Antibody-hapten Complexes Research Articles

Immunization of chickens with an agonistic monoclonal anti-chicken CD40 antibody–hapten complex: Rapid and robust IgG response induced by a single subcutaneous injection

Producing diagnostic antibodies in chicken egg yolk represents an alternate animal system that offers many advantages including high productivity at low cost. Despite being an excellent counterpart to mammalian antibodies, chicken IgG from yolk still represents an underused resource. The potential of agonistic monoclonal anti-CD40 antibodies (mAb) as a powerful immunological adjuvant has been demonstrated in mammals, but not in chickens. We recently reported an agonistic anti-chicken CD40 mAb (designated mAb 2C5) and showed that it may have potential as an immunological adjuvant. In this study, we examined the efficacy of targeting a short peptide to chicken CD40 [expressed by the antigen-presenting cells (APCs)] in enhancing an effective IgG response in chickens. For this purpose, an immune complex consisting of one streptavidin molecule, two directionally biotinylated mAb 2C5 molecules, and two biotinylated peptide molecules was produced. Chickens were immunized subcutaneously with doses of this complex ranging from 10 to 90μg per injection once, and relative quantification of the peptide-specific IgG response showed that the mAb 2C5-based complex was able to elicit a strong IgG response as early as four days post-immunization. This demonstrates that CD40-targeting antigen to chicken APCs can significantly enhance antibody responses and induce immunoglobulin isotype-switching. This immunization strategy holds promise for rapid production of hapten-specific IgG in chickens.

Use of antibody–hapten complexes attached to optical sensor surfaces as a substrate for proteases: Real-time biosensing of protease activity

Fluorescent antibody protein (IgG) was attached to the surface of an integrated optical glass waveguide chip via specific binding to a covalently attached hapten and used as a substrate for the measurement of protease activities. Exposure of the optical chip to proteases resulted in digestion of the bound fluorescent antibody molecules and proportional decrease in the detectable fluorescence resulting from loss of fluorescence from the evanescent field. The bound fluorescent antibody protein was used as a unique universal protease substrate in which the combined biological activity and fluorescence signal were the basis of measurement. The action of proteases was monitored in real-time mode where the gradual decrease in evanescent fluorescence was recorded. The chip was regenerated by complete digestion of the antibody substrate by excess pepsin and recharged by incubation with a fresh sample of the labelled antibody. The biosensor was used to detect activity of several proteases including a bacterial protease preparation, Pronase E. The linear range of measurable Pronase E activity was from 0.03 to 2 units/mL. A measurement cycle took 40 min for samples with high protease concentration (>or=0.5 units/mL), when the concentration of the protease was less measurement times up to 100 min were required. The method demonstrates the principle of a new mode of real-time biosensing of proteases. The modular integrated optical glass waveguide biosensor system used in this study is compact and controlled by a laptop computer and could easily be miniaturised and utilized as a true probe device for detecting proteases with potential applications in a wide range of areas including research, clinical diagnostics, biotechnology processing and food and detergent manufacturing industries.

The novel FluoroChrome ImmunoAssay — (FCIA). The role of molecular environment upon molecular structure exemplified by constriction of a flourescence-photochrome flanked by two proteins

A rapid, sensitive, and quantitative novel immunoassay [FluoroChrome ImmunoAssay, FCIA] technique was developed which auspiciously combines both the high sensitivity of fluorescence measurements with the high specificity of an antibody. As opposed to existing immunoassays, FCIA is performed without separation of antibody-bound haptens from those that are free, and utilizes fluorescence measurements from widely available standard commercial fluorimeters. FCIA is based on the hypothesis that an appropriately designed stilbene-antigen analogue probe will suffer considerable steric hindrance to trans-cis photoisomerization when bound within the combined constraints of both an antibody binding site and a second globular protein. Specifically, an appropriately designed 2,4–dinitrophenyl-hapten derivative of fluorescent trans-4,4′-diaminostilbene (DAS), was squeezed between two large globular proteins: lysozyme (Lys) from one side, and anti-2,4,6-trinitrophenyl antibody (antiTNP) from the other side, in order to provide the desired constricted environment to restrict trans/cis-stibene isomerization within the antiTNP-DNP-DAS-Lys adduct. As was theoretically predicted and then experimentally verified, the trans-cis photoisomerization rate for the bound probe was found to be markedly inhibited, compared to that expected for the free probe in solution. The fluorescence-photochrome labeled probe was competitively displaced from the antiTNP binding site in the presence of the picric acid hapten, and photoisomerization then commenced to produce the fluorescence-silent cis-stilbene diastereomer. The process of association and dissociation of a hapten-antibody complex was readily monitored by the fluorescence technique in the presence of both antibody-bound and free haptens.

Change of the unbinding mechanism upon a mutation: A molecular dynamics study of an antibody–hapten complex

We study forced unbinding of fluorescein from the wild type (WT) and a mutant [H(H58)A] of the single-chain variable-fragment (scFv) anti-fluorescein antibody FITC-E2 by molecular dynamics simulations using various pulling techniques. A large number of long simulations were needed to obtain statistically meaningful results as both the wild type and the H(H58)A mutant unbinding occurs through multiple pathways, often with metastable intermediates. For the wild type, the rate-limiting step in the unbinding process corresponds to the breaking of the non-native interactions characteristic of a specific intermediate. The H(H58)A mutation disfavors the occurrence of this intermediate. Two events where the hapten partially unbinds in the absence of pulling force are observed in extensive equilibrium simulations of the wild type, and their analysis indicates that forced unbinding and spontaneous unbinding proceed along similar pathways. The different unbinding mechanisms observed in the simulations suggest a possible reason for the difference in the experimental off-rate between the two antibodies. We predict mutations that are expected to modulate the occurrence of the unbinding intermediate. For two such new mutants [H(H58)A and S(H52)A], our predictions are validated in silico by additional simulations. The accompanying paper in this issue by Honegger et al. reports the X-ray structure of FITC-E2 with a derivative of fluorescein, which was used as the starting conformation for the work presented here.

Quantum chemical study on the affinity maturation of 48G7 antibody

In this study, we calculate the binding energies of hapten with the germline and mature forms of the 48G7 antibody. The energies are obtained using a combined method of a linear-scaling quantum chemical calculation (known as MOZYME) and a continuum solvent model (known as COSMO). The calculations successfully reproduced the experimental fact that the binding energy increases on going from the germline to mature forms. Detailed analysis indicated that such affinity improvement is mainly due to the electrostatic improvement caused by the following mutations: Asp/His L 55, Glu/Lys H 42, Asn/Asp H 57, Gly/Asp H 66, Asn/Lys H 77 in the heavy (H) and light (L) chains. An interesting finding is that the intermolecular charge transfer and electronic polarization effects significantly contribute to the binding energy between the hapten and the antibody. Furthermore, the frontier orbitals of the antibody are localized at the hapten binding site. These results imply that the orbital interactions play an important role in stabilizing the antibody–hapten complexes. In conclusion, quantum mechanical calculation such as MOZYME is indispensable for in silico drug screening.

Conserved sequence and structure association motifs in antibody-protein and antibody-hapten complexes.

In this paper, we present the association requirements across a wide variety of antibody-antigen complexes. Phylogenetic analysis clearly indicates the representative nature of our structural dataset. Antigen molecules range from small-molecule haptens to complete protein structures. Common association motifs identified include five conserved tyrosine residues and a single conserved arginine residue from CDR-H3. Further, specificity is refined by a diverse array of antibody-antigen electrostatic interactions that maximize complex specificity. Through analysis of calculated pKa shifts on antigen binding, we find that these interactions are conserved at 23 alignment 'hot-spot' positions. Despite consistent roles in defining substrate specificity, 16 hot-spot positions are conserved less than 50% of the time. On the other hand, because of the conserved functional role of these positions, mutant screening at hot-spots is more likely to result in increased antigen specificity than elsewhere. Therefore, we believe these results should facilitate subsequent antibody design experimentation.

Detection strategies for catalytic antibodies

This paper discusses the detection of antibody catalysis using soluble test substrates. Antibodies raised against a transition state analog of a chemical reaction typically show dissociation constants for the antibody–hapten complexes in the range of 10 −9–10 −7 M. If hapten binding is transferred to catalysis as measured by the transition-state dissociation constant K TS= K M/( k cat/ k uncat)= k uncat/( k cat/ K M), this corresponds to the concentration of antibody necessary to double the apparent rate of the reaction. This sets a lower limit for the detection of catalysis if no additional effects are present to induce catalysis. The concentration of antibodies in hybridoma cell culture supernatants (5–50 μg/ml) meets this requirement. The use of high-throughput screening (HTS) for catalysis together with ELISA to select hybridomas leads to the isolation of not only one, but also many catalytic antibodies. As examples, the application of HTS for catalysis using fluorogenic reactions to isolate retro-Diels-Alderase and pivalase catalytic antibodies useful for prodrug activation chemistry are discussed.

Probing ligand recognition in the decarboxylase antibody 21D8: implications for the catalytic mechanism.

Antibody 21D8, which was elicited with a naphthalene-1,5-disulfonate hapten, catalyzes the medium-sensitive decarboxylation of 5-nitro-3-carboxybenzisoxazole. Structural studies on the hapten-antibody complex show that the active site contains two anion binding pockets separated by a hydrophobic region. To gain further insight into the ligand binding and catalytic mechanism of 21D8, six site-directed mutants were prepared, four for investigating the role of each of the two hapten sulfonate binding sites and two for examining packing interactions between bound ligands and the binding pocket. With the exception of an Arg(L46)Met substitution in the more deeply buried sulfonate binding pocket, modification of the active site resulted in reductions in catalytic efficiency (k(cat)/k(uncat)), ranging between 3- and 23-fold. Importantly, and contrary to predictions based on computational docking experiments, the differential effects of the individual mutations on the K(m), K(TS), and K(product) parameters suggest that only substrate binding modes which place the carboxylate group in the more solvent-exposed sulfonate binding site are catalytically relevant. Such an orientation would permit a potentially significant interaction between the developing oxyanion in the transition state and the side chain of Arg(L96). Incomplete desolvation of the carboxylate in this orientation may also help explain the modest efficiency of 21D8 compared to the most accelerating aprotic dipolar organic solvents.

Probing Ligand Protein Binding Equilibria with Fluorescence Fluctuation Spectroscopy

We examine the binding of fluorescent ligands to proteins by analyzing the fluctuation amplitude g( 0) of fluorescence fluctuation experiments. The normalized variance g( 0) depends on the molecular brightness and the concentration of each species in the sample. Thus a single g( 0) measurement is not sufficient to resolve individual species. Titration of the ligand with protein establishes the link between molecular brightness and concentration by fitting g( 0) to a binding model and allows the separation of species. We first apply g( 0) analysis to binary dye mixtures with brightness ratios of 2 and 4 to demonstrate the feasibility of this technique. Next we consider the influence of binding on the fluctuation amplitude g( 0). The dissociation coefficient, the molecular brightness ratio, and the stochiometry of binding strongly influence the fluctuation amplitude. We show that proteins with a single binding site can be clearly differentiated from proteins with two independent binding sites. The binding of fluorescein-labeled digoxigenin to a high-affinity anti-digoxin antibody was studied experimentally. A global analysis of the fluctuation amplitude and the fluorescence intensity not only recovered the dissociation coefficient and the number of binding sites, but also revealed the molecular heterogeneity of the hapten-antibody complex. Two species were used to model the molecular heterogeneity. We confirmed the molecular heterogeneity independently by fluorescence lifetime experiments, which gave fractional populations and molecular brightness values that were virtually identical to those of the g( 0) analysis. The identification and characterization of molecular heterogeneity have far-reaching consequences for many biomolecular systems. We point out the important role fluctuation experiments may have in this area of research.

Automated Docking of Ligands to Antibodies: Methods and Applications

Many approaches to studying protein–ligand interactions by computational docking are currently available. Given the structures of a protein and a ligand, the ultimate goal of all docking methods is to predict the structure of the resulting complex. This requires a suitable representation of molecular structures and properties, search algorithms to efficiently scan the configuration space for favorable interaction geometries, and accurate scoring functions to evaluate and rank the generated orientations. For many of the available methods, tests on experimentally known antibody–antigen or antibody–hapten complexes have appeared in the literature. In addition, some of them have been used in predictive studies on antibody–ligand interactions to provide structural insights where adequate experimental information is missing. The AutoDock program is presented as example of a method for flexibly docking ligands to antibodies. Applying parameters of the second-generation AMBER force field, three antibody–hapten complexes (AN02, DB3, NC6.8) are used as new test cases to analyze the ability of the method to reproduce experimental findings. The X-ray structures could be reconstituted and the corresponding solutions were ranked with best energy score in all cases. Docking to the free instead of the complexed NC6.8 structure indicated the limits of the rigid protein treatment, although fairly good guesses about the location of the binding site and the contact residues could still be obtained if conformational flexibility was allowed at least in the ligand.

Molecular dynamics and free-energy calculations applied to affinity maturation in antibody 48G7

We investigated the relative free energies of hapten binding to the germ line and mature forms of the 48G7 antibody Fab fragments by applying a continuum model to structures sampled from molecular dynamics simulations in explicit solvent. Reasonable absolute and very good relative free energies were obtained. As a result of nine somatic mutations that do not contact the hapten, the affinity-matured antibody binds the hapten >10(4) tighter than the germ line antibody. Energetic analysis reveals that van der Waals interactions and nonpolar contributions to solvation are similar and drive the formations of both the germ line and mature antibody-hapten complexes. Affinity maturation of the 48G7 antibody therefore appears to occur through reorganization of the combining site geometry in a manner that optimizes the balance of gaining favorable electrostatic interactions with the hapten and losing those with solvent during the binding process. As reflected by lower rms fluctuations in the antibody-hapten complex, the mature complex undergoes more restricted fluctuations than the germ line complex. The dramatically increased affinity of the 48G7 antibody over its germ line precursor is thus made possible by electrostatic optimization.

A simple method for determining KAs of both low and high affinity IgG antibodies

A rapid and convenient method for measuring affinity constants ( K A) of IgG antibody–hapten complexes is described. A key advantage of this method is its suitability for quantification of both low and high affinity interactions. A comparison is made of the K As of the low affinity anti-phosphocholine (PC) antibody T15 ( K A=2.9×10 5 M −1) and five heavy chain complementarity determining region 2 (HCDR2) mutant antibodies expressed as IgG2b transfectants. As a demonstration of the general applicability of the technique, colchicine binding to the high affinity monoclonal IgG2a antibody C44 ( K A=1.5×10 9 M −1) is measured also; thus the assay is applicable over a four-log range of affinities. The assay, based upon use of fixed Staphylococcus aureus Cowan I strain cells as an adsorbent for antibody-radiolabeled antigen complexes, is conducted over a range of hapten concentrations at constant antibody concentration. The K A is obtained by Scatchard analysis.

A structural basis for transition-state stabilization in antibody-catalyzed hydrolysis: crystal structures of an abzyme at 1.8 Å resolution

The three-dimensional structure of a catalytic antibody, 6D9, has been solved as a complex with a transition state analog. The structure was determined from two different crystal forms, and was refined at a resolution of 1.8 Å. The antibody 6D9, which was induced by immunization with the phosphonate transition state analog 3, hydrolyzes a prodrug of chloramphenicol monoester 1 to generate the parent drug 2. The kinetic studies have shown that the antibody is catalytic by virtue of the theoretical relationship between the affinity for the transition state and the catalytic efficiency ( k cat/ k uncat= K S/ K TSA). The crystal structure makes it possible to visualize the theoretical relationship. A side-chain (N ϵ) of His L27Dis placed in a key position to make a hydrogen bond to the phosphonate oxygen of the transition state analog with a distance of 2.72 Å, suggesting a hydrogen bond to the oxyanion developing in the transition state of the hydrolysis. There are no catalytic residues, other than the histidine, around the phosphonate moiety. In addition, in the antibody-hapten complex, the hapten bears a folded conformation and the two stacked aromatic rings are buried deep in the antigen-combining site through aromatic-aromatic interaction with Trp H100Iand Tyr H58. The conformation of the bound hapten suggests that the antibody binds the substrate to change the conformation of the ester moiety to a thermodynamically unstable E-form, thereby making it easy for the substrate to reach the transition-state during catalysis. These observations reveal that the catalytic mechanism is explained purely on the basis of the stabilization of the transition state. The refined high resolution structures reported here are envisaged to have an impact on the understanding of other hydrolytic antibodies, since their haptens share some unique features with the hapten used in this study.

Surface properties of antigen-antibody complexes.

In this paper, the authors show that liquid-liquid partition chromatography in an aqueous two-phase system offers unique possibilities of comparing the overall surface properties of intact antibodies in solution before and after binding of antigen. The authors demonstrate that the surface properties of antigen-antibody complexes are dependent on the variable regions of the antibodies, the nature of the antigen and/or possible conformational changes induced by antigen binding. Thus, each antigen-IgG antibody pair formed one type of complex with respect to the exposed dominant surface. The antigen-binding sites of IgG antibodies were exposed and dominant even after binding of hapten or hapten-carrier. In contrast, the antibody-combining sites were concealed upon protein binding and the exposed surfaces of the protein-antibody complexes were related mainly to those of the antigen. IgA1, IgA2, IgE and IgM formed, in comparison to the IgG, hapten-antibody complexes which exhibited surface properties that could be related to both the antigen-binding sites and Fc parts of the antibodies. Moreover, the results indicated that antigen-induced conformational changes occurred in either IgA1, IgA2, IgE, or IgM, but not in IgG1, -2, -3 and -4, making the surfaces of their heavy chain constant regions more similar.

Critical evaluation of the applicability of capillary zone electrophoresis for the study of hapten-antibody complex formation

The applicability of capillary zone electrophoresis (CZE) for the determination of association constant of an hapten-antibody complex with values as high as 10 7 (mol/1) −1 was investigated (dissociation constant in the nmol range). As a reference method the well known, enzyme-linked immunosrbent assay (ELISA) was selected. The study describes the optimisation of the experimental conditions of the CZE technique. The CZE measurements were optimised according to an experimental design. The results of the CZE and ELISA methods are compared giving consideration to the reproducibility (repeatability) of the two methods.

Development of a regenerable amperometric immunosensor for 7‐hydroxycoumarin

AbstractA regenerable immunosensor, based on a competitive assay principle and utilizing electrochemical detection, was developed for the determination of 7‐hydroxycoumarin. Horseradish peroxidase (HRP) labeled anti‐7‐hydroxycoumarin antibody was employed in the determination, and the enzyme activity was measured as a function of current due to the reduction of hydrogen peroxide in the presence of a mediator (potassium ferrocyanide). Sensor preparation involved chemical immobilization of 7‐hydroxy‐coumarin‐BSA conjugate on a glassy carbon electrode surface using a water soluble carbodiimide, 1 ‐ethyl‐3(3‐dimethyl‐aminopropyl) carbodiimide, as coupling reagent in the presence of N‐hydroxysuccinimide. The competition between the free and immobilized forms of the hapten for their HRP labeled antibody in solution allows for the quantitation of free 7‐hydroxycoumarin. The sensor can be regenerated by simply immersing the electrode tip in acidic media. Under these conditions the hapten‐antibody complex was selectively disrupted without adversely affecting the sensor performance. The conditions for removing the non‐specific adsorption of the enzyme conjugate were also studied. The results showed that the detection range obtained for the hapten was physiologically relevant.

The immunological evolution of catalysis.

The germline genes used by the mouse to generate the esterolytic antibody 48G7 were cloned and expressed in an effort to increase our understanding of the detailed molecular mechanisms by which the immune system evolves catalytic function. The nine replacement mutations that were fixed during affinity maturation increased affinity for the transition state analogue by a factor of 10(4), primarily the result of a decrease in the dissociation rate of the hapten-antibody complex. There was a corresponding increase in the rate of reaction of antibody with substrate, k(cat)/k(m), from 1.7 x 10(2)M(-1) min(-1) to 1.4 x 10(4)M(-1) min(-1). The three-dimensional crystal structure of the 48G7-transition state analogue complex at 2.0 angstroms resolution indicates that one of the nine residues in which somatic mutations have been fixed directly contact the hapten. Thus, in the case of 48G7, affinity maturation appears to play a conformational role, either in reorganizing the active site geometry of limiting side-chain and backbone flexibility of the germline antibody. The crystal structure and analysis of somatic and directed active site mutants underscore the role of transition state stabilization in the evolution of this catalytic antibody.

Efficient aldolase catalytic antibodies that use the enamine mechanism of natural enzymes.

Antibodies that catalyze the aldol reaction, a basic carbon-carbon bond-forming reaction, have been generated. The mechanism for antibody catalysis of this reaction mimics that used by natural class I aldolase enzymes. Immunization with a reactive compound covalently trapped a Lys residue in the binding pocket of the antibody by formation of a stable vinylogous amide. The reaction mechanism for the formation of the covalent antibody-hapten complex was recruited to catalyze the aldol reaction. The antibodies use the epsilon-amino group of Lys to form an enamine with ketone substrates and use this enamine as a nascent carbon nucleophile to attack the second substrate, an aldehyde, to form a new carbon-carbon bond. The antibodies control the diastereofacial selectivity of the reaction in both Cram-Felkin and anti-Cram-Felkin directions.

Drug-protein conjugates: preparation of triamcinolone-acetonide containing bovine serum albumin/keyhole limpet hemocyanin-conjugates and polyclonal antibodies.

A radioimmunoassay has been developed for the quantitation of triamcinolone-acetonide (TAAc) at the picogram level. For use of TAAc as an antigenic epitope first the drug was hemisuccinoylated at C-21 as confirmed by 13C-NMR- and mass spectroscopy after derivatization. This hapten was conjugated to the carrier-protein bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH) by different amide-bond generating methods (imidazolide-, carbodiimide-, carbodiimide/sulfo-N-hydroxysuccinimide-, mixed anhydride-method) yielding antigens of quite different conjugation number, solubility and usefulness. The mixed anhydride-method yielded most useful soluble conjugates bearing 0.3-31.5 mol TAAc per mol carrier-protein. Coupling by the carbodiimide-method yielded insoluble conjugates, inappropriate for antigen synthesis in hapten immunoassays because of formation of coupling agent modified residues and crosslinking of the carrier-protein. Specificity of the antisera obtained by immunization with TAAc-BSA and TAAc-KLH was assessed by isolation of the soluble hapten-antibody complex and a RIA protocol was developed providing a detection limit of 200 pg (0.46 pmol) TAAc/ml sample.

Crystal Structure of a Catalytic Antibody with a Serine Protease Active Site

The three-dimensional structure of an unusually active hydrolytic antibody with a phosphonate transition state analog (hapten) bound to the active site has been solved to 2.5 A resolution. The antibody (17E8) catalyzes the hydrolysis of norleucine and methionine phenyl esters and is selective for amino acid esters that have the natural alpha-carbon L configuration. A plot of the pH-dependence of the antibody-catalyzed reaction is bell-shaped with an activity maximum at pH 9.5; experiments on mechanism lend support to the formation of a covalent acyl-antibody intermediate. The structural and kinetic data are complementary and support a hydrolytic mechanism for the antibody that is remarkably similar to that of the serine proteases. The antibody active site contains a Ser-His dyad structure proximal to the phosphorous atom of the bound hapten that resembles two of the three components of the Ser-His-Asp catalytic triad of serine proteases. The antibody active site also contains a Lys residue to stabilize oxyanion formation, and a hydrophobic binding pocket for specific substrate recognition of norleucine and methionine side chains. The structure identifies active site residues that mediate catalysis and suggests specific mutations that may improve the catalytic efficiency of the antibody. This high resolution structure of a catalytic antibody-hapten complex shows that antibodies can converge on active site structures that have arisen through natural enzyme evolution.