Chapter 7 Immunoassays for environmental analysis

D. Barcel6 (Editor)/Sample Handling and Trace Analysis of Pollutants: Techniques, Applications and Quality Assurance © 1999 Elsevier Science B.V. All rights reserved. 287 Chapter 7 Immunoassays for environmental analysis A. Oubifiaa, B. Ballesterosa'b, p. B o u C a r r a s c o b , R. G a l v e a , j. G a s c 6 n b , F. I g l e s i a s a, N. S a n v i c e n s a a n d M . - P . M a r c o a aDepartment of Biological Organic Chemistry, IIQAB-CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain bDepartment of Environmental Chemistry, IIQAB-CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain CONTENTS 7.1 7.2 7.3 7.4 7.5 7.6. 7.7 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Types of immunoassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.1 Direct competitive assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.2 Indirect competitive assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.3 Labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.4 Other types of immunoassay configurations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.1 C h e m i c a l structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.2 Antibody production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.2.1 Polyclonal antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.2.2 Monoclonal antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.2.3 Recombinant D N A antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hapten design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4.1 Effect of the chemical structure of the immunizing hapten . . . . . . . . . . . . . . . . . . . . . . . . 7.4.1.1 Effect on the IA specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . 7.4.1.2 Effect on the IA detectability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4.2 Effect of the chemical structure of the competitor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4.2.1 Effect on the IA detectability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4.2.2 Effect on the IA selectivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . Conjugation procedures using proteins or enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5.1 Conjugation strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5.2 Cross-linkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immunoassay features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6.1 Detectability and sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6.2 Precision . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6.3 Accuracy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6.4 Specificity and cross-reactivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Matrix effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.7.1 Specific interferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.7.1.1 Cross-reactivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.7.1.2 E n z y m e inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.7.2 Non-specific interferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.7.2.1 Other non-specific interferents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 291 291 292 292 293 304 304 305 306 307 308 308 308 309 310 3 l0 310 312 313 313 317 318 320 320 321 321 322 322 322 322 324 324 A. Oubi~a et al. / Immunoassays 288 7.7.3 Application of IAs to the analysis of environmental matrices . . . . . . . . . . . . . . . . . . . . . 7.7.3.1 Water samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.7.3.2 Soil samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.7.3.3 Food samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.7.4 Biological monitoring by IA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.7.5 Solutions to overcome the effect of the matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.8 Validation studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.9 Conclusions and future developments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ABBREVIATIONS 2,4-D Ab Ag Ag* anti-IgG AP B B0 nexcess BSA CA cDNA CDR CLIA CONA CR CV DDT DMSO EIA ELIFA ELISA EMIT EPA ESI-MS ET Fab Fc FIA FIIA FILIA FT-IR GC-FID GC-MS GO HPLC HRP IA IC50 Ig 2,4-dichlorophenoxyacetic acid antibody antigen labeled antigen antibody generated against immunoglobulins alkaline phosphatase absorbance zero control absorbance standard excess absorbance bovine serum albumin coating antigen complementary deoxyribonucleic acid complementary determining region chemiluminescence immunoassay conalbumin cross-reactivity coefficient of variation 1,1,1-(trichloro)-2,2-bis(p-chlorophenyl) ethane dimethylsulfoxide enzyme immunoassay enzyme-linked immunofiltration assay enzyme-linked-immunosorbent assay enzyme multiplied immunoassay techniques Environmental Protection Agency electrospray ionization mass spectrometry enzymatic tracer antibody binding site crystallized fraction fluoro immunoassay flow injection immunoassay flow injection liposome immunoassay Fourier transform infrared spectrometry gas chromatography-flame ionization detection gas chromatography-mass spectrometry glucose oxidase high-performance liquid chromatography horseradish peroxidase immunoassay concentration inhibiting 50% of the absorbance produced at zero-dose immunoglobulin 324 326 327 327 328 328 329 330 331 A. Oubi~a et al. / Immunoassays KLH LC-DAD LC-PCN-FD LDD LIA LIC MAb MALDI-TOF-MS mRNA OVA PAb PAH PBS PCB PCDD PCDF PCR RAb RIA SFE SPE-LC-DAD TG TNBS /3G 289 keyhole limpet hemocyanin liquid chromatography-diode array detection liquid chromatography-postcolumnreaction-fluorescence detection least detectable dose liposome immunoaggragationassay liposome immunoreaction assay monoclonal antibody matrix-assisted laser desorption/ionization time-of-flightmass spectrometry messenger ribonucleic acid ovalbumin polyclonal antibody polyaromatic hydrocarbon phosphate-buffered saline polychlorinated biphenyl polychlorinated dibenzo-p-dioxin polychlorinated dibenzofuran polychain reaction recombinant DNA antibody radioimmunoassay supercritical fluid extraction solid phase extraction-liquid chromatography-diode array detection tyroglobulin trinitrobenzenesulfonic acid /3-galactosidase 7.1 I N T R O D U C T I O N Environmental contamination is recognized as a worldwide problem. Part of this problem is caused by the application of hundreds of different compounds that are being used as pesticides in agriculture, horticulture and forestry. Inherently, they show a certain degree of toxicity and especially the less degradable, more persistent compounds present a problem. Residues of pesticides have been found in all kinds of environmental samples. Much research has been done to develop new and improve existing methods for pesticide analysis. On the other hand, a large number of industrial compounds such as aromatic compounds, polyaromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and phenolic compounds are highly toxic and widespread environmental pollutants. The methods generally used to measure pesticides and industrial pollutants are based on chromatographic techniques involving extraction, extensive purification and often application of derivatization procedures. Therefore, experienced personnel and expensive equipment are required in order to carry out this work. Immunoassays (IAs) are analytical tests that utilize antibodies (Abs) as specific recognizing elements. Their use for both qualitative and quantitative analysis has proved to be one of the most productive technological contributions to medicine and fundamental life science research in the twentieth century. IA technology is not new, since the principles of IAs were first expounded by Rosalyn Yalow and Solomon Berson in 1959 [1] determining insulin in blood. Since then, there has been an exponential growth not only in the range of applications to 290 A. Oubiaa et al. / Immunoassays which it has successfully been applied, but also in the number of novel and ingenious assay designs. Such technology has not been confined to medical diagnosis, finding applications also in the forensic, veterinary, pharmaceutical, food and environmental sciences. Ercegovich [2,3] first proposed the use of immunochemical techniques for environmental analysis in the 1970s, mainly focusing on radioimmunoassay (RIA). Some years later, Hammock pointed out on the potential and advantages of using enzyme immunoassays (EIAs) for the environmental monitoring of pollutants. Over the following years a great number of articles have appeared describing the development of EIAs for detecting trace amounts of chemicals in the environment [4-12]. However, the acceptation of IAs for environmental applications by analytical chemists has been rather slow, although some authors have noted the potential of these technologies [13-15]. It has been only during the 1990s that the scientific community and regulatory agencies have started to evaluate and recognize the advantages of IAs [16,17]. Nowadays, an important number of IAs for pesticides are commercially available (for the features of some of these assays see Tables 7.2 and 7.3, respectively). The US Environmental Protection Agency (EPA) has validated some of these assays and included 13 of them in the SW-846 methods list (see Table 7.4). IAs exploits the ability of Abs to selectively and reversibly bind organic molecules. The other key reagent in most environmental IAs is the labeled ligand or competitor. IAs owe their versatility to the immune system ability to produce Abs in response to virtually any foreign molecule. For small analytes, such as pesticides, the compound of interest must be conjugated to a large carrier molecule, such as bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), etc., to render it immunogenic. However, even if the method appears to be very easy and simple, an understanding of its basic principles is required to make a good use of an IA. Special attention has to be given to the cross-reactivity (CR) phenomena, matrix effects and to the data interpretation and validation procedures. In summary, immunochemical methods are powerful analytical techniques that can answer many questions concerning environmental contamination. Both the availability of commercial IA test kits and the fact some IAs have already been included in the list of SW-846 methods of the EPA indicate that this technology is finding its place in the environmental area as an effective screening method to complement other analytical techniques. Although it is a relatively new technique for pesticide residue field-testing and for the screening of industrial pollutants, IAs appear to hold great promise. Additional advantages are the reduction of the costs caused by shipping negative samples and the possibilities to provide test results without delays. Most of the IA tests currently described are designed to be sensitive enough to assay water samples directly without concentration or clean-up steps. Future developments on immunosensor systems are a great promise for future automation of these environmental immunodetection strategies. An immunosensor is an analytical device consisting of an immobilized biological component in intimate contact with a transduction device that converts the immunorecognition event into a quantifiable electrical signal. When the Ab interacts specifically with the antigen (Ag), the generated physicochemical changes are sensed, in this case, electronically. The aim of this chapter is to provide an overview of the potential of IAs as environmental analytical methods. Immunosensors will only be briefly mentioned, since more A. Oubi~a et al. / Immunoassays 291 information is provided in Chapter 22. We will describe the types and components of IAs (Sections 7.2 and 7.3) as well as the steps involved in their development and the criteria used for their developpoment (Sections 7.4-7.7). Finally, Section 7.8 wiil discuss aspects related to the validation of immunochemical techniques. 7.2 TYPES OF IMMUNOASSAYS From all types of IAs, we can mainly distinguish between competitive versus noncompetitive IAs and homogeneous versus heterogeneous IAs [18-24]. In the homogeneous assays, there is no separation between the free and bound phase before the detection step. Usually, the binding of the Ag to the Ab or vice versa modulates the activity of the reporter enzyme or label. The homogeneous assays are very suitable for monitoring processes due to their shorter analysis time compared to the heterogeneous assays; however, inconveniences such as matrix effects and insufficient sensitivity are often observed. Moreover, not all the Ags, especially low molecular weight Ags, are able to modulate the activity of the enzyme, which explains the reduced impact of these assays in the environmental field [7,25-27]. Heterogeneous formats have found a broader application to many fields and also to different kinds of analytes. One of the immunoreactants is bound to a solid support making possible the separation between the bound and free phases without the need of modulating the activity of the label. When the analyte is a large molecule with two or more epitopes, the non-competitive IAs are the most common and easy to develop. For example, the immobilized Ag is recognized by the Ab and the immunoreaction is detected by a second labeled Ab. In another configuration, an excess of labeled Ab is used to detect the Ag captured by another Ab bound to the solid surface. This format is very popular and the assays are known as sandwich-type assays. Restrictions of this configuration are that the Ag must have epitopes and that usually both Abs should have their specificity directed to a different one. However, most of the environmental pollutants are small molecules that cannot simultaneously interact with two Abs or be immobilized directly on a solid phase. In those cases competitive IAs should be employed. Non-competitive IAs have nevertheless been used for the detection of soil-bound pesticides The general strategy of competitive assays is based on the competition of the free Ag with a fixed amount of labeled Ag (Ag*) for a fixed and limiting amount (low concentration) of Ab. At the end of the reaction the amount of labeled Ag and subsequently the free Ag is determined. The most usual configurations are shown in Fig, 7.1 and are briefly described below; however, readers are referred to other publications for a more extensive description of the different types of IA formats [28]. 7.2.1 Direct competitive assay Ab coating format. An equilibrium is established between the Ab bound to the solid surface (either directly or through orientating reagents such as antibodies generated against immunoglobulins (anti-IgG) or protein A [29]), the analyte and the analyteenzyme tracer which are in solution. After the main incubation step, the unbound reagents 292 A. Oubiaa et al. / Immunoassays are washed away and the amount of label bound to the solid phase by the Ab is measured. A decrease in the signal is directly proportional to the amount of analyte present. Ag coating format. This is based on the competition between the immobilized Ag (or surface derivatized analyte) and the analyte for a fixed small amount of labeled Ab. 7.2.2 Indirect competitive assay This works under the same principle as the Ag coating format, but the concentration of the analyte is measured in this case indirectly by the quantitation of bound Ab with a second labeled Ab. 7.2.3 Labels The labels used to quantify the immunoreaction can be of different nature. Enzymes are the most common labels used in IAs for environmental analysis. Enzymes frequently used are horseradish peroxidase (HRP), glucose oxidase (GO), alkaline phosphatase (AP) and /3-galactosidase (/3G). These specific enzymes react with a convenient substrate, producing a chromogen often absorbing in the visible region. Their extremely high catalytic power amplifies the signal caused by the immunoreaction, increasing the sensitivity of the IAs. EIAs consist, thus, of a two-pronged strategy: the reaction between the immunoreactants (Ab with the corresponding Ag) and the detection of that reaction using enzymes coupled to the reactants, as indicators or labels. EIA techniques can be divided in two main groups: enzyme multiplied immunoassay techniques (EMIT) and enzyme-linked immunosorbent assay (ELISA). The first one is a competitive homogeneous IA. Few EMITs have been described for environmental monitoring of pollutants. As an example a homogenous IA has recently been developed for the pyrethroid permethrin reaching a detection limit of about 2-5 ng ml-l by modulating the activity of the enzyme amylase [25]. ELISAs are the most popular EIAs used on environmental analysis (see Table 7.1). They are competitive heterogeneous assays which can be performed immobilizing one of the reagents on a variety of solid supports (tubes, microtiter plates, plastic-baked nitrocellulose membranes, magnetic particles, etc.). Because of their potential for processing simultaneously many samples, ELISAs have become popular for the rapid screening of organic pollutants in the environment, as it is shown by the numerous reviews and articles [20,21,30-38]. Examples of ELISAs for all types of pesticides (organophosphorus [3942], carbamates [12,43-45], triazines [46-51]) and some industrial contaminants (PAHs [52], PCBs [53], phenols [54-56]) can be found in the literature. In RIA the emission of radiation of an isotope is used to detect the immunoreaction. Radioligands, especially those that emit gamma radiation, can be rapidly, conveniently, and sensitive counted. One of the most commonly used isotopes is 1251, which provides high specific radioactivity in comparison to tritium or ~4C-labeled ligands and also because it does not require using scintillation fluids. Picogram-level determinations can be usually reached. These kind of IAs were the first developed assays for environmental monitoring [2,57]. Thus, RIAs have been described for S-bioalletrin [58], PCDDs [59], parathion [60], PCBs [61 ], paraquat [62], benomyl [63], etc. Nevertheless, because of the precautions that have to be taken when manipulating radioactive substances, the generation of radioactive residues and the adverse health effects produced, others types of labeling substances have gradually replaced radioactive labels. A. Oubi~a et al. / Immunoassays 293 Fluoro immunoassay (FIA) and chemiluminescence immunoassay (CLIA) use fluorescent labels such as fluorescein, rhodamine or rare earth quelates such as Eu(III), Tb(III), Sm(III) chelates and chemiluminescent labels such as luminol [64,65]. The former are faster and have more precision than RIA [35], but the latter present a considerable loss of the luminescent quantum yield of the label after the coupling reaction. Another inconvenience is the presence of matrix components catalyzing the luminescent reaction. In the case of fluorescent labels, the sensitivity may be limited by the background noise of some samples [66]. To enhance detection in most FIAs for pesticides, the fluorophore is generated enzymatic ally rather than using them directly as labels and therefore can also be considered as EIAs. FIAs have often been adapted to flow-immunoassay sytems (see Section 7.2.2). FIAs and CLIAs have been described for many analytes such as dichloprop [67], 2,4-dichlorphenoxyacetic acid (2,4-D) [27], diclofop-methyl [68], triazines [65,6971], triasulfuron [72], etc. Liposome-based IAs use liposomes containing usually water-soluble fluorescent or electroactive molecules [73]. These liposomes are used to label either the Ag or the Ab depending on the IA format used (see Fig. 7.1). For example, in one of these assays [74]; a solution containing a mixture of alachlor and alachlor tagged dye-containing liposomes are allow to migrate through an anti-alachlor Ab zone, on a plastic-backed nitrocellulose strip, where competitive binding occurs. Unbound liposomes continue migration to a liposome capture zone, where they are quantified either visually or by densitometry. The amount of liposome-entrapped dye that is measured in this zone is directly proportional to alachlor concentration in the sample. A liposome immunoassay was developed for PCBs analysis in a nitrocellulose strip with a detection limit of 2.6 pmol [75] (see Fig. 7.2). The assay takes place in just 23 min and the measurement of color intensity is carried out visually or with a desktop scanner. Similarly, this kind of label has frequently been used on immunosensor configurations by combining it with electrochemical or optical transducer systems [76,77]. Other perspectives include the use of organometallic markers such as Cr(Co)3, Mn(CO)3, Co2(CO)6, etc., and their detection by Fourier transform infrared spectrometry (FT-IR) [78,79]. The differences encountered on the spectra profiles of these labels prompts to the possibility of developing multianalyte metalloimmunoassay procedures. Similarly, it has been suggested recently to use mass spectromety as a detecting system [80]. The approach of the mass spectrometry IAs would consist in the microscale immunoaffinity capture of target Ags followed by mass-specific identification and quantitation using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Mass spectrometric detection of Ags is unambiguous, as Ag signals are observed at characteristic mass-to-charge values in the mass spectrum. Moreover, another important aspect of such mass-specific detection is the ability to use a single assay to screen biological systems for the presence of multiple, mass-resolved Ags. This strategy has been used for the detection of mycotoxins. 7.2.4 Other types of immunoassay configurations The solid support used to immobilize the immunoreagents determines some of the formats used. IAs are mainly carried out in 96-well polystyrene, polyethylene, polypropylene or polyvinyl microtiter plates. Polystyrene tubes are also quite popular as well as A. Oubiaa et al. / Immunoassays 294 o,I t"q 0 0 ¢,q r~ [., Z < b-, 0 0 © < Z ,.--, tt'~ O,I tt~ tt'3 rio Z © > Z ~q u., © ¢',1 0 t",l r~ o ~ t--- eq o o.--,,,6 ¢,q < [.., Z < < < < < < << < ca, © ,..~.s~..z~..,~..~..,~..~ ~ [.., © << < << < < < < < < < << < ,..d ,-d < ~Z [.., © © © .~ .,..~ ~ ~ o ,.c:l A. 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Oubifia et al. / Immunoassays I I I I c',l ~-, I c-,I ~- c',l ~ I I I 0~ 4- , I I I I I .i ~., TM 301 I I I ~., o~~o~o~o~~o~~oo~~~o~ ~8 °,.~ 00 i c5 I # i c5 I I o i i c5c~ i I ~ I i c5 = i c5c5o,i E ~ o o o ~ ~ d d d d o d o o o o ~ o o o ~ o ~ o o o o ~ o d d d o o d d o o d d d d d d d d d 0 0 0 o °,..~ o I O0 o~ ~'h ~'h ¢',1 ~ ~ O ,'-'~ ~', 0 ¢',1 ,.Q © i © 0 i c',l ~ 8 0 .= ~ ~o ~ ~o ~ .~ ~Z~ ~ ~ o o ~ + o 0 =~ ~o ~.~ o ~ 302 A. Oubifia et al. / Immunoassays I~ ~ 0 0 0 0 Cxl 0 0 0 0 I I I I I I 0 ~ ~ t",l 0 © ? 0 0 I I I I 0 • I o I 0 o ° I I I I I I I ::I. n~ ¢; I I I ~.-,1 r¢~ c-,I • ° ¢xl I I I I I o~ c,l o o I x o I °,..~ o 7 o I I Z I ~ I °~ I I 7 © •" "~ 0 0 © < 0 0 © d ~2 < 0 o 0 (..) °,..~ .,,.~ 303 A. Oub#Ta et al. / Immunoassays TABLE 7.4 EPA IA METHODS USEFUL FOR THE SEPARATION, DETECTION AND QUANTITATION OF ORGANIC POLLUTANTS IN DIFFERENTENVIRONMENTALMATRICES Method Immunoassay Matrix 4010A 4015 4016 4020 4030 4035 4040 4041 4042 4050 4051 4500 4670 PCP 2,4-D 2,4,5-T PCB TPH PAH Toxaphene Chlordane DDT TNT RDX Mercury Triazines Water and soil Water and soil Soil Soil and oil Soil Soil Soil Soil Soil Water and soil Water and soil Soil Water a a Submitted. polystyrene, latex or polycarbonate beads. In this case, separation of free and bound reagents is performed by centrifugation. Magnetic beads are also frequently used and several IAs for pesticide determination are commercially available in this format (see Table 7.1) that uses a magnet for the separation step. Immunoreagents can also be immobilized covalently to amino- and carboxy-modified surfaces by standard coupling methods. IAs may as well be performed in membranes as dipsticks or immunofiltration assays. These assays are usually applied for field-testing and the analysis can be performed in a few minutes. The test principle is the same as for the microtiter plates but the reaction time is much shorter due to the high surface area of the membrane and the short distance between the reaction partners. The liposome immunomigration strip assays previously mentioned are examples of these kinds of assay [75,81]. Similar assays have also been described for triazines [82-85], mycotoxins [86,87], marine toxins [88], etc. Polarization fluoroimmunoassay (PFIA) measures the increase of fluorescence polarization when a specific Ab binds a fluorophore-labeled hapten. Similarly, a decrease in the signal is observed when a free analyte competes with the labeled hapten for binding to the Ab. PFIAs has been described for several pesticides [89] such as simazine [90], atrazine [26], propazine [70], dichloprop [67], 2,4-D [27], etc. Enzyme-linked immunofiltration assay (ELIFA), consists in coating the Abs on a membrane instead of a plastic surface. In this way, it has been claimed that detectability can be greater due to the major adsorbance of the proteins on the membrane. Furthermore, time analysis is shorter than in other IAs because the different solutions are sequentially dropped-on. Flow injection immunoassays (FIIA) apply the characteristic unsegmented continuous flow from FIA techniques to automate IA procedures. This configuration improves test time and reliability by maintaining the same detectability. For review articles on FIIAs see [91-93]. FIIAs have been described for a variety of pollutants such as triazines [51,84,94- 304 A. Oubiaa et al. / Immunoassays A Direct Competitive ELISA vv,¢v oo I v vvll Coating Antigen ~ Analyte IgG B Indirect Competitive ELISA °@ IgG-HRP AntilgG-HRP Enzyme Tracer o~ TMB Substrate C Sandwich ELISA Fig. 7.1. Scheme of the heterogeneus IA configurations most commonly employed (see text for description). From Marcop et al. [313]. 96], explosives [97], PCBs [98], etc. Using a similar concept, flow injection liposome immunoassays (FILIA) have been reported for the herbicide imazethaphyr [99]. 7.3 ANTIBODIES 7.3.1 Chemical structure Abs are the key reactives of the immunochemical techniques. Abs are polypeptides belonging to the immunoglobulin (IgG) family. The Abs used in most of the immunochemical techniques are IgGs formed during the humoral response of the immune system of the mammals when in contact with a foreign substance or Ag. The IgGs (MW 150 000 Da) are monomers of Igs formed by two pairs of polypeptidic chains interconnected by disulfide bonds (see Fig. 7.3). Two chains contain approximately 450 amino acid residues and are known as the heavy chains, contrary to the light chains with only 212 amino acid residues. Both light and heavy chains are divided into constant and variable regions. The constant region or crystallized fraction (Fc) has the same amino acid sequence in all the lid 305 A. Oubifia et al. / Immunoassays (a) (b) 8 cm'~' ~k, 3 cm I C1 ~.75 cml R1 :::-. C2 •"~,,a R2 it,'.'. r [PCB] I ~ FLOW 1 I [PCB] Fig. 7.2. (a) Liposome immunocompetition assay (LIC); R1, liposome/PCB competition zone. (b) Liposome immunoaggregation assay (LIA); R2, liposome-antibody aggregation zone. C1, C2, antibiotin capture zones. Agreed to and accepted by ACS Copyright Office [75]. sequence of this region, unique on each IgG produced by a single B-cell clone. This amino acid sequence and the spatial conformation of the binding site determine the specificity of the molecule. The interaction between Ab and Ag is reversible and it is stabilized by electrostatic forces, hydrogen bonds, hydrophobic and Van der Waals interactions. 7.3.2 A n t i b o d y p r o d u c t i o n Several types of Abs are used for immunochemical techniques. The most common Abs used on IAs for environmental analyses are polyclonal antibodies (PAb) and monoclonal Antigen binding sites ../ ~-.~ H2N Fab NH2 I - - Fc CDRs: ComplemetarityDetermining Regions (hypervariableregions) sugars HO0~ 10. Fv Fab Fig. 7.3. Structure of immunoglobulin G (IgG) molecules. F(ab')2 306 A. Oubiaa et al. / Immunoassays antibodies (MAb) (see Sections 7.3.2.1 and 7.3.2.2). The preparation of MAbs by in vitro immunization [100-102], using lymphocytes in a culture media, has arisen as an interesting approach. Thus, studies performed using transgenic plants as living bioreactors for the production of important biomolecules, such as MAbs, could be promising [103,104]. Finally, DNA technology is an exciting strategy for producing MAbs or fragments [104-106]. Although few IAs based on recombinant DNA antibodies (RAb) have been described, the potential advantages of using this approach are widespread in the near future. In addition to the recombinant DNA approach, Ab fragments keeping the Fab region have been obtained by using specific enzymes (pepsin: F(abl)2 and Fc fragments; papain: 2 Fab and 1 Fc fragment). Paralog peptides (MW 1000-2000 Da) of the CDRs can also be produced chemically by solid-phase synthesis [107]. Finally, Ab fragments can also be obtained by using reducing agents that dissociate the disulfide bonds to generate free sulfhydryl groups used on some immobilization procedures. As an example, mercaptoethylamine reduces the disulfur bond of the hinge region without dissociating the heavy and light chains [ 108]. 7.3.2.1 Polyclonal antibodies PAbs are obtained by immunizing mammals with the target Ag. The more common animals immunized are rabbits, mainly when large volumes of serum are not required, but many other species can be used such as goats, pigs, sheep or cows. Immunization protocols are well established [ 109]. Intradermal injections of the immunogen mixed with an adjuvant, to enhance the immune response, are carried out each 4 or 5 weeks. Generally, after 3-6 injections no increase in the Ab titer is observed. The collected antiserum can be used TABLE 7.5 ADVANTAGES AND LIMITATIONS OF IAs Advantages Limitations Wide applicability Haptens can be difficult to synthesize Can be vulnerable to cross-reacting compounds Possibility of non-specific interferences Requires independent confirmation Inappropiate for small sample loads or multi-residue determinations High development costs Lack of acceptance, conservative attitudes Sensitive and specific Suitable to field use Usually reduced sample preparation Easy of use Rapid High sample throughput Simultaneous analysis of multiple samples Low cost of analysis For certain polar compounds, IAs are the choice A. Oubit~a et al. / Immunoassays 307 directly as immunoreagent, although in some cases IgGs are purified, often by precipita, tion with saline solutions or by affinity chromatography. The immune response is a complex process where molecular recognition and communication episodes between specialized cells of the immune system take place thorough certain mediators to produce a cascade of events leading to the multiplication of specific clones of B-cells in the serum. In the immune system, a single B-cell produces a single type of IgG, which recognizes a little part of the Ag molecule. The antiserum contains a heterogeneous mixture of IgGs that recognize the global structure of the Ag with high specificity and sensitivity. For more extensive information on the mechanisms involved in the immune response, the reader is addressed to other documents [110-113]. However, it is important to note that the sequence of molecular recognition events involved in the immune response determines that Ags below certain molecular size are not able to trigger an immune response. Many environmental pollutants are organic molecules with molecular weights below 1000 Da and consequently when they are administered to animals do not elicit antibodies. To raise Abs to them, it is necessary to chemically synthesize an appropriate hapten (see Section 7.4) and to couple it to a carrier, usually a protein. The avidity and specificity of the antiserum is determined by the chemical structure of the immunoconjugate (see Section 7.4.1.1), the immunization schedule, and also the particular immune system of the animal used. For this reason, it is recommended to immunize two or three animals with each Ag. The variability of the PAbs features from one animal to each other can be a limitation when a constant supply of identical antisera is required, for example, for commercial purposes. 7.3.2.2 Monoclonal antibodies In contrast with PAbs, MAbs contain a unique defined IgG molecule that is produced by a single B-cell clone. The production of MAbs is well documented [104,114,115]. In this case, the mammals of choice are mice. After immunization, the mouse spleen is removed, and the spleen cells are fused with tumorgenic B-lymphocytes as myeloma cells for its immortalization. After fusing, the mixture is divided into many culture wells and allowed to growth. The presence of specific Abs in each well is tested, generally using a competitive ELISA, and the positive wells are further cloned. The operation is repeated until a B-cell clone producing Abs with the desired properties is isolated. The screening process may become tedious and time-consuming, increasing thus the cost of Ab production. MAbs production presents the advantage that, theoretically, it provides an unlimited amount of Abs with identical affinity for the Ag. Another advantage is the possibility to screen for those clones of Abs with the desired pattern of specificity. However, the widespread idea that MAbs provide more sensitive and selective IAs than PAbs is not true. Therefore, before addressing the Abs preparation, one should balance the cost of the process regarding the potential applicability of these Abs. 7.3.2.3 Recombinant DNA antibodies This approach for obtaining Abs consists on the isolation of the genes encoding the 308 A. Oubiaa et al. / Immunoassays desired polypeptidic fragment from the Ab and their introduction into an expressionvector system (i.e. Escherichia coli). In general, the whole process would include the isolation of messenger ribonucleic acid (mRNA) from hybridoma, spleen cells or lymphocytes, synthesis of complementary deoxyribonucleic acid (cDNA) by reverse transcriptase, amplification of the RNA-DNA hybrid by polymer chain reaction (PCR) using suitable primers, ligation of cDNAs obtained in a bacterial plasmid vector, transformation of competent host cells and expression and screening for desired Ab fragments [ 116-120]. The advances achieved during the last years in the field of recombinant DNA technology, and the progressively deeper knowledge of the molecular structure of Abs and their interaction mechanism with the Ag, have allowed the production of Ab libraries generated by this technique. Only five to ten amino acid residues of each chain from the Fab region form the binding pocked of the Ab. The DNA technology would allow receptor design for each particular Ag, modeling a priori their size and composition, meaning the characteristics of the active site. Although at the moment, very few recombinant Abs (RAbs) fragments for environmental contaminants have been described (triazines [118,120,121], parathion [122], dioxin [123], etc.), the economic factor is one of the advantages of this technique over the conventional methods of obtaining PAbs and MAbs. PAbs are easy to obtain but they are limited by the batch production; in contrast, MAbs are expensive to maintain and screen. Recombinant DNA libraries would allow a cost-effective production of Ab, moreover on a high scale. 7.4 HAPTEN DESIGN This is the most crucial step on the development of an immunochemical technique for pesticides or other low molecular weight environmental pollutants. Specificity and selectivity of an immunochemical technique are mainly determined by the Ab [ 124-130] and the chemical structure of the competitor used as CA or enzyme tracer. Many examples in the literature prove that an appropriate hapten design determines the features of the resulting antibodies. A hapten is a molecule analogous to the target analyte and properly functionalized to allow covalent attachment to a carrier compound. 7.4.1 Effect of the chemical structure of the immunizing hapten Immunizing hapten design is the key step in the development of IAs. As mentioned above, their selectivity and detectability are mainly determined by the quality of the resulting Abs. The immunizing hapten should represent a near perfect mimic of the target molecule in chemical structure, spatial conformation, electronic distribution and hydrophobic properties. However, sometimes characteristic portions of the molecule are sufficient to generate valuable antibodies. It is advisable to avoid modification of the immunogenic groups in the target molecule and/or introducing new ones, as this supposes an alteration of its structural, geometrical and electronic properties, and consequently a reduction of the sites for potential molecular recognition. Nowadays, it is possible to make theoretical calculations and computer models in order to predict the more appropriate chemical structure of the hapten [ 139-142]. Examples found in the literature demonstrate that the chemical function used for the A. Oubiaa et al. / lmmunoassays 309 conjugation should be separated of the most important moiety of the target analyte by a spacer arm in order to avoid carrier hinderance. A 3-6 atom spacer length has often been considered as optimum size for the spacer [44,46,131-134], although not many exhaustive studies have addressed this point. Moreover, IAs have been described when Abs had been raised using haptens with shorter spacer arms [ 135,136]. The spacer arm should preferably replace a carbon hydrogen bond. Ideally, a spacer arm should be a chain of methylenes terminated by a functional group. The presence of bulky or other functional groups or heteroatoms may lead to the generation of antibodies versus the spacer arm and consequently to a poor recognition of the free analyte in the competitive ELISA [137,138]. 7.4.1.1 Effect on the IA specificity • i ,. . . . Different criteria have to be considered when analyte-specific Abs are desired. Thus, the exposure of the most characteristic part of the target compound should be maximized, attaching the spacer arm to the appropriate place. On the other hand, if a class-analyte assay is intended, the common part of the molecules has to be chosen for maximal exposure to the immune system. By using this criterion, Skerritt and Lee [39] obtained Abs and IAs showing different specificities against pyrethroids (see Fig. 7.4a). When the attachment took place in site Y, the characteristic groups of each pyrethroid molecule were exposed to the immune system and analyte-specific Abs were obtained. Similarly, classspecific Abs were obtained by attaching the spacer arm by site X thus maximizing the exposure of the phenoxybenzyl moiety which is common to several pyrethroid insecticides. Haptens with the linker placed in the middle of the molecule (site Z) were also synthesized by Lee et al. [134,143], and the resulting assays compared to those obtained from haptens prepared by site X and Y were the most sensitive (see Table 7.6). In this case a high recognition of the dihalovinylcyclopropane moieties was observed, suggesting that the Ab specificity may be directed toward this portion of the molecule rather than the aromatic groups. This fact was explained by the authors as a result of the greater distance existing between the 2~-vinyl carbon of the cyclopropane moiety to the coupling point than the C3 ~ of the phenoxybenzylgroup. Goodrow et al. [129] studied three possible handle attachment sites for haptens of arylurea herbicides monuron and linuron (see Fig. 7.4b). The preparation of a hapten with a spacer arm in site A is synthetically easier than the others, but also the resulting molecule mimics the target analyte very well. The linker hardly modifies its properties while keeping the aromatic ring far enough from the attachment site without altering the urea functional group. The features of the developed immunochemical assays have confirmed the predicted advantage of this hapten (see Table 7.7) [131,144]. Newsome and Collins [145] prepared immunizing haptens by introducing a linker in site C. The resulting polyclonal antisera showed a very poor specificity since many other chemical structures containing the dimethyl urea moiety were recognized in the assay. Finally, site B immunizing haptens probably produced important variations of the electronic and hydrophobic characteristics of the molecule. Additionally, the possibility of establishing hydrogen bonds in this position of the molecule has been altered. These haptens had been used as competitors by other authors [146], observing an important decrease of their recognition by the Ab. 310 A. Oubi~a et al. / lmmunoassays 7.4.1.2 Effect on the IA detectability Knowledge on which are the most important groups of the molecule that can participate establishing non-covalent interactions with the Ab is important in order to obtain highaffinity antibodies. Low-affinity antibodies are expected to render IAs with low detectability. Thus, in our group, Ballesteros et al. [ 139] analyzed the potential participation to establish bonds with the Ab of the most important functional groups of the Irgarol 1051 molecule (see Fig. 7.4c). Three haptens differing in the attachment site of the linker were synthesized and the resulting Abs were screened to develop competitive ELISA. Computer molecular modeling studies were used to explain the differences encountered between the different immunizing haptens. This study demonstrated the prevalence in this case of noncovalent hydrophobic interactions stabilizing the analyte-Ab immunocomplex. Thus, the immunizing haptens (linkers in sites H and G) keeping the tert-butyl group rendered several IAs with inhibition concentration at 50% of B/Bo (IC50) below 0.3 txg 1-~. In contrast, the hapten lacking this group gave IAs with ICs0s above 1.0 Ixg 1-~ (linker in site F). Another example is the case of the development of an immnoassay for the herbicide bromacil. Antibodies were generated by using haptens having the spacer arm in two different positions: through a carbon atom or blocking an amino group of the uracil ring. In this case the conversion of a secondary amine to a tertiary one reduced the possibilities of establishing non-covalent interactions with the Ab and thus, these antibodies rendered an IA showing lower detectability [ 147]. The same explanation can be given for the difference in sensitivity encountered with the IAs developed for urea herbicides [131,144] (see above). Antibodies obtained by coupling fenitrothion to the carrier protein through the nitro group led to assays with very poor detectability while the best assay used antibodies raised to a hapten coupled through the thiophosphate group, which suggests participation of the nitro group in stabilizing the immunocomplex [148-150]. Oubifia et al. (unpublished results) failed to obtain a competitive IA to determine 4-NP when using Abs that had been raised from a hapten blocking the phenolic group of the molecule. 7.4.2 Effect of the chemical structure of the competitor Competitors are those haptens used to prepare coating antigens (CAs) and enzyme tracers. Contrary to the immunizing haptens, requirements regarding similiarities with the target are not so strict. In fact, there has often been claimed that certain heterology may favor assay sensitivity [44,47,131,151 ]. In this way, the equilibrium constant defining the formation of the immunocomplex competitor-Ab would be lower than the one directing the reaction between the analyte and the Ab. Optimal heterologous system are usually accomplished by the screening behavior of several haptens coupled to enzymes or proteins. These haptens may have different degrees of heterology depending on the variation of the chemical structure and the length and/or position of the spacer arm. 7.4.2.1 Effect on the IA detectability Despite the heterology concept above mentioned, its application is not always general. Thus, Galve et al. [ 142] obtained a good assay for trichlorophenol by employing a homologous enzyme tracer. However, Abad et al. [43] improved the detectability of an ELISA A. Oubifia et al. / Immunoassays 311 (a) SITE Y l 0 RI PYRE]HROID IN SEC TICIDES SITE X SITE Z (b) SITE C SITE A RI-/~J l R2 PHENYLUREA ~ICIDES | SITE B (c) SITE H SITE F \ ? SITE G Fig. 7.4. Possible attachment sites to design immunizing haptens for different pesticides. (a) Pyrethroid insecticides, (b) phenylurea herbicides and (c) Irgarol 1051. for carbaryl by using a heterologous hapten conjugate. But the same authors [152] produced MAbs to 1,1,1-(trichloro)-2,2-bis(p-chlorophenyl) ethane (DDT) where that heterology approach did not improve the limit of detection of the assay. One explanation could be that in the first case, only slight modifications were made on the length and position of linker while on the second one, changes were more severe since only a part of the total structure of the molecule was employed as competitor. Against that, a greater detectability was obtained on an IA for chlorpyrifos developed by Manclfis and Montoya [153] by employing heterologous conjugates consisting in just a part of the complete analyte chemical structure. Aside of all these contradictory results, our experience 312 A. Oubi17a et al. / Immunoassays TABLE 7.6 LEAST DETECTABLE DOSE (LDD) AND PERCENTAGE OF CROSS-REACTIVITY (CR) OF DIFFERENT PYRETHROID INSECTICIDE IAs a Pyrethroids Permethrin RI R2 H Attachment site b CI~~H LLD %CR Ref. 1-10 [262] (Ixg 1-1) Site X 1.50 Site Y 20.0 a Ci ~ Deltamethrin H Br~~H B r ~ Deltamethrin CN - [125] 0.40 Site Z Br~~H 5.0 B r ~ a -- [125] 0.20 a The IAs used different sources of Abs raised against haptens with the linker placed on different sites. b See Fig. 7.4 for chemical structures of the haptens and positions of the attachment sites. suggests that high-affinity antibodies may be able to render excellent assays even under homologous conditions while the detectability of IAs using Abs with low affinity for the analyte may be increased by using heterologous competitors. Thus, from the three immunizing haptens evaluated to raise Abs against Irgarol 1051, we observed that those raised against hapten with the linker in sites H and G afforded IAs with high detectability using homologous or quasi-homologous competitors. In contrast, hapten with the linker in site F only afforded acceptable assays when the chemichal structure differred to a greater extent from that of the analyte [ 139] (see Fig. 7.4c and Tables 7.8 and 7.9). 7.4.2.2 Effect on the IA selectivity Some authors claim that the chemical structure of the immunizing hapten is the main fact influencing IA specificity, while the chemical structure of the competitor mainly TABLE 7.7 DETECTABILITY OF PHENYLUREA HERBICIDE IAs a Phenylureas Rl R2 R3 Attachment site b IC50 (Ixg 1-1) Ref. Diuron C1 C1 CH3 C1 C1 H C1 CH3 CH3 2.30 n.c. 0.50 16.0 [ 144] Monuron Diuron Site Site Site Site A B A C [ 131 ] [ 145] a The IAs used distinct sources of Abs raised against different haptens. b See Fig. 7.4 for chemical structures of the haptens and positions of the attachment sites, n.c., no competition. A. Oubifia et al. / Immunoassays 313 Table 7.8 EFFECT OF THE CHEMICAL STRUCTURE OF THE COMPRTITOR HAPTEN IN THE IA DETECTABILITY FOR THE IRGAROL 1051a Immunogen Group a HRP-tracer b Antisera ICs0s (p~g/1)c <0.1 I s/'~'/¢c~H-K['I'I ~,,,N.~~j~" NH-- HN II 4eKLM III IV 0.1-0.3 2a 2b 13-14-15 13-14-15 2c 2d 2e 13 13-15 2f 13-15 4a 4b 4d 4e 4c 15 15 15 0.3-1 1-10 15 14 13-15 14 ¸ 13-15 13-15 13-14 13 13 >10 14 14 14 14 14 14 Groups I, II, III and IV represent increased homology with the target analyte. b See Table 7.9 for the chemical structures of the competitor haptens used as HRP tracers. c The numbers 13, 14 and 15 designatethe differentantisera obtained againstthe immunogen4cKLH. It can be observed how the chance for assays with high detectability is higher when the homology is lower. a defines IA detectability. Other authors [55,146] have in contrast suggested that different antisera-coating Ag combinations may modify the IA selectivity. In our laboratory, Oubifia et al. [55] analyzed the selectivities of three IAs for 4-nitrophenol using Abs raised against the same hapten and a battery of different haptenized CAs. The cross,reactivity studies showed a high affinity for compounds with important chemical similarities to the target analyte (as a hydroxyl and nitro group in p a r a orientation), but depending on the coating Ag used, the cross-reactivity of some analytes, such as 2,4-DNP, could change from 16% to 703%. In fact, it was possible to develop an IA for 2,4-DNP using Abs raised to 4-NP that was able to recognize specifically 2,4-DNP even in the presence of 4-NP in the sample [ 154]. In spite of that, these authors also point on the important influence that animal immunoresponse variability may have on the properties of the resulting IA. 7.5 CONJUGATION PROCEDURES USING PROTEINS OR E N Z Y M E S 7.5.1 Conjugation strategies There are many proteins and enzymes to conjugate the hapten. The most frequently used as either immunogens or CAs are KLH, tyroglobulin (TG), conalbumin (CONA), BSA and ovalbumin (OVA). Similarly, HRP or AP are the most frequently used enzymes on heterogeneous IAs. Methods used for protein conjugation have been extensively reviewed [155,156] and Table 7.10 shows a summary of some common coupling strategies. These reactions are usually made in a liquid phase, although solid-phase strategies yield excellent conjugation and easy purification procedures [157-159]. 314 A. Oubi~a et al. / Immunoassays TABLE 7.9 CHEMICAL STRUCTURES OF THE ETs SCREENED DURING THE DEVELOPMENT OF IAs FOR IRGAROL 1051 Irgarol 2a 2b 2c 2d 2e 2f 4a 4b 4c 4d 4e Rl R2 R3 NHBu t NHEt NHPr i NHEt NHPr I NHBu t NHPr c NHEt NHEt NHBut NHBu t NHPr c NHPr c NH(CH2)3COOH NH(CH2)3COOH NH(CH2)sCOOH NH(CH2)sCOOH NH(CH2)3COOH NH(CH2)3COOH NHPr I NHEt NHPr c NH(CH2)3COOH NH(CH2)3COOH SCH3 C1 C1 C1 C1 C1 C1 S(CH2)2COOH S(CH2)2COOH S(CH2)2COOH SCH3 SCH3 The functional group of the spacer arm of the hapten governs the selection of the conjugation method to be used. Different functional groups are possible, but the most frequently used is the carboxylic group. The amino group has also been used in the production of Abs for mycotoxins making use of the Mannich reaction [ 160,161 ] or for the thiocarbamate herbicide molinate [12] using a diazotization method. Similarly, the alcohol group has been used to prepare the immunogen of deoxynivalenol [ 162]. Li et al. used a hapten halide to prepare an immunizing hapten for 4-NP [ 163]. The use of thiolated proteins has also been reported in the case of ceftiofur sodium [ 133]. Thiolated proteins are then able to react with electrophilic groups of the hapten molecule. Amino-modified dextrane has also been used as coating Ag to develop an IA for mycotoxins [164]. The amino groups react with the carboxylic groups of the hapten molecules. It is advisable to use a different conjugation method for the immunizing hapten and the competitor. In this way, the secondary products that may be formed will not interfere in the assay reducing the background noise. Gendloff et al. developed different IAs for mycotoxins, using the same tracer but different methods of conjugation since undesired conjugation reactions using carbodiimides and the mixed anhydride method are possible [165]. Aside from the conjugation method used it is important to know the stability of the resulting bond and the yield of the coupling reaction. Thus, it has been reported lower Ab titers when an ester group was used to conjugate the hapten to the protein [ 166]. Similarly, imines formed when haptens possessing an aldehyde group react with the amino group of the lysine residues should be reduced with cyanoborohydride to obtain a stable secondary amine [ 167]. Regarding conjugation yield, it has been reported that a high ratio of haptens A. Oubi~a et al. / Immunoassays 315 "a = © I c~ = e,i 0 0 0 o •E <~6 ~.~ © = ~1 o ~ 9 z~ z k2 < ~, 0 ~2 ,..el ~ ",~ . ~0 , ~ z ~ ~ .c ~ =?s 4z ~ ° ~ o ~ o ~ Z .~ ,.~ ~ •~ ~ ~ o ~ o ~2 ~ ss~g .. ~.~~.~o x © x~ a~ 0 -~ ..~ © ~ = © © eq Z 0 0 0 Z I eq Z I o g~ 0 . ~ 316 A. Oubiaa et al. / Immunoassays eq eq o zz z ~,~ ~: ~~, o =6 z z 9 oo i z n., i z I I i I O0 ~ o t",l o 0 ° ,..~ 0 ~ 6 z ! o o ,..~ f2~ m i., f2~ ~ o ~o O ~ ~ .~ ~ o .= ~ ~, ~.~ ._ 0 ,~ .~ ~ ~ .~ o5 r~ "~ 0 ,.~ .~ ~.~.~ ¢~ , . 0 0 ¢) ~ I:~ 0 r~ m _ m m o < | ~ • ,.2, z " ~ o o .~ d, z I | z d. o o .1 < o o o "" A. Oubi~a et al. / Immunoassays 317 to protein for the immunogens increases the strength and specificity o f the immune response, but for competitors, a moderate value is more desirable. However, this consideration is not of general application, since excellent Abs\have been obtained when few moles of hapten per mole of protein were attached. Thus, in gur group high-affinity Abs for atrazine were obtained immunizing with a conjugate with f~w hapten molecules attached (control BSA conjugate hapten density was 4.8) [51]. With these Abs, an ELISA was developed, able to detect as low as 9 ng 1-1 in water samples. On the other hand, an ELISA for 4-NP was developed with a detection limit of 0.61 txg 1-1 using a coating Ag with a hapten density of 24.2, much greater than that of the immunogen (2.4 of hapten density) [55]. Verification of the coupling reaction is mainly accomplished by spectrophotometry, although this method is not useful when the hapten does not have a convenient cromophore. Moreover, measurements are often inaccurate producing an overestimation when molecules of the analyte remain non-covalently trapped into the tertiary structure of the protein. Electrophoresis offers a qualitative estimation that the conjugation has taken place whenever the hapten produces a change on the electrophoretic properties of the protein [133,168,169]. Radiolabeled haptens [11,170] have also been used, but labeled haptens are not always available and its use generates handling and waste disposal problems. Trinitrobenzenesulfonic acid (TNBS) [141] is used to evaluate free lysine residues. Studying amino acid composition [171,172] gives a quite accurate idea of the extent of the conjugation reaction. ELISA techniques [146,173] have also been used for hapten density determination but the values encountered may be underestimated if part of the covalently attached haptens are not available for Ab recognition. During the last few years, mass-spectrometric techniques have been introduced, since they allow molecular weight estimation of high molecular weight compounds such as proteins. In this context, electrospray ionization-mass spectrometry (ESI-MS) [170,174,175] and matrix-assisted laser desorption mass spectrometry (MALDI-MS) [176] have provided reliable results. An advantage of MALDI-MS, described by Wengatz et al. [177], is that this method detects only covalently bound haptens whereas haptens bound by adsorption may also contribute to the signal in the UV-spectrum. All these methods present advantages and disadvantages, but their applicability is often limited when the carrier molecules have a high molecular size (i.e. KLH > 2000 kDa). 7.5.2 Cross-linkers Sometimes, the total synthesis of a hapten is a tedious and costly process. The expense of a long synthetic procedure with the quality of the Abs obtained by means of an easier way must be balanced. In these cases, if the analyte structure has a suitable functional group, cross-linkers can be used. Cross-linkers are hetero- or homobifunctional molecules. Its function is to make a bridge between the analyte and the carrier, allowing its connection and avoiding steric hinderance. They can be used in many cases and only require the presence of appropriate functional groups in the analyte chemical structure (Table 7.11). Cross-linkers make the immunogen preparation easier, but they present the disadvantage of blocking important antigenic determinants in the target analyte. This strategy has been use to prepare haptens of complex molecules such as natural toxins, the antihelmintic agent hygromicin or sodium cetiofur [178-180]. 318 A. Oubiaa et al. / Immunoassays 7.6 IMMUNOASSAY F E A T U R E S In an ELISA format based on a competitive configuration (see Section 7.2), the photometric determination of the enzyme activity by absorption is related to the analyte concentration via a dose-response curve such as that represented in Fig. 7.5. Such calibration curves are constructed with standard analyte concentrations and have a sigmoidal shape with a linear portion. When the analyte concentration is very low, the equilibrium is in favor of a high amount of enzyme conjugate linked to the Abs, and the corresponding absorbance is maximal. The working range of the calibration curve is defined by the lower and the upper limits that can be exploited. Within this range, the change in absorbance correlates with the analyte concentration. At a higher concentration than the upper limit, the assay is saturated, and an increase in the analyte concentration no longer has an effect. Many experimental dose-response curves can be found in the literature, or are provided with commercial kits. The most common representation gives the variation of the absorbance (B) using a logarithmic abscissa for the concentration. However, to allow direct comparison of several standard curves, the absorbance data can be normalized between 100%, which corresponds to a zero control absorbance (B0), and 0%, which corresponds to a standard excess absorbance (Bexcess)Ideas on IA data processing have been well developed since at least the early 1970s and it is fair to say that all the main questions were settled by around 1975 [181-183], although there were some important contributions in the early 1980s. Various mathematical transformations have been proposed for a better linearization of the standard curves [ 184,185]. In theory, there are a very large number of choices of curves and models; in practice there are perhaps eight distinct methods which fall into three groups [ 184]. Some companies that sell IAs provide microprocessors that automatically convert IA optical readings to sample concentration, and the transformations are given in the commercial characteristics of the kit. One representation often selected for the microprocessors has the form logit versus log concentration logit(%B/Bo) = ln[(%B/Bo)/lO0 - (%B/Bo) ] but the most used transformation is the four logistic parameters model [ 182] defined by the following equation: y= (A - D)/[1 + (x/C) 8] + D where y is the response (absorbance) and x is the dose. The values A and D correspond to the upper (maximum absorbance) and lower (theoretical non-specific binding (NSB( factor) asymptotes of the curve, respectively. The parameter B (slope factor) is related to the slope of the curve at the inflection point. Parameter C is the dose that corresponds to the center, or inflection point, of the curve. With a dose-response curve it is possible to characterize the IA regarding detectability and sensitivity. For quality control, precision, accuracy and specificity, further experiments are required that will be described below. However, special attention also has to be paid to the material employed to run the assays such as microtiter plates, pipettes, batch of the biological reagents, stability, storage conditions, buffers, etc. A. Oub#ia et al. / Immunoassays 319 TABLE 7.11 LIST OF SOME OF THE MOST FREQUENTLY USED HOMO- AND HETEROBIFUNCTIONAL CROSSLINKERS Homob ifunctional 1. Amino group 1.1. Bis-imidoesters (Bisimidates) 1.2. Bis-N-succinimidyl derivates 1.3. Aryl halides 1.4. Acylating agents 1.4.a. Diisocyanates and Diisothiocyanates 1.4.b. Sulfonyl Halides 1.4.c. Bis-Nitrophenol Esters 1.4.d. Acylazides 1.5. Dialdehydes 1.6. Diketones 2. Sulfhydryl group 2.1. 2.2. 2.3. 2.4. Mercurial reagents Disulfide forming reagents Bismaleimides Alkylating agents 2.4.a. 2.4.b. 2.4.c. 2.4.d. 2.4.e. 3. Carboxyl group 3.1. Bisdiazomethylene derivatives 4. Phenolate and imidazolyl group 4.1. Bisdiazonium reagents 5. Guanidinyl group 5.1. p-Phenylene diglyoxal Heterobifunctional 1. Amino and sulfhydryl group 1.1. N-succinimidyl derivatives 1.2. p-Nitrophenyl esters derivatives 1.3. Imidoesters 1.4. Acyl azides and acyl chlorides 1.5. Aryl and alkyl halides 1.6. Haloketones 2. Carboxyl and ether sulfhydryl or amino group 2.1. Diazoacetyl groups 3. Carbonyl and sulfhydryl group 3.1. Alkoxylamino groups Bishaloacetyl derivatives Dialkyl halides s-Triazines Aziridines bis-Epoxides 320 A. Oubiaa et al. / Immunoassays Bo tu co ' ~ m Prreocifisl/eoi n ~ p t-~ ~ ,ft. t.m ~ log Analyte Concentration Fig. 7.5. Typical sigmoidal dose response as measured by the absorbance of the solution after incubation of a fixed concentration of antibodies and enzyme conjugates, with increasing concentration of analyte. Definition of usual parameters: limit of detection or least detectable dose (LDD), concentration inhibiting 50% the signal at zero-dose (IC50). B0 and Bexcess are the absorbances of the zero control solution and of the standard excess solution. 7.6.1 Detectability and sensitivity Although both terms are often confused, sensitivity is defined by the capacity to distinguish slight changes in the concentration, corresponding to the change in response (absorbance) per unit of reactant (analyte concentration). In contrast, detectability of an IA is usually expressed either as the limit of detection or as the smallest concentration of the analyte that produces a signal which can be significantly distinguished from zero for a given sample matrix with a stated degree of confidence. The minimum detectable concentration is often known as the least detectable dose (LDD) (see Fig. 7.5). Although there is no standardized way of defining the detectability, there is a general consensus in favor of selecting the dose which inhibits 10% of the binding of the Ab with the enzyme tracer at 90% B/Bo. The LDD is then often measured using the standard samples in pure water or buffer. The minimal detectable dose has also been described as two or three times the standard deviation from the mean measurement of the blank dose signal. Many scientific papers refer to the detectability of their assays as the IC50 value. 7.6.2 Precision The precision of an IA is defined as the extent to which replicate analysis of a sample agrees with each other. The reproducibility is the ability to yield the same result within analysis, between analysis and between operators. According to the non-linear shape of the dose-response curve, the variance is non-uniform, and the experimental errors increase towards the two limits of the measuring range, especially in the non-linear parts (see Fig. 7.5). Therefore, the precision should be given calculating the standard deviation per percent of the coefficient of variation (CV) vs. concentration. The highest precision is A. OubitTa et al. / lmmunoassays 321 obtained for concentrations close to the concentration obtained at B/Bo -- 50%. The precision profile is the best way to determine the dynamic range of an assay. 7.6.3 Accuracy The accuracy of an assay reflects its ability to measure true values for an analyte. The dose-response curves are usually constructed with standard solutions. Another easy test consists of preparing several dilutions of a real sample and simply measuring them. Ideally, in the absence of interfering substances, the standard curve should be parallel to the curve obtained by diluting the sample within the working range. The most used method is to calculate recoveries by spiking real sample with known amounts of the target analyte. However, the best evaluation of the accuracy is obtained by comparing the results obtained from real samples with other validated analytical techniques. 7.6.4 Specificity and cross-reactivity We mentioned before (Section 7.4.1.1) that the chemical structure of the immunizing hapten is very important for the specificity of the IAs. The specificity of an assay reflects its ability to produce a measurement of the analyte to be determined in the presence of other compounds. Cross-reactivity is defined as a specific interference related with the Ab performance and it is best described as the inability of the assay Ab to discriminate flawlessly between the analyte and a related molecule. Depending on the chemical structure of the hapten used for immunization and the class of chemicals under investigation, Ab recognition of compounds similar to the analyte is frequently observed. Therefore, it should be determined which compounds cross-react and to which degree [186]. CR of a particular Ab determines its applicability. Immunoassays with low CR values are suitable for single compound analysis. In contrast, a group-specific assay requires high CR values. Thus, the selection of the Ab depends on the purpose of the application. Several methods to calculate CR have been described and reviewed, but the most prevalent is the 50% displacement method, originally introduced by Abraham [187]. In this method, the concentration values of standard and cross-reactant necessary to displace 50% of the bound tracer are compared. A ratio of the resulting concentrations can be referred to as %CR at the IC50. %CR -- (IC50 analyte/ICs0 crossreactant) x 100 Depending on the slope and shape of the response curve the percentage of CR (%CR) may be different at different displacement levels. Regarding CR measurements, it is worth mentioning that in real environmental situations, a single analyte is not to be expected, but various analytes will be found together with the target compound. In this sense, the measurement of the CR should approach the real environmental situation as closely as possible. In clinical chemistry, Miller and Valdes [188] reported a model that closely approaches real situations by measuring the CR of an analyte in the presence of cross-reactants. It was proposed that one should apply various doses of cross-reactants, each in the presence of various doses of standard. This approach has been adapted to environmental CR studies of IAs for atrazine [189] and for chlorpyrifos-ethyl [190,191 ]. The effect of varying the concentration of the cross-reactant at a fixed standard concentration was analyzed. CR estimation, determined in the absence 322 A. Oubi~a et al. / Immunoassays of the target analyte did not correlate with the degree of interference caused by a crossreactant in the presence of the standard. As an example, Table 7.12 shows the calculated CR of 2-benzimidazolyurea respect to carbendazim and benomyl as standards. Knowledge of CR can be a valuable tool for analyzing a cross-reactant using an ELISA designed to monitor a different analyte, provided that this analyte is not present in the sample. In this way, a commercial ELISA for detecting parathion was used to study the disappearance of fenitrothion (a cross-reactant) under real environmental conditions in rice crop waters [192]. 7.7 MATRIX E F F E C T The matrix effect could be defined as an induced deviation from theoretically predicted assay parameters, caused by constituents or properties of the sample other than the analyte in a real sample. In many cases the interfering substances are completely unknown [ 193]. The ultimate objective of any IA is to obtain correct results when measuring a particular analyte in a real sample; however, there are some factors, included in the matrix effect notion, which in case of being uncontrolled, can be avoided from achieving this aim. Because the IA actually is an in vitro biochemical reaction, many physicochemical conditions can influence IA performance, i.e. pH, type of buffer, reaction mixture constituents, ionic strength, temperature, etc. All these factors may have an influence, not only on the Ab-Ag interactions, but also on the enzyme performance and therefore, should be the subject of evaluation studies. Interferences on IA methods can be categorized into two major classes: 1. Specific interferences (those substances which affect binding of Ag by competing for the specific binding sites on the Ab) 2. Non-specific interferences (those which affect the binding event between the Ab and the Ag, or the enzyme activity in a general way) 7.7.1 Specific interferences 7. 7.1.1 Cross-reactivity This referrs to those kinds of sample components which are recognized by the Ab. Usually these interferences can be predicted knowing the chemical structure of the immunizing hapten. However, specific studies have to be made which have been already discussed in Section 7.6.4. 7. 7.1.2 Enzyme inhibitors They are sample components with a direct and specific inhibitory effect on the enzyme label. For instance, in the case of HRP, primary alkyl hydroperoxides in the sample will act as substrates for the enzyme label, inducing catalysis. Since HRP is a suicidal enzyme inactivated by the catalytic process, these peroxides of the samples can inflict a variable loss of activity in the enzymatic tracer. Furthermore, it has been observed an inhibition by several anions like azide, which inhibits the peroxidase by binding to the heme group of the enzyme [194]. Therefore, azide should not be added as antimicrobial agent to buffers A. Oubiffa et al. / Immunoassays 323 o O ~9 o O [© I I I © I o O ¢..) z < ~ ~-~ 0 00 . ~ N © © < z < O O tt'3 © < I I I i ddc~d I ~c~ r.t3 o z z O N < z O z 8 O < 0 e~ ~C) ~ ~=~,-~ I~- ~ oo ~:~C~ O o z D < "=~ r~ f < N < ~9 z © . ,...~ I < =L 0 o z C~ v b q} < 0 O O ,-"~ O O O O ,'-~ O O <p {D ",~ [-.. ~ O N r..) b b~ 0 O 0 %) {1,} ! t",,I o 324 A. Oubiaa et al. / Immunoassays used in EIA with a peroxidase tracer. Some cations such as Ca 2+ can lead to an activation of the peroxidase [ 186]. 7.7.2 Non-specific interferences These types of interference are due to other components that bear no structural resemblance to the analyte at all or physicochemical parameters, such as ionic strength, pH, salts, organic compounds, etc. An increase of ionic strength usually leads to a reduction of electrostatic interactions between Ab and Ag, because charges taking part in those interactions are shielded by the ions present [195]. The IA performance in solutions with different ionic strength should be checked, since it may change from one analyte to another depending of the predominating forces present in the immunocomplex. Thus, an IA for atrazine [51], tolerated PBS concentration between 0.1 and 1.0 M of PBS (see Fig. 7.6) while IAs for phenolic compounds were very much influenced by the ionic strength of the sample [55,142,154]. On the other hand, non-covalent forces can also be affected by pH. Some assays are very sensitive to pH effects, while others perform well in a wide range of pH. For example, assays for phenolic compounds were completely inhibited below pH 5.5. [55,163] and worked very well at basic pH. In contrast, some triazine IAs, such as the ones for atrazine and Irgarol [139,196], have been reported to work very well between pH 2.5 and 10.5 (see Fig. 7.7). 7. 7.2.1 Other non-specific interferents A troublesome class of non-specific interferents are very low molecular weight molecules such as chaotropic ions (SCN-, I-, Br-, C1-), which may alter the 3-dimensional structure of the Ab or break the superficial interactions Ab-Ag. Other interferents are organic acids like propionic or acetic acid that have been reported to break Van der Waals interactions [ 195]. ELISAs for carbofuran and chlorpyrifos showed a high tolerance to the presence of a wide range of inorganic cations and anions in water and soil samples [197,198]. On the other hand, humic substances may bind non-specifically to the Ab, leading to false-positive values. Furthermore, humic acids may interact with pesticides by mechanisms involving either physical sorption or chemical reaction [199,200]. When coupling IA with extraction procedures, it is necessary to consider the organic solvents used during a previous clean-up step as potential interferents of the IA test. In an ELISA for carbaryl, the tolerance level to organic solvents was studied, showing that moderate levels of such solvents have a clear influence in the assay (see Fig. 7.8) [201]. Other authors have reported the same kind of interferences due to organic solvents [ 197,202]. However, the extent and direction of the solvent effect may be different from assay to assay. Thus, Sugawara et al. [203] reported the positive effects of dimethylsulfoxide (DMSO) over the detectability of an IA for dioxins. 7.7.3 Application of IAs to the analysis of environmental matrices Since pesticides are widely employed, it is expected to find them inside a high number of matrices, mainly water, soil and food, but it is also possible to check the 325 A. Oubi~a et al. / Immunoassays 0.8 0.6 Bo • IC5o - Bo/IC5o - 0,4 - 0.2 - 0.0 - ob o L¢ PBS concentration (M) Fig. 7.6. Effect of the ionic strength on the atrtazine IA performance. From Gasc6n et al. [51]. presence of these compounds even in human fluids and tissues. Regarding water and soil matrices, probably the most studied types of matrix, almost all the possible interferences which have been previously referred can be found (salts, pH, cross-reactants, enzyme inhibitors, humic substances, etc.) and almost every pesticide has been tested in this matrix, e.g. atrazine [199], nitrophenol [55], organochlorine compounds such as endosulfan [141], organophosphorus pesticides such as chlorpyrifos [198] or diazinon [202], etc. •¢" °=° 1.25~....~ " pH 2.5 1.00~~k~,,,~ ~---......~~'~ " " pH 3.5 pH5.5 [ N"%,"%, •,, ¢n ° °5° I 0 on "~0-2 • 10-1 10 0 pH7.8 pH 8.5 101 [Irgarol], nM Fig. 7.7. Effect of the pH on the IA for Irgarol 1051. The ELISA is operative between pH 2.5 and 10.5. Only a small decrease of the sensitivity and of the B0 was observed at acidic pHs whereas no significant changes occurred at basic pHs. Assays were run simultaneously on three different ELISA plates. Agreed to and accepted by ACS Copyright Office [139]. 326 A. Oubiaa et al. / Immunoassays [ICsobuffedlCsosolvent]xl O0 120 100 ! 80 60 40 20 1 2 4 8 10 16 20 30 40 50 60 70 % solvent in buffer Fig. 7.8. Presence of small amounts of organic solvents in the ELISA alters the characteristics of the assay. Acetone, acetonitrile and poly(polypropylene glycol) 1000 produce a dramatic negative effect on the sensitivity of the carbaryl assay. Of the solvent tested, only methanol seemed to be tolerated when the concentration remained below 10% (v/v) in the assay buffer. Datum points represent the average of three wels. Coefficients of variation averaged are less than 2% and the standard deviations are shown for methanol. (~) Poly(propylene glycol) 1000; (+) acetone; (m) acetonitrile; (O) methanol. Agreed to and accepted by ACS Copyright Office [201]. 7. 7.3.1 Water samples IAs have been intensively used for the determination of pesticides in surface [11,204], rain [205,206] and ground water [207-210]. Mouvet et al. [211] evaluated IA operational features (cross-reactivity, detectability and reproducibility) of several IA kits for the determination of triazines in surface and ground water. Within-assay coefficients of variation (CV) were below 7%, while inter-assay CV was always lower than 20%. Results obtained with different water matrices were compared to those obtained by gas and liquid chromatography (GC and LC) to obtain the best correlation for the surface water samples. In a similar study, Thurman et al. [212] used a commercially available IA to carry out analysis of triazines in surface and ground-water samples comparing the results with those obtained by gas chromatography-mass spectrometry detection (GC-MS) after solid-phase extraction (SPE), obtaining correlation coefficients greater than 0.90. Carbaryl and its main degradation product 1-naphthol were also determined in wellwater samples from Almeria (Spain) for several months using ELISAs previously developed for these analytes [ 136,201,213]. The results matched very well with those obtained when applying the EPA method 531.1. In May-June the carbonyl levels of some wells exceeded the upper limit of 0.1 txg 1-~ established by the European Community for drinking waters, corresponding to the time when applications of pesticides started in that region. Levels were again low in July, indicating either movement of the pollution A. Oubitia et al. / Immunoassays 327 or degradation of the pesticide. Thus, 1-naphthol was detected specially after field treatments in July-August. Seawater samples have also been analyzed by immunochemical methods. Atrazine was analyzed in estuarine and coastal waters by a magnetic particle IA (High-Sensitivity RAPID ELISA, Ohmichron Corp.) and by on-line solid phase extraction-liquid chromatography-diode array detection (SPE-LC-DAD) [196]. It was noted in this study that repeated injections of samples with a high salinity (15-35 g 1-1) caused broadening of the peaks in the chromatogram and therefore problems for quantifying small atrazine concentrations. This was attributed to a lifetime reduction of the precolumn because of the high salt concentration. The chromatographic method showed high precision and was more accurate since recovery values were always close to 100%. However, for high salt content samples this was only true when frequent renewal of the precolumn was performed. In contrast, several replicates of the same sample could be performed by IA since it was not affected by extreme salinity conditions. Similarly, the antifouling agent Irgarol 1051 has been determined in enclosed seawaters of the Mediterranean Spanish Coast by an ELISA recently developed in our laboratory [139,214]. A monitoring survey was performed using ELISA and on-line SPE-LC-DAD during 1996/97 with monthly sampling at the same coastal area. There was a good agreement between both techniques when measuring levels ranging from 0.007 to 0.325 ~g 1-1. 7. 7.3.2 Soil samples Several pollutants have been analyzed in soil by immunochemical techniques (triasulfuron [215,216], triazines [85,217-219], PCBs [53,220,221], chlorpyrifos [222,223], pentachlorophenol [224], bromacil [17], etc). In this matrix, pollutants have to be extracted with organic solvents although on a few occasions water has also been used for very polar compounds. As mentioned above, the effect of the organic solvent over the IA features has to checked. Usually IAs tolerate small amounts of solvents which are miscible with water and the detectability is good enough to allow dilution of the solvent extract. Supercritical fluid extraction (SFE) is an excellent alternative to be combined with IA analysis. Wong et al. compared SFE with solvent extraction of parathion and 4-nitrophenol from soil. The extracts could be measured directly by IA without any solvent exchange. Bound pesticide residues in soil have also been detected by immunochemical methods. Some pesticides bind organic matter of soil, mainly humic and fulvic acids and cannot be analyzed by common extraction methods. Ulrich et al. [225] developed a non-competitive sandwich IA for the analysis of bound residues based on a humic acid Ab and a triazine Ab. Humic acids were extracted from the soil, bound to the plates and the non-extractable triazine residues were detected by HRP-labeled atrazine Abs. Similarly, Dankwardt et al. [200] used a competitive IA for the investigation of bound residues. 7. 7.3.3 Food samples IAs have been applied to monitor a wide variety of environmental pollutants in food matrices. For instance, carbofuran [226], aldicarb [227], thiabendazole [228] and azinphos-methyl [229] have been analyzed in fruit juices. For example, a validation of a 328 A. Oubiaa et al. / Immunoassays carbaryl IA has been reported for the analysis of banana, carrot, green beans, orange, peach and potato extracts [230]. The analyses were carried out diluting the methanolic extracts 1:50 in PBS buffer. This dilution factor had negligible effect in the ELISA reaching sensitivities varying from 3.9 to 5.7 Ixg 1-~ in these conditions. An excellent correlation was observed when compared the results with those obtained by liquid chromatography-diode array detection (LC-DAD). However, it has been demonstrated that analysis can also be directly performed in fruit and vegetable juices by just diluting them with the assay buffer and adjusting the pH of the final solution [231,232]. Aldrin and dieldrin [233], paraquat [234] and benzoylphenylurea insecticides [146] have also been analyzed in milk. A dipstick IA using MAb was used for the determination of atrazine in milk and other food samples yielding excellent recoveries [83]. The total assay time was 25 min and the dynamic range was between 0.3 and 10 lxg 1-~. 7.7.4 Biological monitoring by IA Monitoring of human exposure to pesticides and other environmental pollutants has been carried out by IA measuring parent chemical and/or metabolites, protein or DNA adducts. Tissues can be analyzed but usually body fluids such as blood, milk, urine, sweat or expired breath are preferred to assess individual exposure. Most of the IAs developed have sufficient detectability for biomonitoring. Thus, an IA to analyze 1-naphthol was developed showing and IC50 of 72 txg 1-1 [136]. However, the urine of agricultural workers and formulators in contact with carbaryl had levels of 1naphthol ranging from 0.07 to 1.7 mg 1-1 and 6.2 to 78.8 mg 1-1, respectively. An IA for 3,4,5-trichlor-2-pyridinol, the main metabolite of chlorpyrifos in urine, was developed with an IC50 of 0.12 txg 1-1 [ 151 ]. The effects of the urine matrix on the detection of p-nitrophenol, a metabolite of parathion, by ELISA have been reported [235]. The presence of urine during the Abanalyte interaction increased the apparent IC50 value and inhibited color development. However, because of the high detectability of these IAs, analysis was still possible by diluting the urine with the assay buffer until a 5% concentration value was reached. When testing the matrix effect of the urine in an IA for determining metabolites of naphthalene it was found necessary to dilute the urine [135], although in this case the IC50 was not affected. However, the authors suggested overcoming this effect by changing the ionic strength of the assay buffer. IAs for other urinary metabolites have been reported such as the mercapturic acid conjugate of atrazine [173,236], hydroxyatrazine [48], paraquat [62], picloram [62], etc. 7.7.5 Solutions to overcome the effect of the matrix There are several examples of successful countermeasures to overcome the matrix effect in IAs, such as sample purification, dilution, high-performance liquid chromatography (HPLC), stepwise addition and other changes in assay format. When developing a new IA or testing an analyte in a matrix with unknown effects, possible problems due to interferences should be determined in order to find the most suitable countermeasures. Classically, this analysis has been carried out by comparing a standard curve to another one in which the substance supposed to cause some interference is incorporated. If there is no matrix effect, no differences should be observed between the parameters of both curves. A. Oubiga et al. / Immunoassays 329 Another possible method is to measure fortified samples and compare the expected and measured recovery values. These strategies are only possible if a blank sample matrix is available. In an IA with interferences, recovery suffers from deviations (it should be around 100%) and differences between observed and measured values usually decrease with dilutions. Diluting the sample makes it possible to avoid a negative effect from the matrix. As an example, in an IA for aldicarb determination, it was not possible to eliminate the matrix effect of lemonade juice extracts unless the samples were diluted 50 times or greater [227]. In the same way, honey samples being analyzed using a flow-through immunosensor had to be diluted to 1 g 1"1 [237]. In a MAb-based ELISA for the determination of carbaryl in apple and grapes juices, a sample dilution of 1:64 of apple juice was found to be suitable for ELISA, but it was not sufficient to analyze grape juice [231]. However, matrix dilution has a direct influence over the assay sensitivity. Using clean-up procedures such as solid-phase or liquid-liquid extraction it is possible to remove interfering substances from samples while preconcentrating the target analyte, if desired. The procedure is then time-consuming and handling of many samples becomes more complicated. In this case, solvent compatibilities with IA should be checked. Matrix effects have also been dealt with by adding known amounts of standard to the sample and extrapolating the result to zero-added standard (running a standard curve in the matrix of interest). After characterizing the dependence of observed signal on analyte concentration in that matrix, one can determine which analyte concentration produces the signal where no standard is added. This approach relies on an observed constancy in the relationship between relative absorbance and concentration for product extracts, which implies that standard curves run in the matrix are parallel to that run in the standard buffer. IA color development depends on the logarithm of the concentration, so adding a known amount of standard to a blank sample has much greater effect on the absorption than adding the same amount of standard to a positive sample. Because of this, the ratio of absorbances of a standard added to unaltered sample can be used to classify a sample as positive or negative [238]. 7.8 VALIDATION STUDIES The process of validating a method cannot be separated from the actual development of the method conditions, because the developer will not know whether the method conditions are acceptable until validation studies are performed. The development and validation of a new analytical method may therefore be an iterative process. Results of validation studies may indicate that a change in the procedure is necessary, which may then require revalidation. During each validation study, key method parameters are determined and then used for all subsequent validation steps [239]. Although there is general agreement about which type of studies should be done, there is great diversity in how they should be carried out [240]. The literature contains diverse approaches to performing validations [241-243]. The acceptance of IAs is dependent upon the demonstration of quality and validity compared to more traditional methods. The validation of results given by IA is usually performed by comparison with chromatographic methods, when available, and if possible with real samples. The validation guide- 330 A. Oubi~a et al. / Immunoassays lines depend on whether the IAs are used to complement the traditional analytical methods or to replace them. For quantitative methods, confirmation of the limits of quantitation, delineation of the quantitative range, evaluation of interferences and estimation of the accuracy and precision with field samples are required. When a sample pretreatment (extraction, clean-up, etc.) is required, it should be included in the method validation and described in the written procedure. Information to be included in written immunological analytical methods can be found in Mihaliak et al. [244], who describe the guidelines for using IAs in support of pesticide registration. Immunochemical methods are particularly well adapted to environmental fate studies, i.e., of aquatic and terrestrial field dissipation, ground water, and run-off studies. Environmental surveys provide a good opportunity of comparison with chromatographic measurements in validation studies. Most of these validation studies using real samples contaminated by the analyte are devoted to atrazine, since this herbicide is found everywhere in the world and is, of course, included in most environmental surveys [34,196,204, 211,219,245-250]. A good example corresponds to a survey of 750 water samples collected from four streams in USA, with a set of 224 of them which have been also analyzed by GC-MS [249]. No false negative was observed and only 5.5% of the assays gave a false positive using ELISAs. Good correlation between both techniques was obtained in similar studies performed with alachlor [246,248,251,252], metolachlor [253] and carbofuran [197]. Marco et al. [213] also validated two IA methods for environmental monitoring of carbaryl and 1-naphthol in ground water samples with liquid chromatrography-post column reaction-fluorescence detection (LC-PCR-FD). Validation studies for analytes less commonly detected in environmental matrix are performed with spiked samples. Oubifia et al. [223] evaluated an ELISA for the determination of chlorpyrifos-ethyl and compared the result given by IA with those obtained by automated on-line solid-phase extraction followed by LC-DAD in spiked estuarine waters, yielding a correlation of 0.991. Good correlations have been found using IA for the determination of chlorothanil as compared with the gas chromatography flame ionization detection (GC-FID) method, and for carbendazim as compared with LC determinations [254]. 7.9 CONCLUSIONS AND FUTURE DEVELOPMENTS IA has proven to be a very useful technique for the screening of contaminating substances in environmental samples. In spite of this IAs are not free from limitations (see Table 7.5) and are not yet fully accepted methods. The increasing number of target substances for which assays have been developed and the enormous number of publications of this topic will help in introducing them into the analytical laboratories. The strength of this technique lies in the possibility to screen a large number of samples (screening methods) within a short time at low costs. Further promising developments are multi-analyte immunochemical systems wherein more than one compound or group of compounds can be detected simultaneously. Much effort should be put into the development of continuous measurements, such as FIIA and immunosensors [255]. The IA-based dipsticks are other topical and relatively simple sensing devices [83]. Also, new strategies for Ab production are being developed. As a A. Oubi~a et al. / Immunoassays 331 result of more stringent rules for the use and handling of experimental animals, conventional Abs will increasingly be replaced by Abs that can be produced with in vitro methods. Genetically engineered Ab appears to be very attractive because its selectivity and affinity can be tailored by site-directed mutations without requiring new immunizations [256]. A promising goal is the completely synthetic production of binding proteins or other synthetic receptors that are fitted to the structure of the analyte by molecular design. The use of libraries guarantees that bottleneck Ab production be closed. 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