6 Molecular Antibody Technologies for Biosensors and Bioanalytics

Karl Kramer, Georg Mahlknecht and Bertold Hock

Center of Life Sciences, Technical University of Muenchen, Freising, Germany

1 INTRODUCTION

Biosensors take advantage of biomolecular interactions, which are applicable to various analytical areas.1,2 Sensitivity and selectivity of bioanalytical systems essentially depend on the properties of the biorecognition elements to be used for analyte binding. Biological structures required for the selective receptor unit of the sensor are usually derived from subcellular components and include in most of the studies reported so far on enzymes, antibodies, hormone receptors, nucleic acids, and membrane components. These are complemented with biorecognition elements organized on a higher functional level such as organelles or entire cells. The latter are giving access to additional information beyond the receptor ligandbinding event. Immunochemical assay systems employing Abs as binding components are effective tools for the analysis of a wide variety of analytes ranging from low molecular weight xenobiotics (e.g., herbicides, insecticides) to complex proteins (e.g., structural elements of pathogenic microorganisms, disease associated protein misfolding e.g., Creutzfeld Jacob Disease or tumor associated antigens). However, the main strength of antibodies is a potentially high afnity and selectivity toward the target molecule. Despite optimism for the potential of biosensors, there are many examples successfully

tested in a laboratory or at prototype level but only few of them entered the analytical praxis. This has been hampered by several obstacles. With respect to Ab immobilization on transducers, stability, sensitivity, and susceptibility to matrix effects are frequently mentioned.3 One of the most remarkable developments in Ab production techniques emerged in the last two decades, when scientists started to produce Abs in vitro. Avoiding the vertebrate in vivo system offered new facilities, since this approach was considered to solve several problems encountered with Ab obtained from sera or by the aid of hybridoma technology. In this chapter state of the art Ab technologies are briey introduced and major achievements are summarized. Aspects, which still suffer from the current technological standard and therefore need further development are discussed. Finally, limitations of the molecular Ab approach are indicated, which are already apparent.

1.1

The Antibody Scaffold

The key reaction at the recognition part of immunosensors is based on the interaction between the Ab molecule and the corresponding antigen as ligand. The potential of immunosensors is crucially determined by the selectivity and afnity

Handbook of Biosensors and Biochips. Edited by Robert S. Marks, David C. Cullen, Isao Karube, Christopher R. Lowe and Howard H. Weetall. 2007 John Wiley & Sons, Ltd. ISBN 978-0-470-01905-4.

BIOLOGICAL AND MOLECULAR RECOGNITION SYSTEMS

of the individual Ab applied for the detection of a particular ligand. Ab molecules are members of the immunoglobulin superfamily. They consist of two identical heavy chains and two identical light chains (Figure 1). Two antigenbinding Fab fragments are assembled each by the complete light chain combined with the corresponding part of the rst two N-terminal heavy chain domains. Two Fab fragments are linked via exible hinge regions with the crystallizable fragment Fc , which harbors the remaining constant heavy chain domains CH 2 and CH 3. The globular structure of Ab domains is caused by the characteristic immunoglobulin fold.4 Antiparallel -strands form a typical double layer in each domain, which is stabilized by hydrophobic interactions and conserved intradomain disulde bonds. The antigen-binding site is localized at the N-terminal moiety of the variable regions. Owing to the frequency of sequence variation between different Ab molecules, sections with hypervariable or complementarity-determining regions (CDR) and conserved or framework regions can be distinguished for these domains.5,6 Each of the variable (V) regions contains three CDR loops that are embedded into four sections of the framework region. In addition to their elevated sequence variability, most of the CDR loops are characterized by signicant differences in length.7 Antigen contact sites are mainly provided by distinct amino acid residues of the three CDRs of each V domain. The composition and conformation of the six CDRs (three from the light and three from the heavy chain V domain) in each paratope determines the topography of the antigen-binding site and, hence, the recognition of the ligand. Additional interactions by residues of the framework region are reported for discrete ligands.8 Binding energy between the paratope and the antigenic epitope involves hydrogen bond, hydrophobic, van der Waals, and coulombic interactions as well as calcium bridges.9 Since these multiple forces are exclusively effective on short molecular distances, they require a complementary topography at the Abantigen interface. The afnity and selectivity of an Ab is thereby dened by the total of the intermolecular forces and by the degree of complementarity. At the beginning of genetic engineering of biosensors, Ab were predominantly prepared as

monovalent fragments either in the scFv10,11 or Fab format.12 Fab fragments are comprising the entire Ab light chain and the corresponding part of the Ab heavy chain. The scFv fragments are reduced to the variable domains of the Ab heavy and light chain, which are connected by an articial peptide linker (Figure 1). The proper functioning of biosensors essentially depends on the immobilization of the respective ligands, Ab, or coating conjugates on the sensor surface, their correct orientation, and homogeneity. Recombinant approaches are also intensively used for the addition of functional structures, for example, for the attachment of anchor groups. Furthermore, Ab fragments that are fused with marker proteins offer the advantage of a reduced number of assay steps in the analytical test format. The genetically engineered fusion of the Ab binding function with marker enzymes was already reported in bioanalytics.13 The corresponding vector was developed to contain an alkaline phosphatase and generic restriction sites for the convenient one-step cloning of scFv fragments isolated from Ab library repertoires. Similarly, a gene encoding green uorescent protein was inserted into a vector harboring a picloram-specic Ab fragment.14 The resulting uobody avoids the enzymesubstrate reaction for colorimetric detection that is required in a conventional analytical format with substrate turnover. Finally, fusion proteins can offer new strategies of bioanalytical assays. The assay utilizes the antigen-dependent reassociation of Ab variable domains and concomitant complementation of the enzyme -galactosidase (-gal),15 in order to enable a noncompetitive homogeneous immunoassay for small target molecules instead of the standard competitive heterogeneous enzyme-linked immunosorbent assay (ELISA). In a proof of principle approach, the reassociation of two fusion proteins was monitored by restoring the enzymatic activity. The rst protein consists of an anti-4-hydroxy-3-nitrophenylacetyl variable heavy chain (VH ) fragment fused to an N-terminal deletion mutant of -gal (VH ), and the second protein correspond to the variable light chain (VL ) fragment fused to a C-terminal deletion mutant of -gal (VL ). Upon mixing of the reagents with the sample, an analytedependent increase in reassociation and thus, in

MOLECULAR ANTIBODY TECHNOLOGIES FOR BIOSENSORS AND BIOANALYTICS

VL

VH

VL

VH

Tag

scFv CL CH1

VH VL

Fab

1 CH CL

L1 L2

H3

H1

H2

CH2 Carbohydrate

L3

CH3

Antibody (IgG)

VH gene FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4

VL gene FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4

Figure 1. Domain structure of native IgG antibody molecule (top left) and genetically engineered single domain antibody (sdAb), single-chain Fv (scFv), disulde stabilized Fv (dsFv) and Fab fragments. Some of the fragments are genetically fused to afnity tags, for example, Strep tag (Strep), E tag (E). Middle right: Ribbon model of the VH and VL domain with pesticide analyte complexed in the Ab binding site (L1-3: VL CDR1-3; H1-3: VH CDR1-3). Bottom: Segmentation of VH and VL encoding genes into framework regions FR1-4 and the hypervariable complementarity-determining regions CDR1-3. The ribbon model was kindly provided by S. H rsch. o

enzymatic activity was observed. Compared to the corresponding heterogeneous open sandwich ELISA, a 1000-fold improvement in sensitivity was attained.

1.2

The Evolutionary Cycle

The evolutionary cycle is the central aspect of Ab engineering in order to alter the Ab afnity

BIOLOGICAL AND MOLECULAR RECOGNITION SYSTEMS

prole. Applying the Darwinian principle of variation and selection in vitro elegantly circumvents extended immunization periods as required in classical Ab production schemes.16,17 The rationale behind the concept evolved as modern evolutionary theory in the rst half of the twentieth century, when population biologists started to describe the natural selection of gene populations. They established mathematical models for the evolution of genes under the inuence of recursive rounds of mutation, recombination, and selection.18 Applying principles of natural selection to the evolution of genes in the laboratory, fundamental evolutionary theorems predict that the tness of a gene will evolve most rapidly in a population of high genetic variability, which is exposed to selection pressure.19 The methodology of choice in a directed evolution experiment is therefore to construct a library of variant genes, and screen or select improved variants from the protein products of these genes. The principles of directed evolution in vitro are very similar to the mechanisms of somatic mutation in vivo. The Ab repertoire of the primary immune response in vivo is predominantly the result of recombining germ line genes of the V, D, J clusters (i.e., the genes constituting elements of the nally expressed VH and VL genes). The primary response yields Ab with generally low afnities. Thereafter, Ab variable genes of the primary repertoire are modied during the secondary immune response in the microenvironment of lymphoid germinal centers by somatic hypermutation. Physiologically appropriate variants are subsequently selected from this pool of mutant immunoglobulins upon their improved afnity to the antigen.20 In vitro, Ab genes are subjected to iterative evolutionary cycles of mutation and selection, until they meet the requirements for the designated application (Figure 2). Frequently applied in vitro methods for sequence diversication include random and directed nucleotide alterations or recombination techniques.21 Random techniques are for instance, based on the introduction of point mutations by error prone polymerase chain reaction (PCR). Nucleotides throughout the entire gene are hence randomly exchanged.22 In contrast to random methods, oligonucleotide-directed procedures insert synthetic sequences into the gene of interest at specic sites, for example,

strand overlap extension23 or megaprimer-based PCR.24 Recombination techniques are based on randomized recombination of gene segments or complete genes (e.g., Ab VH and VL encoding genes). Ab gene repertoires encoding highly functional diversity can be subsequently selected either by physical or genetic techniques. The most frequently applied physical selection methods comprise phage display,25,26 cellular display on the surface of bacteria or yeast,27,28 ribosome display,29 and mRNA display.30 These procedures are all characterized by the physical linkage of proteins and their encoding genes, which can be reamplied for further processing. In phage and cell surface display the Ab library is fused to virus coat or cellular membrane proteins of virions and cells.31 In contrast to phage or cell surface display, ribosome display does not depend on a transformation step for creating a selectable library of protein variants. Here the Ab library is fused to a C-terminal tether and transcribed into mRNA in vitro, which subsequently recombines with ribosome particles. Following in vitro translation, functional proteins are processed in the selection step. Even more advanced methods are based on RNA display, where the procedure omits the presence of instable ribosome complexes.32 For the selection of the most favorable binding proteins the ligand is prevalently immobilized on a solid phase, for example, at the surface of a polystyrene tube, microtiter plate well or pin and incubated with the phage library. Complexes that recognize the ligand are subsequently eluted, after nonbinding variants have been removed by washing. Since selection at the surface of a solid phase may put problems on the procedure, selection strategies were developed for in solution separation of ligand-binders. Binders of biotin-labeled ligands can be tackled using streptavidin-coated magnetic beads.33 Another strategy, the selectively infective phage (SIP) technology is based on the fusion of receptor protein and C-terminal domains of the pIII coat protein in the phage display system.34 Recombinant phages displaying the Ab fragments on their surface are lacking the protein domain, which enables the infection of bacteria. Infectivity is exclusively restored upon the specic interaction of the displayed Ab and the ligand,

MOLECULAR ANTIBODY TECHNOLOGIES FOR BIOSENSORS AND BIOANALYTICS

Coupling of Ab genotype with phenotype

MRNADNA

Identification of best variants

Bioanalytical application

V L

Tag
Figure 2. Principle of directed evolution. J: joining element.

which is coupled to the protein domain for host infection. All physical selection methods require signicant quantities of the ligand in order to perform selection and screening, whereas genetic methods rely on the in situ synthesis of, and subsequent interaction between, binding ligand and target to confer a selectable phenotype. The receptor and a peptide- or protein-ligand are synthesized in vivo and interact in the host cell.

Various yeast hybrid systems have been designed, which utilize the transcriptional activation mechanism for selection.35 For example, a DNA-binding domain of the transcription activator can be fused to the N-terminus of the receptor protein, whereas the activation domain of the transcription activator is fused to the potential ligand. The DNA-binding domain binds to a promoter, but transcription is exclusively activated, if the DNA-binding domain is connected to the

V L

Generation of Ab encoding gene repertoire

Recursive evolutionary cycles

Selection by analyte-driven evolutionary pressure

BIOLOGICAL AND MOLECULAR RECOGNITION SYSTEMS

activation domain by the interaction of receptor and ligand. If no receptorligand binding occurs, the reporter gene transcription is not triggered. The concept effectively facilitates the distinction between cells harboring potent and weak binders. A similar concept is pursued by the protein complementation assay.36,37 In this case, the gene coding for an essential enzyme (e.g., dihydrofolate reductase or -lactamase) is deliberately cleaved into two fragments. Each of the resulting domains is fused to either the receptor or the ligand. The resulting fusion proteins are coexpressed and the enzyme restores functionality, if the domains get into close contact via the receptorligand interaction. Finally compartmentalization strategies using the not-natural compartments of cells as mentioned before, but articial water-in-oil emulsions can be essentially considered as in vitro methods. Cellular compartimentalization is mimicked by adding an in vitro transcription/translation reaction mixture to a stirred suspension of mineral oil and surfactants, which results in an emulsion with mean droplet diameter similar to that of bacterial cells.38 A mixture of two genes, the M. HaeIII gene (which encodes DNA methyltransferaseHaeIII) and the folA gene (which encodes DHFR), were transcribed and translated in the aqueous compartments. Then the emulsion was broken, and the DNA in the aqueous phase was subjected to cleavage by HaeIII endonuclease followed by amplication by PCR. Because the DNAmethyltransferase methylated HaeIII-cleavage sites in droplets that contained the M. HaeIII gene, only the M. HaeIII gene would survive the cleavage by HaeIII and be amplied by PCR. After one round of selection, a mixture of M. HaeIII to folA gene with a ratio of 1 : 1000 was enriched to a 1 : 1 ratio.38 Since all of the steps in in vitro compartmentalization are performed in vitro, this approach has the potential to reach the limit at which the DNA is the limiting reagent in library size.39

1.3

Primary Antibody Repertoires for Bioanalytics

A very common approach to generate Ab diversity is the cloning of natural Ab gene repertoires, which are isolated from donor organisms, for example, mice. The corresponding repertoire is expected to

be unbiased, which means that the immune system of the donor organism has not been challenged by an antigen. These nave repertoires theoreti cally harbor Abs for any target structure, however, at a moderate afnity level for the majority of binding molecules. In contrast, biased Ab repertoires can be cloned from immunized sources. The latter strategy benets from in vivo mechanisms of the immune system, since Ab variable genes encoding the antigen-binding domains are modied during the secondary immune response in the microenvironment of lymphoid germinal centers by somatic hypermutation. Appropriate variants are subsequently selected from this pool of mutant immunoglobulins upon their improved afnity to the antigen.47 Therefore, immunizing an organism with a specic antigen serves as an in vivo preselection of potent Ab genes for the immunogen. We developed a strategy to generate a groupselective library representing a large natural Ab gene pool. This library was designed to include a range of s-triazine-specic Abs in order to facilitate the selection of Abs against dened members of the s-triazine family.40 For this purpose, Absecreting B cells were derived from Balb/c mice, which had previously been immunized with different s-triazine immunogens including derivatives of atrazine, ametryn, terbuthylazine, deethylatrazine, and simazine. B cells secreting s-triazineselective Abs were separated from unspecic B cells by means of immunomagnetic separation. This method takes advantage of the membranebound receptor molecules on the B cell surface, that is, transmembrane protein complexes that share their ligand-binding characteristics with the secreted Ab. These surface receptors can be tagged by target molecules covalently linked to paramagnetic particles. After magnetizing the particles in a magnetic eld, specic B cells were removed from bulk cultures by magnetic force.41 Ab-encoding genes from magnet-bound B cells were subsequently cloned into a phage display system. The resulting library comprised all target relevant sequences that have been included in the starting B cell repertoire. The group-selective Ab library was then used to select Ab variants selective for s-triazines containing a tertiary butyl group (BUT), that is, terbutryn and terbuthylazine as well as those s-triazines bearing an isopropylamino residue (IPR), that is, atrazine and propazine. Specic

MOLECULAR ANTIBODY TECHNOLOGIES FOR BIOSENSORS AND BIOANALYTICS

phages were enriched by three repetitive cycles of selection applying immunoafnity chromatography with BUT derivative-coated and IPR derivative-coated columns. Binding studies for the characterization of selected Ab clones were essentially performed by an ELISA and an optical biosensor. The clones could be selectively displaced by atrazine as indicated by the corresponding calibration curves (Figure 3). Reaction kinetics of the three best binding clones for triazine herbicides containing tertiary butylamino and isopropylamino groups were determined with the BIAcore 2000 sensor. Figure 4 shows representative sensorgrams for the binding of a recombinant Ab, derived from clone BUT-4, to a tertiary butyl derivative-ovalbumin conjugate or ovalbumin as a control. The binding constants KD ranged from 1.2 108 M for IPR-7 to 8 108 M for BUT-56, which corresponds to the afnity level of maturated Abs that are obtained during the secondary immune response.40 In a second approach, we employed the fully synthetic human combinatorial Ab library HuCAL (MorphoSys AG, Martinsried, Germany) as a primary immune repertoire for the selection of pathogen-directed Ab variants. The library is based on consensus sequences for each of those seven VH and VL germline families, which are most frequently used in the human immune response. Diversity is created by replacing the VH and VL CDR3 of the master genes by CDR3 library cassettes, generated from mixed trinucleotides42
Association

450 400 350 300 RLU 250 200 150 100 50 0 0 100 101 102 l1] 103 104 IPR-7 IPR-32 BUT-4 BUT-8 BUT-56

s -Triazine [g

Figure 3. Calibration curves of IPR and BUT clones for the analysis of atrazine and terbutryn, respectively, by direct heterogeneous competitive ELISA. IPR and BUT clones were selected by phage display technology employing s-triazine derivatives, which contain isopropylamino (IPR) and butylamino (BUT) residues. RLU: Relative luminescence units.

and biased toward natural human Ab CDR3 sequences.43 Specic clones were selected from the HuCAL library by means of phage display for the detection of several food-born pathogens. The stability of selected Ab fragments was investigated with respect to their thermal stability, which is a rough indicator for their long-term storage properties, for example, immobilized on sensorchips, and their molecular integrity at unfavorable temperature conditions. For this purpose Ab fragments
Dissociation

160 140 120 100 RU 80 60 40 20 0 20 200

28 nM

14 nM 7 nM 3.5 nM 1.8 nM 0.9 nM

200

400 Time (s)

600

800

1000

Figure 4. Sensorgrams of scFv binding (clone 4). CM5 chips were coated with 4000 RU terbuthylazine-ovalbumin conjugate (channel 2) or ovalbumin (channel 1) as a control using amine coupling. Afnity measurements were performed at 25 C with 128 nM of active scFv (diluted in PBS containing 0.005% Tween 20) for 3 min using a ow rate of 30 l/min.

BIOLOGICAL AND MOLECULAR RECOGNITION SYSTEMS

of selected clones were incubated for 24 h at various temperatures and their functionality was tested by ELISA. The corresponding results for the pathogen-selective Abs are presented in Figure 5. Almost all Ab fragments were stable up to temperatures of 37 C and 50 C, respectively. Most of the Abs showed reduced or even completely lost ligand binding at higher temperatures. These observations are consistent with analogous experiments performed with conventional monoclonal Abs derived from hybridoma cultures in our laboratory. However, one out of the six fragments depicted in Figure 5 retained full functionality even after incubation at 80 C, which indicates an exceptional stability with respect to the considered protein family. Sequence alignment with human germline genes revealed that the immunoglobulin VH genes of the clones providing the highest stabilities could be predominantly assigned to the VH 3 gene family. This is at least a hint that the stability of Ab fragments may be an inherent structural feature of particular germline families. These results are well in line with investigations of the biophysical properties of Ab fragments performed by other groups employing the HuCAL library. Ab

fragments containing the variable domain combinations H33 and H53 showed superior stability. Combination with light chains exhibited also high levels of stability depending on the particular amino acid sequence of the CDR-L3. Another issue in Ab engineering is the availability of the antigen. We encountered this problem, when raising Abs against genetically modied organisms (GMO). Since posttranslational processing in plants is signicantly different from analogous processes of bacteria physiology, we avoided cloning and expression of the GMO antigen in Escherichia coli. However, antigen was only available at low percentage of purity, which would have affected all subsequent processes, for example, immune response biased to immunogen contaminants, inefcient screening, and problematic identication of positive clones. Applying conventional Ab synthesis protocol by using animals as Ab source, rough samples of ground our from GMO corn had to be extracted and puried chromatographically. However, yield of puried antigen turned out to be poor and the procedure itself very laborious. In order to circumvent the problems raised by the lack of appropriate antigen quantity and purity, human germline gene Ab
80 C 65 C 50 C 37 C 22 C 4 C

SaBa/6H

Sasig Da/12D

Lmb/1B

Esco Heb/4A

CjBa/1F

Bace L2a/2c 0 20 40 60 80 100

Binding activity (%)

Figure 5. Thermal stability of pathogen-selective Ab clones isolated from the HuCAL library. Ab fragments were incubated for 24 h at temperatures ranging from 480 C. Binding activity was subsequently evaluated by measuring the maximum and minimum signal in ELISA according to the formula: (Amax Amin temperature treated Ab)/(Amax Amin of untreated control) 100%.

MOLECULAR ANTIBODY TECHNOLOGIES FOR BIOSENSORS AND BIOANALYTICS

libraries (Tomlinson)44 were used as a primary repertoire for a subtractive selection strategy. Each panning round started with a selection against our extracts derived from non-GMO soybean. Thereby, all Abs directed against components of the extract were retained in the panning vessel. Unbound clones were transferred to a second panning vessel coated with our extracts from GMO plants. Since the majority of Abs directed against non-GMO protein in the plant extract have been effectively removed in the prepanning step, GMO selective Abs were now the remaining fraction, which could bind specically. Thus several clones from the primary library repertoire could be identied, which were appropriate for GMO monitoring. Figure 6 gives an example for the performance of the clone G7 in a simple ELISA format. By the selection method several parameters of the nal Ab characteristics were considered. The subtractive selection procedure enabled the isolation of Abs against a nonpuried antigen. Furthermore, the amount of antigen applied in the panning corresponded exactly to that present in sample extracts of GMO plants. Thus, the afnity level of Ab variants was precisely adjusted to the needs in GMO monitoring. Third, since panning was performed in plant sample extracts, the

selected clones recognize conformation of GMO proteins, even after they have been processed for sampling. Finally, interference by sample components (for instance, non-GMO soy bean proteins, extraction solvents) were considered by the selection protocol too.
1.4 Secondary Antibody Repertoires for Bianalytics

1 OD450 nm 0.8 0.6 0.4 0.2 0 0 2.5 Content GMO (%)

Figure 6. ELISA for GMO detection in crude our extracts from genetically engineered soy bean based on recombinant scFv fragments. Percentage of contamination with our derived from GMO plants is indicated. The GMO protein corresponds to CP4- EPSPS (i.e., 5-enolpyruvylshikimate-3-phosphate synthase from the Agrobacterium sp. strain CP4), which confers tolerance to the glyphosate herbicide.

The gene repertoire of the group-selective Ab library presented in the preceding text was employed for subsequent optimization of individual Ab molecules by directed evolution.45 It was expected that this library would substantially facilitate the engineering of desired Ab specicities and afnities to any member of the triazine group without the need of new immunizations. The Ab clone IPR-7 was used as a template for the optimization process. This clone was initially selected from the group-selective library (see Figure 4) and provided the highest afnity to s-triazines bearing an IPR, that is, atrazine and propazine (Table 1).40 Chain shufing was applied as a directed, recombinatorial approach for improving the afnity of this clone. At the outset the light chain of the template Ab IPR7 was shufed with the heavy-chain repertoire of the group-specic library, and subsequently selected by phage display. Then, the heavy chain of the best binder (IPR-26) was shufed against the library light-chain repertoire. The kinetic data of the Ab variants are detailed in Table 1. The equilibrium dissociation constants of the Ab variants are approaching the typical KD level of afnity matured Ab in vivo. The optimized variant IPR-83 showed a 17-fold increase in afnity as compared to the template Ab IPR-7. Interestingly, sequence analysis of the shufed clones revealed a bias of amino acid substitutions
Table 1. Association rate constant ka , dissociation rate constant kd and equilibrium dissociation constant KD for IPR obtained from the scFv clones IPR-7, IPR-26 and IPR-83. The values for ka , kd and KD were measured utilizing the BIAcore 2000 system

Clone IPR-7 IPR-26 IPR-83

ka (M1 s1 ) 1.38 10 2.10 105 6.73 105

5

kd (s1 ) 1.75 10 1.93 103 5.02 104

3

KD = kd /ka (M) 1.27 108 9.20 109 7.46 1010

10

BIOLOGICAL AND MOLECULAR RECOGNITION SYSTEMS

from the template IPR-7 to the optimized variant IPR-83 in the 5 moiety of the V genes including the rst two CDRs and the anking frame regions.45 This is in contrast to a series of Ab optimization experiments that are primarily targeting on the variation of the CDR3 regions at the 3 moiety of the Ab gene.46,47 The VH CDR3 region is generally considered to constitute the key determinant for the antigen selectivity.48 However, the distribution of sequence alterations obtained in the presented Ab optimization is consistent with proposed models for mutational mechanisms during the secondary immune response in vivo.47,48 In addition, experimental data obtained from in vivo immune repertoires are conrming individual sites at the V genes that are prone to hypermutation. These mutational hot-spots for afnity maturation are strategically located at the CDR1 and CDR2 rather than in the CDR3 loop.49,50 Therefore, the applied in vitro optimization strategy resulted in a distribution of sequence alterations, which is tting very well into the current knowledge of natural afnity maturation. The applicability of the optimized Ab variant IPR-83 was tested by measuring environmental samples. IPR-83 was applied to determine atrazine contaminations of soil samples collected in Southern Germany. Although atrazine has been banned in Germany by the European Community in 1991, environmental contaminations have been observed during the last years due to illegal applications. The corresponding threshold for atrazine is 100 g kg1 soil. The immunochemical analysis

was complemented by HPLC analyses as reference method for validation (Table 2). The ELISA measurements were consistent with the HPLC data within the experimental error.45 Thus, the engineered scFv mutants proved to be suitable for the application in environmental analyses under real sample conditions.

2 DISCUSSION AND OUTLOOK

Table 2. Atrazine analyses of soil samples by ELISA employing the mutant antibody IPR-83. Validation was performed by HPLC

Soil sample 1 2 3 4 5 6 7 8 9

ELISA atrazine (g kg1 ) 43 4 323 8 35 8 91 13 126 4 141 6 65 5 108 11 121 3

HPLC atrazine (g kg1 ) 49 3 313 12 33 3 85 10 120 21 135 14 59 8 115 9 113 7

[HPLC data were kindly provided by Dr. J. Lepschy, Bayerische Landesanstalt f r Bodenkultur und Panzenbau, Freising, Germany.] u

The principles of directed evolution in vitro are very similar to the mechanisms of somatic mutation in vivo. The Ab repertoire of the primary immune response in vivo is predominantly the result of recombining germ line genes of the V, D, J clusters (i.e., the genes constituting elements of the nally expressed VH and VL genes). The primary response yields Abs with generally low afnities. Thereafter, Ab variable genes of the primary repertoire are modied during the secondary immune response in the microenvironment of lymphoid germinal centers by somatic hypermutation. Physiologically appropriate variants are subsequently selected from this pool of mutant immunoglobulins upon their improved afnity to the antigen.20 The primary repertoire in vitro is established by cloning synthetic or natural Ab genes as described above for the fully synthetic HuCAL and the group-selective library, respectively. The former one is a commercially available library. Other examples of accessible primary repertoires include the Tomlinson I and J Ab libraries.44 Some of these primary libraries are very large (containing more than 1010 different Ab variants) and therefore considered to be universal, which suggests that the corresponding Abs are covering a huge panel of various specicities. However, even at its best these libraries will represent no more than a basic immune repertoire. The optimization by means of molecular evolution strategies or alternatively by rational design probably remains therefore an integral part in the synthesis of appropriate Abs for the majority of immunochemical applications in environmental analysis. Therefore, one of the vital goals for the future is the simple and cheap access to evolutionary technologies for tailored binders with predened properties such as selectivity, afnity, stability,

MOLECULAR ANTIBODY TECHNOLOGIES FOR BIOSENSORS AND BIOANALYTICS

11

and more. Massive parallel processing combined with high-throughput strategies as well as highcontent screening will have a benecial impact on this new era of Ab production. The hardware is already available for a straightforward production of rAbs. From automated plating and picking of bacteria colonies up to robotic screening and data processing. Self controlled variation and selection has to be integrated in the concept. For instance, recombination in vivo can be achieved by coupling chain shufing with the phage infection of bacteria as demonstrated for the cre/lox system. An Ab light-chain repertoire in the phage is recombined with the heavy-chain repertoire in the bacteria host by infection.51 Selection of specic Ab variants can be controlled by integrating the SIP technology (see preceding text).34 Currently the potential of alternative Ab molecules is being exploited, a trend, which is mainly driven by the complex patent situation in the Ab area. For instance, single domain Ab (sdAb) are naturally occurring in camelids and sharks.52,53 In addition to Ab domains, so-called Ab mimics have been reported to be amenable to molecular optimization. The tenth bronectin type III domain (10 Fn3),54 a monomeric member of the immunoglobulin superfamily, and the extracellular domain of human cytotoxic T-lymphocyte associated antigen (CTLA4)55 were used as a scaffold for library synthesis. In addition to immunoglobulins and immunoglobulin-like polypeptides, some specic afnity reagents have been selected from small, globular protein scaffolds not related to Ab. For instance, the -helical Z-domain of protein A, designated as afbodies was improved in directed evolution experiments to the nanomolar range.56 Similarly, the bilin-binding protein (BBP), a lipocalin from the buttery Pieris brassicae, was used for library synthesis.57 The protein has a conserved -barrel core formed by eight antiparallel -strands. Peptide loops, which connect the individual strands, confer binding to the ligand. Afnities in the picomolar range for digoxigenin were achieved by selective mutation of these anticalins.58 Another type of protein scaffold is the ankyrin repeat (AR). AR proteins are composed of several 33 amino acid repeats stacked in a row. Each repeat comprises a -turn followed by two antiparallel -helices and a C-terminal loop reaching the -turn of the adjacent loop.59

Synthetic libraries were developed by randomizing amino acid positions at the -turn and the short hinge connecting the two -helices.60 The AR libraries yielded high-afnity binding variants with KD in the nanomolar range against various protein targets. These binding proteins show that there are promising candidates with a potential for environmental analysis, which could provide an alternative for the classical Ab molecule.

ACKNOWLEDGMENTS

The authors would like to thank MorphoSys AG for providing the HuCAL library and the MRC HGMP Resource Centre for giving access to the Tomlinson I + J library. Further thanks are addressed to Mrs H. Geltl and Mrs A. Hubauer for their excellent technical assistance. Financial support was obtained from the EC (ENVIROSENSE ENV4-CT96-0333; IMAGEMO QLK3-CT-200202141) and the Bavarian Government (Project no. BFS 306/98).

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