antibody can detect 16 sulfonylurea herbicides at a con-centration of 0.1 mg=L or lower. Targeted to imidazolinone herbicides, a multi-analyte immunoassay technology was developed by Chin et al. in 2002. In thiswork, a set of haptens structurally resembling the herbicide imazethapyr wassynthesized and used to derive monoclonal antibodies (MAbs) and then to developimmunoassay technology. Their result indicated that the developed multi-immunoassay technology can detect imazethapyr, imazaquin, imazapic, and imaza-mox in the 3–30 ng=mL range, and imazapyr and imazamethabenz-methyl in the300–500 ng=mL range.

Table 4. Examples of multi-analyte immunoassay for herbicides

Targets Format and performance Strategy Reference

S-triazine herbicides (atrazine,

simazine, cyanazine, and

prometon)

ELISA

LOD: sub ng=mL range

Specific

immunoassay

model

Wortberg et al.

1995

S-triazine herbicides (cyanazine,

atrazine, terbuthylazine, and

propazine)

ELISA

LOD: below 0.1mg=LGeneric hapten

method

Bruun et al.

2000

S-triazine herbicides (simazine,

atrazine, and propazine)

ELISA

LOD: ng=L range

Generic hapten

method

Herranz et al.

2008

S-triazine (simazine, simetryn,

ametryn, prometryn, terbutryn,

aziprotryn, atraton, and

atrazine)

ELISA

LOD: undefined

Generic hapten

method

Kolar et al.

2002

S-triazine herbicides (atrazine,

terbythylazine, and ametryn)

Chemiluminescent

immunoassay (CIA)

LOD: undefined

Multi-antibody

strategy

Samsonova

et al. 1999

Atrazine, simazine, and 2,4-D Flow injection

immunoassay (FIIA)

LOD: undefined

Multi-antibody

strategy

Franek et al.

2000

S-triazine herbicides (atrazine and

simazine)

Biosensor

LOD: mg=mL range

Broad-specificity

antibodies

Reder et al.

2003

Sulfonylurea herbicides

(metsulfuronmethyl,

cinosulfuron, triasulfuron,

primisulfuron-methyl,

thifensulfuron-methyl,

chlorsulfuron, prosulfuron, and

tribenuron-methyl)

ELISA

LOD: undefined

Generic hapten

method

Kolar et al.

2002

Sulfonylurea herbicides (16

sulfonylurea herbicides)

ELISA

LOD: 0.1 mg=L or lower

Generic hapten

method

Degelmann

et al. 2004

Imidazolinone herbicides

(imazethapyr, imazaquin,

imazapic, imazamox, imazapyr,

and imazamethabenz-methyl)

ELISA

LOD: ng=mL range

Generic hapten

method

Chin et al. 2002


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Multi-Analyte Immunoassay for Other Pesticides

Pesticides comprise a large group of chemicals, and in addition to theaforementioned widely applied types, numerous others are included. Multi-analyteimmunoassay technology targeted to these pesticides has also been developed byspecialists. For example, a surface plasmon resonance (SPR) based immunoassay wasdeveloped by Yang and Kang (2008) for the detection of several carbamate pesticides(carbofuran, carbaryl, and benfuracarb). Here, the antibody used for the immunoassaywas specific for glutathione-S-transferase (GST), and when antigens were applied to theprotein GST, the detection limit was 2 ng=mL of carbamate pesticide. And, morerecently, a direct competitive ELISA in multi-enzyme tracers format for the simul-taneous analysis of carbaryl and metolcarb in agricultural products was described bySun et al. (2010). Under the optimum conditions, the limits of detection of carbaryland metolcarb were 0.15mg=L and 1.2mg=L, respectively; recoveries of spiked sampleswere in fruit juices and vegetables great than 70%, and the correlations between the dataobtained using multi-enzyme tracers ELISA and HPLC were acceptable.

CONCLUSION AND FUTURE OUTLOOK

Although numerous strategies can be applied to achieve multi-analyte immu-noassay, the generic hapten method was the most widely applied one. The most criti-cal step of develop multi-analyte immunoassay by this method is the selection of thegeneric hapten. Traditionally, this is primarily based on trial and error. For example,a series of haptens was prepared and conjugated to carrier protein for broad-specificity antibody production respectively, and then the desired generic haptenwas selected by comparison of the performance of those antibodies. This procedureis time-consuming and laborious, and sometimes all of the obtained antibodies lackthe required features to develop multi-analyte immunoassay. Recently, computer-assisted molecular modeling (CAMM), which can provide insights into molecularstructure and biological activity that are difficult or otherwise impossible to obtain,provided a useful tool for generic hapten design. Here, several haptens were designedas candidates and then CAMM was used to optimize the energy and calculate thevalences and charges, and then the hapten, which was both structurally and electro-nically most similar to the target analytes, was selected as the immunizing hapten (Xu,Shen et al. 2009). Several attempts have been made to develop multi-analyte immu-noassay for sulfonamides (Spinks et al. 1999; Muldoon et al. 1999), triazines (Delau-nay, Pichon, and Hennion 2003), and nitroaromatic residues (Julicher et al. 1995)with the assist of CAMM. Based on the results of these works, it can be conductedthat CAMM will become a useful tool in the development of immunoassays withbroad selectivity in the future, especially along with the development of computa-tional chemistry and molecular simulation techniques. For more details about theapplication of CAMM to rational hapten design, the review published by Xu, Shenet al. (2009) could be referenced.

Typically, polyclonal or monoclonal antibodies were applied for multi-analyteimmunoassay development. Once prepared, the properties, such as affinity andselectivity, of these antibodies cannot be further modified. However, the newlydeveloped recombinant antibody (rAb) technology, which prepares antibody under


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the genetic level, permits the use of prepared antibody properties for furtherimprovement. For example, a monoclonal fluoroquinolone antibody, 6H7, was tar-geted to random mutagenesis to broaden the specificity of the antibody in develop-ment of a generic assay for fluoroquinolone antibiotics by Leivo et al. (2011). Theresult of this work indicated that the best characterized mutant antibody was capableof recognizing seven of eight targeted fluoroquinolones below maximum residuelimits set by the European Union. It can also be concluded that rAb technology willbecome a useful alternative for broad-specificity antibody preparation and thereforemulti-analyte immunoassay development.

Most of the published research indicated that the sensitivity of multi-analyteimmunoassay is inferior to single-target immunoassays (Liang, X. J. Liu, Y. Liu,et al. 2008; Liang, Y. Liu, Zhu, et al. 2008), which cannot fulfill the requirementof practical application. Traditionally, competitive models are widely applied in pes-ticide immunoassay. However, a theoretical study of Jackson and Ekins (1986)demonstrated that noncompetitive immunoassays are potentially superior to com-petitive immunoassays in terms of sensitivity, precision, kinetics, and working rangesof analytes. Therefore, although it was not widely applied now, noncompetitiveimmunoassay will be a new trend in pesticide multi-analyte immunoassays. Sand-wich ELISA is a widely used noncompetitive immunoassay to determine antigenconcentration. However, it has a fundamental limitation that the antigen to be mea-sured must be large enough to have at least two epitopes to be captured; thus, it can-not be used to measure low molecular weight compounds, such as pesticides. Inorder to overcome these drawbacks, a series of special immunoassay models havebeen developed. These formats are based on chemical modification to the analytes,unconventional antibodies (anti-idiotype antibody, anti-metatype antibody, or anti-body fragment), special separation steps (capillary electrophoresis, affinity columnor membrane), or other elegant tricks (Kobayashi and Goto 2001). Among these,noncompetitive immunoassay models that are based on unconventional antibodieswere our key recommendations here.

The anti-idiotype antibody-based noncompetitive immunoassay was originallydescribed by Barnard and Kohen (1990; Barnard, Karsiliyan, and Kohen 1991) anddenoted ‘‘idiometric assay.’’ This method was newly applied by Niwa et al. (2009) toestablish a noncompetitive-type ELISA for cortisol. The result of this work indicatedthat this assay had an approximately threefold higher sensitivity than the competitiveELISA using the same anti-cortisol antibody and its selectivity was unaffected. In arecent work of our group (He et al. 2011), phage display technology was applied topreparing recombinant anti-idiotype antibodies of O,O-dimethyl OPPs. Althoughthe result of this work indicated that the sensitivity of anti-idiotype antibody-basednoncompetitive immunoassay was almost the same as the traditional competitivecounterpart, the affinity of our prepared anti-idiotype antibody can further improveand therefore improve the sensitivity of the ‘‘idiometric assay.’’ Anti-metatype anti-bodies recognize the antibody=antigen complex but exhibit very low or no affinityfor the antibody or the antigen alone. This remarkable property was cleverly utilizedby Self, Dessi, and Winger (1994) to develop noncompetitive immunoassay for smallmolecules and exemplified by digoxin detection. Results indicated that the sensitivityof this assay is 30 ng=L. Another noncompetitive immunoassay format, named‘‘open sandwich immunoassay (OSIA),’’ was originally described by Ueda et al. in


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1996, and successfully applied in the detection of a series of hapten molecules (Leeet al. 2008; Lim et al. 2007; Ihara et al. 2009; Shirasu et al. 2009; Sakata et al.2009; Suzuki et al. 2007; Yokozeki et al. 2002). This assay is based on the obser-vation that for some antibodies the association of separated VH and VL chains fromthe variable domain of antibody is strongly favored in the presence of antigen. Theprominent superiority of this format makes it easy to realize homogenous assay,therefore, making the processes of detection simpler and more convenient. Typically,these homogeneous assay systems are either based on enzymatic complementation(Ueda et al. 2003), resonance energy transfer [fluorescence (Ueda et al. 1999; Araiet al. 2000; Wei et al. 2006), or bioluminescence (Arai et al. 2001)].

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multi-analyte immunoassay for pesticides: a review

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