Immunochemical techniques: Antibody production for pesticide analysis. A review

ANALmcA CHIMICA

ACTA Analytica Cbimica Aeta 311(1995) 393-405

Immunochemical techniques: antibody production for pesticide analysis. A review Bertold Hock *, Andrea Dankwardt, Karl Kramer, Alexander Marx Technische Universitiit Miinchen at Weihenstephan, Faculty of Agriculture and Horticulture, Department of Botany, D-85350 Freising, Germany

Received 13 September 1994; revised 20 February 1995; accepted 27 February 1995

Abstract Immunochemical techniques supplement traditional analytical methods in an ideal way because they are extremely sensitive, simple, and inexpensive. The most important types presently used for environmental analysis are immunoassays, immunosensors, immunochromatography, and immunolabeling. Representative examples are given. Standardized immuno-

chemical methods for environmental monitoring profit from monoclonal antibodies because of defined selectivities and affinities toward the analyte. A critical step in the production of monoclonal antibodies is the development of efficient screening procedures to identify suitable hybridoma lines. Immunomagnetic beads offer an alternative to conventional screening with immunoassays because the problems of being time-consuming, escaping detection or loosing valuable cells can be solved. This technique is based on tagging surface receptors of hybridoma cells with binding properties of the secreted antibodies by magnetic beads, which are coated with hapten conjugates. In spite of the considerable progress in hybridoma technology, antibody development has entered an entirely new domain because of the potential of recombinant antibodies. Recombinant technology will eventually allow faster production of antibodies with new binding properties (mutant antibodies) and also a stepwise abandonment of animal experiments required for antibody production. The production of phage antibodies for s-triazines is described. Keywords: Biosensors; Immunoassay; Pesticides; Antibodies; Environmental analysis; Review

1. Introduction Antibodies Cabs) have been applied for the first time as tools for binding assays by Berson and YaIow [l]. The main applications were observed in the medical field. Immunochemical approaches to environmental analyses have experienced considerable progress only during the last ten years. This is

* Corresponding author. 0003-2670/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0003-2670(95)00148-4

due to the availability of an increasing number of abs for a variety of xenobiotics, on improved antibody cab) properties, i.e., higher affinities and selectivities toward the analyte as well as improved robustness toward matrix effects, and finally on new applications, especially in the field of immunochromatography and immunosensors (for recent reviews, see Refs. [2-51 ). Considering the great potential of immunochemical methods, it is also important to identify the present limitations. They are mainly seen in two

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factors, the speed of ab production for new analytes and the efforts on standardization of immunochemical methods, which should be coordinated on a worldwide basis. Our laboratory has been involved in the production of abs for pesticides and environmental chemicals during the last ten years (e.g., [6-lo]). In this paper, some recent applications based on our abs are presented, followed by a discussion of the progress in ab development including recombinant abs.

2000 ;

2. Immunoassays 0

The fast progress in the automated determination of pesticides by means of solid-phase extraction combined on-line with either column liquid or capillary gas chromatographic (GC) separation and selective detection [l&12] clearly demonstrates that immunoassays (IAs) cannot compete in terms of the information obtained on the sample composition. The strength of IAs is experienced during screening analyses when a large number of samples has to be assayed in parallel for a single analyte within a short time. This is shown by the following analysis of a large number of soil samples for atrazine in our laboratory. This s-triazine herbicide has been one of the most important herbicides in Germany before its ban in April 1991. It was used for selective pre-emergence and early post-emergence control of annual weeds in crops, primarily corn, and for non-selective vegetation control in non-cropland. Due to its persistence and its application for more than three decades, atrazine and its metabolites have been detected in rain, ground and surface water. The present study has been part of activities directed to soil monitoring to ensure the compliance with the atrazine ban. In this study, which has been carried out in 1993, more than 400 soil samples from corn fields in Bavaria were collected and screened for atrazine residues with an enzyme immunoassy (EIA) based upon sheep abs [13]. The EIAs including several sample dilutions in quadruplicate were carried out within a week as 2-4 persons simultaneously performed the assays. The 15 diluted soil extracts were further diluted 1:150 in order to measure samples

500 pg atrazitdkg

1500 soil (HPLC)

Fig. 1. Comparison of atrazine concentrations in soil extracts determined by EL4 and LC. The extracts were diluted 1:150 prior to the EIA. LC analyses were carried out at the Landesanstalt fiir Bodenkultur und Pflanzenbau.

containing 100 pg/kg close to the middle of the test and to avoid matrix effects. Samples containing concentrations above 300 pg/kg were investigated once more after diluting the original soil extracts 1:500 to shift the working range closer to the middle of the test. We found, that 34 samples (8%) of the investigated soil samples contained more than 100 pg atrazine per kg soil and were considered positive, in other words atrazine is still in use in some cases. According to investigations by Schewes [14], concentrations exceeding this value indicate same-year application of atrazine. Since false negative results are not obtained with immunoassays, the number of samples for validation by liquid chromatography (LC) could be considerably reduced in our studies. This means that, besides all positive samples, only a few negative ones had to be assayed by LC. A close correlation of r = 0.97 was found for the results obtained by the EIA and LC after diluting all samples 1:150 for the EIA (Fig. 1). The slope of 0.86 indicates a slight underestimation by the EIA. A comparison of LC and EIA including the 1:500 dilutions of the samples with concentrations above 300 pug/l also yielded an acceptable correlation of r = 0.96 (not shown). This work underlines the potential of IAs as a

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screening tool even for natural soil samples. At the same time it has been demonstrated that, under the conditions of this report, IAs can be used as a reliable quantitative method.

3. Immunosensors Further progress of the IA technique led to the development of immunosensors (IS). They are commonly considered as biosensors although the future use of recombinant abs, synthetic abs or other synthetic binding proteins may not totally justify this classification. Independent from these considerations, IS have to fulfil the following specifications of true sensors. They are miniaturized measuring devices, which selectively detect analytes and generate reversible and concentration-dependent signals. It is obvious that existing IS do not fulfil all criteria of true sensors. Frequently the selectivity is not absolute, especially in the case of hapten sensors. Secondly, the reversibility allowing continuous measurement does not exist in a strict sense. However, quasi-continuous approaches have been successfully tested starting with flow injection immunoanalysis (FIIA, [15]). A significant step in the direction of true IS is the real time analysis of binding events. This has been realized by applying optical transduction principles such as surface plasmon resonance [16], grating couplers [17] and interferometry [18] and by omitting label-dependent signal generation by enzyme tracers or fluorescent labels. An interesting example has recently been given by Bier and Schmid [17] using a grating coupler IS for pesticide detection. This device allows the estimation of the kinetics of ab binding because the speed and changes of the speed can be recorded. As the transducer has been integrated in a microprocessor-controlled flow system, complete automation of incubation, regeneration and data acquisition is achieved. In this case the binding of a monoclonal antibody (mab) against the s-triazine terbutryn has been studied in the presence of different herbicide concentrations. The immunochemical principle is therefore based on an indirect IA with unlabeled abs. A kinetic interpretation of the data shifted the detection limit to lower analyte concentrations and short-

terbubyn eonc. InMl Fig. 2. grating apparent binding different

Normalized competition curves for terbutryn using a coupler IS (reproduced from Ref. [17]). The change of thickness of the waveguiding film (tF, urn) induced by of the mab KlF4 [8] and the suppression of binding by concentrations of terbutryn is shown.

ened the assay time from 30 to 3 min (Fig. 2). Regeneration of the IS surface was achieved by proteinase treatment and acidic washing to remove the bound abs. It can be foreseen that further developments will lead to multianalyte systems when arrays of different hapten conjugates are available. They can be applied for the binding of mixtures of abs directed against different haptens. In this case even cross-reacting abs may be very valuable because subsequent chemometric analysis can be used for the identification of patterns of related analytes.

4. Immunoaffinity

chromatography

Immunoaffinity chromatography (IAC) is used for isolating and concentrating analytes. It is based on the reversible, biospecific interaction of abs, which are immobilized to an appropriate support material, with their corresponding antigen or hapten. Present applications of IAC are directed toward trace compounds of interest from crude sample mixtures. Consequently their final detection, for instance by LC or GC, becomes easier and more sensitive. Up to now a multitude of IAC applications have been reported although only a very few have been used for pesticides, e.g., [19-211. The following steps have to be optimized before the IAC can be applied as an analytical tool. (1)

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Choice of an appropriate support material. (2) Choice of a suitable antibody. (3) Choice of the elution buffer. The selection of an appropriate support material for a desired application is crucial for an optimal performance of the IAC. The solid phase has to provide a sufficient amount of suitable functional groups for the covalent immobilization of the abs. Covalent coupling compared to adsorptive immobilization techniques reduces the leakage of abs from the solid phase due to several washing and elution steps required during IAC. Moreover the compounds of interest should only be adsorbed by the immobilized abs but not by the support material itself. So far CNBr activated Sepharose has most often been used as support material for IAC applications. However, it has distinct disadvantages such as leakage of immobilized abs, introduction of positively charged groups at neutral pH and toxicity of CNBr [22]. Today a wide range of support materials with various functional groups is available for IAC applications. Abs can be coupled to a solid phase through their amino, carboxyl or carbohydrate residues. O’Shannessy and Wilchek [23] favour the oriented coupling of abs via their carbohydrate groups to the solid phase. In this case the binding sites of the immunoglobulins are not involved in the coupling reaction, as it sometimes occurs when they are coupled via the NH, groups. Therefore both binding sites are accessible for the analyte. If mabs are coupled via their carbohydrates to a hydrazide gel, this does not always result in a higher antigen binding capacity compared to a non-oriented coupling via the amino groups [24]. Mabs were sometimes found to have carbohydrate residues in their Fab part through which

Table 1 Atrazine and terbutylazine Fraction

Sample Flow through Washing buffer eluate Glycine-HCl eluate Washing buffer eluate

the immobilization may occur [24]. According to Scouten et al. [25] abs coupled via amino groups are mainly immobilized at their Fc part, well away from the antigen binding site, because the amino groups of the Fab part are not accessible for coupling. Therefore it is conceivable to get better IAC performance with a non-oriented coupling via the amino groups than with an oriented coupling via the carbohydrate residues. Since abs are the most important and expensive component of IAC, a careful selection of suitable abs is a crucial step. The abs have to be available in sufficient amounts and purity. For this reason mostly mabs are used for IAC. The choice of an ab with relatively wide selectivities facilitates the simultaneous isolation of various analytes out of a crude sample. For instance, atrazine and terbuthylazine could be jointly isolated together by the immobilized mab K4E7 (Table 1) although this antibody cross-reacts with terbuthylazine to a much lesser extent (26%) compared to atrazine (100%) [26]. The third important aspect in the IAC is the choice of a suitable elution buffer. The elution conditions have to be stringent enough to quantitatively remove all bound molecules from the immobilized abs, but not too strong to avoid irreversible denaturation of the covalently attached abs. However, abs covalently attached to a solid phase are much more stable than free abs. Most often a change of the pH is used to elute the bound analyte. Furthermore, chaotropic salts, different ionic strength, denaturants or organic solvents were applied for the elution [27]. To date the IAC was used principally in medicine. As a wide range of abs against pesticides and other

elution from a beaded cellulose ONB-carbonate Experiment

A immunoaffinity

column [Zl]

Experiment

1

2

pg atrazine

pg terbuthylazine

pg atrazine

pg terbuthylazine

4.9 0.0 0.2 4.2 0.2

4.7 0.0 0.2 3.8 0.1

4.9 0.1 0.2 4.1 0.3

4.7 0.1 0.4 3.6 0.1

Samples: 10 ml of a 500 pg atrazine/l+ 500 pg terbuthylazine/l solution; concentrations were determined by LC. Figures are given in pg per fraction.

eluent:

glycine-HCl

(0.2 mol/l,

pH 2.2). Pesticide

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environmental contaminants are available [2] the IAC could also be used for environmental monitoring.

5. Immunolabeling Whereas IAC is designed for the capture and controlled release of analytes, immunolabeling aims at the capture of abs for the tagging of analytes. There are two important applications in the context of environmental and health relevance, the labeling of bound residues and the labeling of DNA adducts. The persistence of pesticides in the soil is partly due to the formation of bound residues, i.e., chemical species which cannot be extracted without significantly altering the structure of these compounds. Capriel and Haisch [28] estimated the portion of non-extractable atrazine residues in soil between 30 and 50% of the applied amount. Most important, the portion bound to soil material does not lose its activity during subsequent years, as it can be made available again for plants grown in this soil [29]. Therefore it is important to obtain information on bound residues and their time-dependent changes. It can be foreseen that screening for bound pesticide residues will become an important issue in the future. We have recently demonstrated that the technique of immunolabeling provides an excellent tool for this purpose [30]. It requires a careful suppression of unspecific ab binding by soil particles. This has been achieved by blocking the unspecific ab binding with unlabeled control immunoglobulins. In addition antigen binding fragments (Fab, cf. Fig. 5) were prepared to remove the Fc parts, which are responsible for a significant part of unspecific binding, and coupled to the fluorescent dye Rose Bengal B. This dye conjugate produced a fluorescence signal which depended upon the amount of atrazine found in native soil samples. Similar techniques were applied by our group for the immunohistochemical localization of bound atrazine residues in plants [31]. Because of the very low concentrations within the tissues, signal amplification by a biotin/streptavidin bridging system and the fluorescent dye phycoerythrine was used yielding an intensive labeling in the plant cell walls and chloroplasts, the major sites of atrazine accumulation. Important practical applications are to be expected

397

in the field of medicine to evaluate the exposure of workers to mutagenic and/or carcinogenic substances that form DNA adducts. Consequently, DNA can be used as a molecular dosimeter for compounds such as acrylic monomers, benzo[ alpyrene, ethylene oxide, nitrosamine, vinylchloride and herbicide metabolites such as alachlor and metolachlor metabolites. Conventional analysis requires DNA extraction, purification and hydrolysis followed by GC-MS analysis of the modified nucleotides. The immunohistochemical approach, which has already been realized in a few cases [32], enables a considerable simplification of the analytic procedure and is expected to expand in the future.

6. Progress in antibody development The examples presented in the preceding sections illustrate the necessity of further expansion of ab production at an increased speed to cope with future demands. Forthcoming applications, especially in the IS field, will mainly rely on mabs and recombinant antibodies (ret abs). 6.1. Monoclonal antibodies The advantages of mabs for environmental analyses are well known. The hybridoma technology [33] guarantees the unlimited production of mabs of the same isotype with constant properties. Although it can be foreseen that mabs may eventually be substituted by ret abs, hybridoma cells are still required, at least for the time being, as sources for mRNA coding for specific mabs. Production of mabs is based on the proliferation of a single ab-producing cell yielding a uniform population of ab of the same type with identical affinities and specificities. Raising mab takes advantage of the vertebrate immune system which is able to make ab against nearly any chemical structure. However, the hapten character of pesticides such as s-triazines requires coupling of derivatives with reactive groups to an immunogenic carrier protein, e.g., keyhole limpet haemocyanin (KLH). After immunizing mice with the appropriate immunogen, the absecreting B-lymphocytes are fused with suitable myeloma cells. The resulting “ hybridomas” are first

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selected in a HAT medium containing hypoxanthine, aminopterin and thymidine. Aminopterin poisons the parent myeloma cells which lack an enzyme in the nucleic acid pathway. The hybridomas can survive the HAT selection by using a saivage pathway, due to the complementation of the genetic defect by the B-lymphocyte genome. In the next step growing hybridoma cell colonies are tested for their ability to produce the desired ab.

with

The classical approach uses competitive IAs for this purpose. Tracer binding by the mabs is tested in the absence and presence of the analyte. Big differences in signal strength (enzyme activities) indicate the two basic requirements for a sensitive IA, (1) strong binding of the analyte and the enzyme tracer by the ab and (2) ready displacement of the enzyme tracer by the analyte. The IA allows the screening of a large number of samples and provides initial infor-

unspecific

surface

v

unspecific surface receptor

*

paramagnetic bead with hapten-protein

hapten-specific surface

receptor

Fig. 3. Principle of immunomagnetic

screening.

receptors

coated conjugate

B. Hock et al. /Analytica

mation about the performance of an ab in this test system. Suitable clones are adapted to serum-free medium and grown in roller bottles where they secrete ab up to 100 mg/ml. 6.2. Immunomagnetic screening A crucial step in the production of mabs is the development of efficient screening procedures to identify suitable cell lines. Super paramagnetic beads offer an alternative to conventional screening exclusively performed with immunoassays to solve the problems of being time-consuming, escaping detection or loosing valuable cells. We have established the technique of immunomagnetic cell separation for the screening of hybridomas which secrete mabs against s-triazines [34]. It is based upon the expression of surface receptors on the surface of hybridoma cells resembling the secreted abs. Producers can be tagged by magnetic beads which are coated with a carrier protein attached to the hapten. After the fusion of spleen cells with myelomas and HAT selection, the beads are bound to those hybridomas presenting hapten-specific mabs and may be jointly removed by a permanent magnet. Fig. 3 illustrates the principle. Several new cell lines have already been isolated. The screening of hybridoma cells by immunomagnetic separation offers several advantages. (1) The success or failure of an entire fusion can be estimated immediately after separation with the aid of a microscope. (2) High affinity hybridomas are preferentially obtained because they will first attach

Table 2 Major developments

in the field of recombinant

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to the beads. (3) As hapten mabs are obtained by immunization with a hapten-protein conjugate, the majority of stimulated lymphocytes will produce mabs against the protein and not against the attached hapten. Coupling the hapten to another carrier protein will leave these undesired cells aside. Therefore, the screening procedure is restricted to a small amount of cells. (4) The field of ret abs will benefit from this technique because a proper choice of starting cell lines is available. 6.3. Recombinant antibodies The reason for the general trend to go one step beyond mabs is the fact that the hybridoma technology does not allow a manipulation of given ab properties. This is the domain of recombinant techniques. They enable for instance the expression of the functional regions of abs in suitable organisms such as E. coli or yeast and the targeted modification of ab binding properties at the DNA level. As mutant abs may be obtained, the circumstantial approach to obtain new abs with different binding properties by means of new immunizations can be circumvented. An extremely powerful strategy is to mimic the immune system of an animal by reproducing the ab diversity in manipulatable microorganisms. Some ab fragment DNA libraries that can be expressed in bacteria and subsequently screened are now available. The techniques required for the synthesis of ret abs originated in the medical field where recombinant techniques were applied for the production of

antibodies

Year

Characterization

Ref.

Hybrid antibodies

1984

1351

Humanized

1986

Combination of mouse V regions with human C regions on the DNA level; gene transfer by plasmids into a selected myeloma cell line CDR replacement (only the hypervariable regions from mice are transferred) Expression of antibody Fv fragments in E. coli Expression of miniantibodies (dimeric Fv fragments linked by amphipathic helices) in E. coli Recombinant ab fragment libraries from H and L chains in lambda phages

Antibody

Antibody

type

antibodies fragments

Antibody libraries and phage antibodies

1988 1992 1989

1361 [371 1381 1391

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humanized abs which are not attacked and destroyed by the immune system. Table 2 lists some of the milestones in the development of ret abs, many of

them were achieved by the groups of Lerner in the USA, Skerra and Pl~ckthun in Germany and Winter in England.

mRNA extraction from hybridoma cells

1 cDNA synthesis

1 Amplification of variable regions

1 Assembly by linker ligation and second amplification linker

Ligation with phagemid scFv-DNA fragment

phagemid BANTAM

515E

1 Transformation of E. coli Fig. 4. Production

of phage antibodies (mewed

after Lee and Morgan [40]).

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The interest of the environmental analysts in ret abs mainly results from the desire to speed up the development of new abs and to become independent from animal experiments. Up to now, different approaches and strategies have been followed, for instance the expression of ab fragments in E. coli using the phagemid vector by the Morgan group [40]

at Norwich (UK) to produce phage abs against the mycotoxins aflatoxin Ml and diacetoxyscirpenol. A similar system was employed by Karu [41] for the synthesis of ret abs against the herbicide diurone. However, the Hammock group [42] at Davis (USA) developed a plasmid vector for the expression of atrazine abs. The latter group also pursues an eukary-

Addition of helper phages and expression

ga

lmmunomagnetic

401

selection of phage antibodies

1 phage rescue

1 ELISA Fig. 4 (continued).

402

B. Hock et al. /Analytica

Chitnica Acta 311 (1995) 393-405 FRl

-CDRl CDR3 CDRZ

mbu

‘\ carbohydrate

Fab

SCFV

antibody

dAb Fig. 5. Architecture

fusion proteins

of IgG antibodies and their fragments.

otic expression system (baculoviruses). But it should be emphasized that ret abs are not yet available for practicable applications, e.g., for pesticide assays with real samples. This has mainly been attributed to difficulties with the proper folding of the gene products and the expression of sufficient amounts of ret abs. We have recently used the phagemid system for the production of phage abs against s-triazines. This system has been originally developed in the group of Winter 1431and was the first one being commercially available. Fig. 4 shows the strategy. It starts with the P6A7

M

extraction of mRNA from hybridoma cells, which were derived from female Balb/c mice immunized with atrazine-, simazine-KLH and ametryn-BSA. Following cDNA synthesis, the ab coding regions are selectively amplified by the PCR reaction. For this purpose short oligonucleotide primers [44] are used, complementary to the flanking sites of the targeted cDNA area, which exhibit relatively conserved nucleotide sequences depending on the species. Since the nucleotide sequences are available in databases, the design of appropriate primers is now well established. In addition, the choice of KlF4

M

K4E7

VH W

0,4 0,6 0,6

1

0,2 0,4 0,6 0,6

VL w

1

0,4 0,6 0,6

1

Fig. 6. Amplification products of scFv derived from three different hybridoma cell lines (K4E7, P6A7, KlF4). marker (100 bp ladder obtained lambda DNA). The caption at the top indicates the volumes of single components

Lanes M, fragment size used for SCFVsynthesis.

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primer determines the type of the antigen-binding fragment (Fab, dAb, Fv, etc., cf. Fig. 5). However, the smallest ab derivative with a complete binding pocket is the Fv fragment. In order to clone and express the Fv fragment, the amplified heavy and light chain variable regions are assembled by the (Gly,Ser), linker sequence and amplified once again. Subsequently, the resulting s&v-DNA fragments shown in Fig. 6 are inserted into the phagemid pCANTAB 5E. Then E. coli bacteria are transformed with recombinant phagemides and subsequently infected with helper phages. The phages pack the foreign DNA and express the Fv fragment as a fusion protein with g3p displayed at their tip. The desired phage abs can be tagged by the antigen in chromatography columns, panning devices or immunomagnetic beads. We prefer immunomagnetic separation because the phages have not to be treated with stringent elutants for their displacement from the solid phase. We observed that E. coli are reinfectable with phages still being bound to the magnetic beads. After rescuing again the selected phages, positive clones are detected by EIA in antigen-coated microtiter plates, using anti-g3p abs. We have recently expressed phage abs, which are capable of specifically binding s-triazine conjugates derived from terbutryn and terbuthylazine. Evidence is obtained from signals that result from labeling bound phages with POD-labeled secondary ab. Absorption signals are proportional to the amount of triazine conjugate immobilized at the solid phase (Fig. 7). Binding of phage abs proves to be specific, it is very low if the hapten-ovalbumin conjugate is replaced by ovalbumin. Further controls were obtained by substituting phage abs by the mab KlF4. The latter one also yields decreasing signals with increasing dilutions of the coat conjugate after challenging with POD-labeled anti-mouse abs. Work is in progress to design proper conditions for the displacement of the phage abs by the analyte. In addition the existing clones will be used for the transfection of a non-suppressor E. coli strain to harvest soluble scFv fragments without the covalent binding to the phage protein g3p. s&v represents the smallest fragment retaining an almost identical antigen binding site compared to the parental abs. Differences are restricted to the artifi-

L

n

KlF4

A 1,5-

l-

085 -

100 w/mL OVA

100

33

10

3,3

1

dmL Terbuttyn-OVA

Fig. 7. Binding of phage antibodies (6C, 2B, 1OE) and mabs against terbutryn (KlF4) to a terbutryn-ovalbumin conjugate immobilized to microtiter plates. OVA represents controls with 100 pg/ml ovalbumin instead of the conjugate. The bars indicate the standard deviation.

cially introduced polypeptide linker, which prevents the associated variable heavy and light chain fragments from dissociation. This is due to the lack of stabilizing disulfide bridges occurring in complete abs. Therefore the scFv are slightly modified molecules and steric hinderance may be the result in certain cases. The group of Hammock [42] solved this problem by expressing an Fab fragment with intact disulfide connections. It was confirmed by slot blot analysis that this fragment specifically reacts with atrazine.

7. Outlook It may be concluded from the preceding discussion that it will be only a matter of time until ret abs become available for practical applications. On the other hand, one should be aware of alternatives that may be capable of supplementing or in some cases even replacing ab technologies. The most promising alternatives are peptides, which can be derived from synthetic chemical libraries having immense diver-

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sity [45], RNA probes, which can be generated by an in vitro selection technology and in principle are capable of binding to any small ligand [46], and guest-host systems, which use synthetic polymers as ligands 1471 and can be produced by molecular imprinting techniques [48]. The final decision, which of the upcoming technologies will be generally accepted in ‘the field of environmental analysis, will essentially depend upon the speed selective ligands with high affinities become available and on the simplicity to prepare such ligands. The merits of the immunochemical approach are not only due to the evolutionary strategy that is used by nature to produce an enormous diversity of abs of high quality, but also to the fact that selection already starts at a high level of optimization.

Acknowledgements We thank Dr. J. Lepschy von Gleisenthall of the Landesanstalt fiir Bodenkultur und Pflanzenbau for his assistance in the preparation of the soil samples and the LC analyses. We are indebted to Mrs. H. Kienberger for her skilful technical assistance for the LC analyses. We are grateful to Prof. R.D. Schmid (University Stuttgart) for his permission to use his data on grating coupler immunosensors.

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