determination of pesticides by enzyme immunoassay
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1061-9348/05/6003- 2005 Pleiades Publishing, Inc
Journal 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 . 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 .
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 , flow-injection analysis , 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  and a method with theuse of a PVC membrane electrode selective for pen-tachlorophenolate  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.
Determination of Pesticides by Enzyme Immunoassay
V. 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 Russia
Received April 9, 2004
Immunochemical 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 CHEMISTRY
DETERMINATION OF PESTICIDES BY ENZYME IMMUNOASSAY 203
The 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 .
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 . 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  and herbicides of the sulfonylurea and benzoylphenylurea groups ) in the testsample.
Immunochemical methods are widely used in ana-lytical practice, various branches of medicine, andmicrobiological and food industries . 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 cases
when 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% .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 ; the use of ELISA for determining pesticides wasalso discussed in . The fundamentals ofELISA and trends in the development of this tech-nique for determining pesticides were surveyed byforeign authors ; procedures for the ELISAdetermination of pesticides in environmental samples 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