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Authors performed investigation on "antigen-antibody" interaction of chicken infectious bronchitis coronavirus (IBV) by a method based on the surface plasmon resonance (SPR). Presence of space-size effect related to a difference between antigen and antibody particle sizes has been theoretically grounded and experimentally proven. Herewith, the difference between responses of the SPR-sensor to specific and non-specific interactions is considerably less (up to 6.3 times) than the expected one (8 - 11 times). An impact of functionalization of sensor's sensitive element surface, as well as acidity of buffer solution on the activity of antigen-antibody interaction was studied here. The difference between sensor's responses to specific and non-specific interactions increased two-fold from 200 to 432ang sec due to this treatment. When changing the acidity of analyzed solution from pH7.3 to pH6.8, the corresponding difference between sensor's responses increased by 6.3 times from 194 up to 1235ang.sec. Thus, an impact of space-size effect on interaction between IBV antigen and specific antibody can be considerably (almost in 3 times) decreased by reducing the acidity of used buffer solution. The results of our investigation can be successfully applied to develop new methods for detection of pathogens and specific antibodies using SPR.

Monoclonal antibodies developed for therapeutic or diagnostic purposes need to demonstrate highly defined binding specificity profiles. Engineering of an antibody to enhance or reduce binding to related antigens is often needed to achieve the desired biologic activity without safety concern. Here, we describe a deep sequencing-aided engineering strategy to fine-tune the specificity of an angiopoietin-2 (Ang2)/vascular endothelial growth factor (VEGF) dual action Fab, 5A12.1 for the treatment of age-related macular degeneration. This antibody utilizes overlapping complementarity-determining region (CDR) sites for dual Ang2/VEGF interaction with KD in the sub-nanomolar range. However, it also exhibits significant (KD of 4 nM) binding to angiopoietin-1, which has high sequence identity with Ang2. We generated a large phage-displayed library of 5A12.1 Fab variants with all possible single mutations in the 6 CDRs. By tracking the change of prevalence of each mutation during various selection conditions, we identified 35 mutations predicted to decrease the affinity for Ang1 while maintaining the affinity for Ang2 and VEGF. We confirmed the specificity profiles for 25 of these single mutations as Fab protein. Structural analysis showed that some of the Fab mutations cluster near a potential Ang1/2 epitope residue that differs in the 2 proteins, while others are up to 15 Å away from the antigen-binding site and likely influence the binding interaction remotely. The approach presented here provides a robust and efficient method for specificity engineering that does not require prior knowledge of the antigen antibody interaction and can be broadly applied to antibody specificity engineering projects.

Structural information pertaining to antigen-antibody interactions is fundamental in immunology, and benefits structure-based vaccine design. Modeling of antigen-antibody immune complexes from co-crystal structures or molecular docking simulations provides an extensive profile of the epitope at the interface; however, the key amino acids involved in the interaction must be further clarified, often through the use of experimental mutagenesis and subsequent binding assays. Here, we describe an in silico mutagenesis method to identify key sites at antigen-antibody interfaces, using significant increase in pH-dependency energy among saturated point mutations. Through a comprehensive analysis of the crystal structures of three antigen-antibody immune complexes, we show that a cutoff value of 1 kcal/mol of increased interaction energy provides good congruency with the experimental non-binding mutations conducted in vitro. This in silico mutagenesis strategy, in association with energy calculations, may provide an efficient tool for antibody-antigen interface analyses, epitope optimization, and/or conformation prediction in structure-based vaccine design.

Despite its relevance to many biomedical fields, relatively little effort has been put into a comprehensible quantitative description of the effect of reaction temperature on the interaction between antigens and their antibodies. In this article, a novel, straightforward mathematical model is proposed, which aims to describe the effect of temperature on antigen-antibody kinetics. The model proposed in this article could hopefully provide clinicians, immunologists, and biochemists with an improved insight into the kinetic effect of fluctuations in reaction temperature on antigen-antibody-dependent processes and therefore into the kinetics of the humoral adaptive immune response.

Vaccine development is challenged by the hierarchy of immunodominance between the target antigens’ epitopes, and the appearance of antigenic variants by pathogen mutation. The strength and breath of antibody responses relies on selection and mutation in the Germinal Center, and on the structural relationship between antigens. Computational methods for assessing the breadth of germinal center responses to multivalent antigens would greatly speed up vaccine development. Yet, such methods have poorly reflected the 3D antigen structure and antibody breadth. Here, we present Ymir, a new 3D-lattice-based framework for in silico antibody-antigen affinities. Key physiological properties naturally emerge such as affinity jumps, cross-reactivity, and differential epitope accessibility. We validated Ymir by replicating known features of germinal centers dynamics. We show that combining antigens with mutated but structurally related epitopes enhances vaccine breadth. Ymir opens a new avenue for understanding vaccine success based on the structural relationship between vaccine antigens.

Cell surface antigen-specific antibodies are of substantial diagnostic and therapeutic importance. The binding properties of such antibodies are usually evaluated by cell-free assays, in particular surface plasmon resonance (SPR) analysis, or flow cytometry. SPR analyses allow the detailed quantitative and dynamic evaluation of the binding properties of antibodies, but need purified, typically recombinantly produced antigens. It can, however, be difficult to produce the required antigen. Furthermore, cellular factors influencing the antigen-antibody interaction are not considered by this method. Flow cytometry-based analyses do not have these limitations, but require elaborated calibration controls for absolute quantification of bound molecules. To overcome the limitations of SRP and flow cytometry in the characterization of cell surface antigen-specific antibodies, we developed Fn14-specific antibody 18D1 as an example of an antibody fusion protein format that includes the luciferase of Gaussia princeps (GpL), which enables very simple and highly sensitive cellular binding studies. We found that GpL-tagging of the C-terminus of the antibody light chain does not affect the interaction of 18D1-IgG1 with its antigen and Fc-gamma receptors (FcγRs). In accordance with this, the GpL(LC-CT)-18D1-IgG1 antibody fusion protein showed basically the same FcγR-dependent agonistic properties as the parental 18D1 antibody. Similar results were obtained with isotype switch variants of 18D1 and antibodies specific for CD95, LTβR and CD40. In sum, we demonstrate that antibody GpL fusion proteins are easily manageable and versatile tools for the characterization of cell surface antigen-antibody interactions that have the potential to considerably extend the instrumentarium for the evaluation of antibodies.

Despite having limited number of antibody molecules, it is still unclear how immune system recognizes surfeit of antigens. A novel mechanism of antigen recognition was deciphered from the crystallographic analysis of scFv 2D10 bound to disaccharide moiety. High-resolution crystal structure of the complex reveals unusual binding of scFv 2D10 with α1-6 mannobiose resembling supramolecular assemblies. The disaccharide interacts with the scFv 2D10 at two sites independent of canonical sugar binding positions, in two chemically distinct environments. At site A, α1-6 mannobiose lies on two-fold symmetry axis interacting through π-stacking and water network. However, at site B, α1-6 mannobiose exists in two orientations related by quasi-symmetry with direct hydrogen bonds. Although individual one on one interactions are low at both sites, symmetry related molecules contribute significantly for stable binding of the disaccharides, indicated by 2–4 fold lower binding energies. These interactions are facilitated due to relatively high concentrations of receptor and ligand. We propose that the molecular crowding leading to supramolecular associations acts as a specificity determinant and could be naturally exploited to bind diverse antigens. This previously uncharacterized property will considerably enhance the recognition repertoire of antibodies and therefore allows the humoral response to deal with the threat posed by plethora of antigens.

Different methods for the covalent immobilization of specific antibodies and their fragments on a silicon surface with the subsequent formation of immune complexes that consist of an immobilized monoclonal antibody, an antigen molecule, and a molecule of a second monoclonal antibody labeled with gold nanoparticles have been studied. Prostate-specific antigen (PSA), which is a molecular biomarker for prostate cancer, was used as an antigen. A covalent conjugate of the fragments of PSA-specific antibodies with gold nanoparticles has been obtained using the thiol groups of the antibodies. Scanning electron microscopy (SEM) was used for the registration of immune complexes on the surface. The high resolution of the method made it possible to detect individual immune complexes by the presence of gold nanoparticles and to calculate their number. A new method for the chemical modification of silicon by 3-aminopropyltrimetoxysilane (APTMS) and a bifunctional reagent 1,4-phenylene diisothiocyanate (PDITC) has been developed. This method provides a uniform distribution of antigen-binding centers and their availability for the formation of immune complexes. The developed immobilization method is promising for the formation of a biospecific biosensor layer based on silicon nanowires.

This investigation completes the amendment of theoretical expressions for the characterization of antigen–antibody interactions by kinetic exclusion assay—an endeavor that has been marred by inadequate allowance for the consequences of antibody bivalence in its uptake by the affinity matrix (immobilized antigen) that is used to ascertain the fraction of free antibody sites in a solution with defined total concentrations of antigen and antibody. A simple illustration of reacted site probability considerations in action confirms that the square root of the fluorescence response ratio, RAg/Ro, needs to be taken in order to determine the fraction of unoccupied antibody sites, which is the parameter employed to describe the kinetics of antigen uptake in the mixture of antigen and antibody with defined initial composition. The approximately 2-fold underestimation of the association rate constant (ka) that emanates from the usual practice of omitting the square root factor gives rise to a corresponding overestimate of the equilibrium dissociation constant (Kd)—a situation that is also encountered in the thermodynamic characterization of antigen–antibody interactions by kinetic exclusion assay.

The envelope glycoprotein E2 is the major immunodominant protein of the classical swine fever virus and can induce neutralizing antibodies and protective host-immune responses in infected swine. We designed, expressed, and purified multi-epitope protein (GST-BT22) that contains a tandem repeat of the E2 antigenic-determinant residues 693–704, 770–780, and 826–843, each of which is separated by a GGSSGG sequence. In the same manner, we also designed, expressed, and purified a second protein (GST-BT23) that contains a C-terminal sequence consisting of residues 1446–1460 from the classical swine fever virus nonstructural protein NS2-3 separated from the GST-BT22 sequence by a GGSSGG sequence. Western blotting of GST-BT22 and GST-BT23 with serum from a swine that had been experimentally infected with the virus showed that the proteins reacted with anti-serum, whereas GST did not. Surface plasmon resonance was used to quantify the affinities of GST-BT22 and GST-BT23 for serum antibodies (Ka = 4.31 × 108 and 5.01 × 108, respectively). GST, used as a control, was reacted an order of magnitude less strongly than did GST-BT22 and GST-BT23. Surface plasmon resonance, therefore, appears to be a sensitive and precise method for epitope evaluation and can be used to characterize the immunogenicity of a recombinant protein.

Total internal reflection ellipsometry (TIRE) has been applied for the investigation of (i) kinetics of biosensing layer formation, which was based on the immobilization of fragmented and intact antibodies, and (ii) kinetics of antigen interaction with the immobilized antibodies. It has been demonstrated that ellipsometric parameter Δ(t) showed much higher sensitivity at the initial phase of Au-protein and protein-protein interaction, while the parameter Ψ(t) was more sensitive when the steady-state conditions were established. A new method, which taking into consideration this feature and nonlinear change of Δ(t) and Ψ(t) parameters during various stages of biological layer formation process, was used for the calculation of antibody and antigen adsorption/interaction kinetics. The obtained results were analyzed using a model, which took into account partial reversibility during the formation of both antibody and antigen based monolayers. It was shown that the immobilization rate of antibody during the preparation of the sensing layer was similar for the formation of both intact and fragmented antibody based layers; however, the residence time was 25 times longer for intact antibody based layer formation in comparison to that of fragmented antibody based layer formation. On the contrary, residence time of antigen interaction with immobilized antibodies was about 8 times longer for the sensor based on fragmented antibodies. Moreover, it has been determined that the structural differences of immobilized antibodies (fragmented or intact) significantly influence antibody-antigen interaction rate, the major difference being in the residence time of antigen interaction with both types of immobilized antibodies.

In order to meet the requirements of antibody comparison and evaluation, the optimization for the surface modification, ligand immobilization, unspecific blocking and ligand receptor interaction condition is introduced to the biosensor based on imaging ellipsometry (BIE), so that it may serve as an antibody screening platform to evaluate antibody antigen interactions. Two kinds of anti-ricin antibody named pVHHS1 and 5S1R are immobilized in a patterned array format on the silicon surface modified with succinic anhydride, respectively, to form a sensing surface. Ricin and its similar toxin abrin as the reference in solution are delivered, respectively, on the patterned sensing surface for anti-ricin antibody and ricin interaction. The limit of detection value of ricin detected by pVHHS1 and 5S1R is obtained as 1 mu g/ml and 5 ng/ml, respectively. Results show that pVHHS1 and 5S1R specifically bind with ricin but not with abrin, which is in accordance with ELISA's result. (C) 2010 Elsevier B.V. All rights reserved.

The antigen-antibody interaction determines the sensitivity and specificity of competitive immunoassay for hapten detection. In this paper, the specificity of a monoclonal antibody against alternariol-like compounds was evaluated through indirect competitive ELISA. The results showed that the antibody had cross-reactivity with 33 compounds with the binding affinity (expressed by IC50 ) ranging from 9.4 ng/mL to 12.0 μg/mL. All the 33 compounds contained a common moiety and similar substituents. To understand how this common moiety and substituents affected the recognition ability of the antibody, a three-dimensional quantitative structure-activity relationship (3D-QSAR) between the antibody and the 33 alternariol-like compounds was constructed using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) methods. The q2 values of the CoMFA and CoMSIA models were 0.785 and 0.782, respectively, and the r2 values were 0.911 and 0.988, respectively, indicating that the models had good predictive ability. The results of 3D-QSAR showed that the most important factor affecting antibody recognition was the hydrogen bond mainly formed by the hydroxyl group of alternariol, followed by the hydrophobic force mainly formed by the methyl group. This study provides a reference for the design of new hapten and the mechanisms for antibody recognition.

IgG molecules are exposed on a regular basis to acidic conditions during immunoaffinity purification procedures, as well as during the production of some therapeutic immunoglobulin preparations. This exposure is known to induce in them an antigen-binding polyreactivity. The molecular mechanisms and the possible biological significance of this phenomenon remain, however, poorly understood. In addition to the previously reported ability of these modified IgG antibodies to interact with a large panel of self-antigens, enhanced binding to non-self-antigens (bacterial), an increased ability to engage in F(ab')(2)/F(ab')(2) (idiotype/anti-idiotype) interactions and an increased functional antigen-binding affinity are reported here. The newly acquired 'induced polyreactivity' of low-pH buffer-exposed IgG is related to structural changes in the immunoglobulin molecules, and is at least partly attributable to the enhanced role of the hydrophobic effect in their interactions with antigen. Our results suggest that data from many previous studies on monoclonal and polyclonal IgG antibodies purified by low-pH buffer elution from protein A or protein G immunoaffinity columns should be reconsidered, as the procedure itself may have dramatically affected their antigen-binding behavior and biological activity. Low-pH buffer-treated pooled therapeutic immunoglobulins acquire novel beneficial properties, as passive immunotherapy with the pH 4.0 buffer-exposed, but not with the native therapeutic intravenous immunoglobulin preparation, improves the survival of mice with bacterial lipopolysaccharide-induced septic shock.

The identification of antigenic epitopes is important for the optimization of monoclonal antibodies (mAbs) intended as therapeutic agents. MS has proven to be a powerful tool for the study of noncovalent molecular interactions such as those involved in antibody-antigen (Ab-Ag) binding. In this work, we described a novel methodology for mapping a linear epitope based on direct mass spectrometric measurement of Ab-Ag complexes. To demonstrate the utility of our methodology, we employed two approaches, epitope excision and epitope extraction, to study a model system consisting of a Fab antibody fragment with specificity toward the peptide abeta(1-40). In epitope excision, the Fab and abeta(1-40) complex was treated with proteolytic enzymes and the digested complexes were directly monitored by MS under native conditions. Mass differences between the Fab-abeta complex and the Fab control revealed the size of epitope peptides that were bound to the Fab. Using the epitope extraction approach, abeta(1-40) was first digested by Lys-C, and the fragment containing the epitope was selected by Fab binding. Data analysis allowed mapping of the epitope to abeta(16-27) which is in good agreement with previously unpublished data. The utility of the methodology was demonstrated by elucidating the binding epitopes for two full-length anti-abeta(1-40) mAbs.

Immunization with anti-idiotypic (anti-Id) antibodies, used as surrogate antigens, has led to promising results, notably in active immunotherapy of cancers, essentially because it breaks immunological tolerance against self-tumor-associated antigens. The aim of the present study was to provide a proof-of-principle that this vaccination approach could be envisaged also in the field of prion diseases, caused by the accumulation of an aggregated pathological isoform of the highly tolerogenic self-prion protein (PrP), and for which no therapy is available. We investigated the possibility of raising anti-Id antibodies mimicking the human PrP (hPrP), using as immunogens either a peptide derived from the paratope of an anti-PrP mAb or the entire antibody. To this end, we cloned and sequenced SAF61 mAb, an anti-PrP antibody already produced in the laboratory, directed against a critical epitope of PrP involved in the aggregation process. A synthetic peptide (denoted CDR3L) was designed from the identification of a 17-amino-acid sequence encompassing the CDR3 region of the light chain whose hydropathic profile was opposed to that of PrP epitope. CDR3L peptide was directly demonstrated to bind hPrP, confirming the role of hydropathic complementarity in antigen-antibody interactions. When injected into rabbits, CDR3L generated anti-SAF61 anti-Id polyclonal antibodies that exclusively recognized SAF61 mAb but were unable to compete with hPrP for antibody binding. By contrast, immunizations with the entire SAF61 mAb generated anti-Id antibodies specifically competing with soluble or membrane-bound hPrP (in EIA or flow cytometry experiments, respectively) for binding not only SAF61 mAb but also other anti-PrP mAbs directed against similar epitopes, i.e. behaving as "internal images" of this disease-related PrP epitope. These results could open the way to raising PrP-like mAbs, which might serve as surrogate antigens in a new active immunotherapeutic approach to prion diseases.

Using spectroscopic ellipsometry as a surface plasmon resonance (SPR) detector, we studied the antibody-antigen interaction of the Her-2/neu protein from SKBR3 breast cancer cells. Chips were prepared by immobilizing the HER2 antibody on a linker-coated Au surface and the SPR was measured after dipping the chip in SKBR3 bu er and drying. The shifts of the SPR peak at each step of the chip preparation and the corresponding morphology change in the atomic force microscopy images suggested that this method could be useful for the diagnosis of cancerous cells in vitro.

The hepatitis B virus capsid (core antigen) is able to bind to and activate naïve B cells and these become efficient primary antigen-presenting cells for the priming of T cells. We have investigated this interaction by using cryo-electron microscopy, three-dimensional image reconstruction, and molecular modeling to visualize capsids decorated with Fab fragments of a receptor immunoglobulin, and surface plasmon resonance to measure the binding affinity. By both criteria, the mode of binding differs from those of the six monoclonal anti-core antigen antibodies previously characterized. The Fab interacts with two sites approximately 30 A apart. One interaction is canonical, whereby the CDR loops engage the tip of one of the 25 A spikes that protrude from the capsid surface. The second interaction is non-canonical; in it, the Fab framework contacts the tip of an adjacent spike. The binding affinity of this Fab for capsids, K(D) approximately 4 x 10(-7) M, is relatively low for an antibody-antigen interaction, but is approximately 150-fold lower still ( approximately 2.5 x 10(-5) M) for unassembled capsid protein dimers. The latter observation indicates that both of the observed interactions are required to achieve stable binding of capsids by this receptor immunoglobulin. Considerations of conserved sequence motifs in other such molecules suggest that other naïve B cells may interact with HBV capsids in much the same way.

A problem of similarity and differences between so-called polyreactive immunoglobulins (PRIGs) and natural antibodies (NAbs), capable of cross-reacting with some structurally dissimilar antigens, has been considered. The analysis of mechanisms of an unspecific interaction between PRIGs or NAbs and antigens evidences for the fact that essential differences exist between these substances. These differences permit classifying the abovementioned substances as different types of immunoglobulin molecules. The major difference between PRIGs and NAbs may include both the mechanisms of the above mentioned immunoglobulin molecules binding to antigens and their interaction affinity, as well as an absolutely different influence of some low-molecular substances on the efficiency of the interaction with antigens. Relying on the obtained data it can be assumed that, since PRIGs and NAbs have fundamental differences, they may perform not only similar but also different functions of the immune system.

Anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies bearing λ1 chains are known to possess fine specificity, referred to as heterocliticity, which causes these antibodies to bind to hapten analogues such as (4-hydroxy-3-iodo-5-nitrophenyl)acetyl (NIP) and (4-hydroxy-3,5-dinitrophenyl)acetyl (NNP) with higher affinity than to the autologous hapten, NP. They also show preferential binding to the phenolate form of hapten than to the phenolic form. We address here the question of whether affinity maturation accompanies in the fine specificity of these antibodies by analyzing the interaction between NP1-, NIP1-, or NNP1-hen egg lysozyme and anti-NP antibodies that possess different association constants to NP using a surface plasmon resonance biosensor. We measured interactions at various pH values and found that heterocliticity as well as preferential binding to the phenolate form of hapten were most prominent in a germline antibody having immature affinity and that fine specificity becomes less evident, i.e., anti-NP antibodies become more specific to the immunizing antigen, NP during the process of affinity maturation.

We prepared IgG and IgM with identical combining sites to a hapten, (4-hydroxy-3-nitrophenyl)acetic acid (NP), and used surface plasmon resonance to evaluate the association constants (Ka) in interactions of these antibodies (Abs) with antigens (Ags) which differed in the size of carriers and NP valence as well as in the stoichiometry of Ag to Ab in the immune complexes. It was found that IgM was unable to form an Ag1Ab1 complex with the highly haptenated Ag, NP(18.6)-bovine serum albumin (BSA), such that one NP(18.6)-BSA molecule was held by multiple contacts with Fab arms from five subunits, although IgM was capable of forming an Ag4Ab1 complex in which each subunit was bound to one NP(18.6)-BSA molecule. IgM was superior to IgG in interactions with large Ags of low hapten density. The Ka values of IgM to these Ags were estimated to be approximately 1x10(9) M(-1), about 20-fold higher than those of IgG. Reduction of inter-subunit and inter-chain disulfide bonds resulted in a decrease in Ka values to large Ags but no change in those to small Ags.

New approaches for the measurement of antibody affinity by ELISA are suggested and considered theoretically. It was shown that not only more precise and more convenient in comparison to that suggested earlier, but also more informative graphical representation of the experimental data in the appropriate coordinate could be used for evaluation of antibody affinity. The following cases were considered: (i) determination of antibody affinity for one kind of univalent antibodies, (ii) determination of antibody affinity for one kind of bivalent antibodies, (iii) determination of antibody affinity for two kinds of univalent antibodies, which are in a mixture, and (iv) determination of antibody affinity for two kinds of bivalent antibodies, which are in a mixture. Advantages and disadvantages of the different approaches are discussed.

We determined thermodynamic and kinetic parameters for the antigen-antibody interaction using a group of anti-(4-hydroxy-3-nitrophenyl)acetyl monoclonal antibodies whose differences in amino acid sequences had arisen only from somatic hypermutation. These monoclonal antibodies were considered to have originated from a common ancestor clone and to represent progression along the affinity maturation pathway. The kinetic measurements showed that both association and dissociation rate constants of the antigen-antibody interaction decreased during maturation. Thermodynamic measurements revealed that an increase in affinity was obtained by an increase in entropy without any significant change in enthalpy. These results suggested that the mechanism for the antigen-antibody interaction shifted from a "zipper" type to a "lock-and-key" type during antibody evolution.

In the present study the atomic force microscope (AFM) was used to image the surface morphology of red blood cells (RBC) for the first time. The AFM yielded very reproducible images without appreciable modifications of the sample surfaces. In addition to this topographical imaging, we have developed an experimental approach to measure the binding strength between antibody (anti-A), and the RBC antigen A, when reversible bonds between specific molecules such as antigen and antibody mediate the adhesion. The experimental results suggest that the procedure established here may be used for specific antibody detection. This study has also enhanced our understanding under physiological conditions of molecular interaction in particular antigen–antibody.

Human adenoviruses (HAdVs) have been implicated in a wide range of diseases affecting primarily the respiratory, ocular and gastrointestinal systems. A rapid and efficient method for the detection of HAdV hexon antigen is described using carbon nanotube (CNT) sensors. Anti-HAdV antibody was immobilised on the reverse surface of a CNT sensor. As a control, non-specific mouse IgG was immobilised on another CNT sensor. I-V(gate) curves were measured after incubation of various concentrations of recombinant HAdVs hexon antigen with anti-HAdVs antibody-immobilised or non-specific mouse IgG-immobilised sensors. The curves showed a positive shift that was dependent on the hexon antigen concentration in the anti-HAdV antibody-immobilised sensor, whereas no such shift was observed in the non-specific mouse IgG-immobilised sensor. The sensitivity of the CNT sensor method was greater than that of enzyme-linked immunosorbent assay. Hence, this method offers a new tool for HAdV detection by analysing antigen-antibody interactions.

The influence of twin 20, lysozyme and protamine on the capability of polyreactive immunoglobu­lins (PRIG) to attach to various antigens was investigated. Twin 20 can inhibit the binding of PRIG to antigens on immunological plates but lysozyme and protamine can enhance it. As far as the mixture of the optimal concentrations of lysozyme and protamine cannot increase PRIG-antigen interaction in comparison to the optimal dose of protamine, we have concluded that the mechanism of their effect on PRIG binding is similar. Of special interest­ is the fact that twin 20 at optimal concentration of lysozyme or protamine does not decrease PRIG binding to various antigens but, on the contrary, increases PRIG-antigen interaction.

The purpose of this experiment is to illustrate how antigen-antibody interactions can be applied to the study of evolution. Antigens are substances that stimulate an immune response from the body. There are two general categories of antigens: pathogens and allergens (Karp 1996). Pathogens include viruses, bacteria, fungi, protozoan parasites and toxins. Allergens include pollen, spores, and food additives. Antibodies are globular proteins produced by B lymphocytes that bind to an antigen and target the antigen for destruction. Antibodies are located in interstitial fluid and plasma and are mostly effective against pathogens that have not invaded host cells. The Ouchterlony test is a double diffusion method on an agar plate that clearly shows any reaction between antigens and antibody (Ouchterlony & Nilsson 1973). Antigens and antibody are inoculated onto an agar plate into separate wells where they diffuse through the medium. As long as the pore sizes within the agar gel substance are large enough, an assumption is made that diffusion of randomly moving particles can freely occur. The agar must be an inert material that does not react with the testing materials. If the antibody recognizes and binds to an antigen on the plate, a white precipitate will form. The precipitate itself is too large to continue to diffuse-once a precipitate is formed, it is stationary. The antigens for this experiment are albumins from various ungulates: horses, pigs, cows and sheep. Albumins are small globular proteins isolated from blood. Albumin is produced by the liver and participates in homeostasis by influencing the osmotic balance of plasma. Antiserum is a liquid containing antibodies that have been exposed to a specific antigen. For example, antihorse serum includes antibodies that will react against antigens isolated from a horse. Antisera were purchased from Sigma, a chemical supply company (PO Box 14508, St. Louis, MO 63178, USA; 800-325-3010). Students learn in lecture and lab that an outstanding characteristic of antibodies is their property of specificity. The general hypothesis being tested is: a specific antigen will react with a specific antibody that has been formed against it, and form a precipitate. Therefore, horse albumin should react only with anti-horse serum, pig albumin with anti-pig, and so on. The results can be used to support the current classification of the ungulates at the order and suborder level. To accomplish this, students must become informed about the following subjects

The third variable loop (V3) of gp120 is a promising AIDS vaccine target because it mediates HIV-1’s contact with coreceptors CCR4 or CXCR5 (1, 2). It is very immunogenic and readily accessible to antibodies (3). Passive immunization with anti-V3 antibodies also shows protection in nonhuman primate studies (4, 5). V3 is generally 35 amino acids in length, beginning with disulfide bond between Cys at position 296 (Cys296) and Cys331 (HXB2 numbering (6)). It can be divided into three structural regions: the disulfide linkage at the base in the gp120 core, the distal crown region of about 13 amino acids which projects ~20 A from the core, and the flexible stem region between the base and the crown (7, 8). The epitopes of most known human anti-V3 mAbs have been mapped to the crown region of V3. Many of these mAbs have been carefully characterized by functional and structural methods. Recent structural studies have demonstrated that although the crown often forms a β-hairpin structure, it can be further divided into three distinct subregions: the band, the circlet, and the arch (8). The arch at the center of the V3 sequence contains the highly conserved 4-residue motif of gp120 residues 312–315, often composed of the sequence GPGR for clade B or GPGQ for nonclade B viruses. The circlet at the middle of the crown has a hydrophobic face and a hydrophilic face; the hydrophobic face contains two highly conserved isoleucine residues (Ile307 and Ile309). The band consists of the often positively charged residues 304 and 305 at the N-terminus and the highly conserved Tyr318 at the C-terminus. Among these subregions of V3 are four conserved structural elements: the arch, the band, the hydrophobic face of the circlet, and the peptide backbone. Anti-V3 antibodies that target these conserved structural elements are broadly reactive (8). Structural studies have also revealed that there are two general modes of antigen recognition for the human V3 mAbs: the ladle mode and the cradle mode. The ladle mode, typified by mAb 447–52D, is one where the antigen binding site is shaped like a soup ladle (9). The bowl of the ladle binds the arch of the V3 crown while the handle, formed by a long CDRH3 in the case of 447–52D, interacts with the N-terminal half of the V3 crown by main-chain interactions. Conversely, the cradle-binding mode, typified by mAbs 2219, is one where the antigen binding site is shaped like a cradle and the V3 crown sinks into the cradle, often burying the hydrophobic face of the circlet (8). Although mAb 447–52D (IgG3, λ) has somatic mutation frequencies of only 5.4% and 2.5% for VH and VL, respectively (10), it harbors a sophisticated antigen binding site. 447–52D has a long CDRH3 of 20 amino acids (Kabat definition) that forms a β-hairpin structure standing tall at one side of the antigen binding site (the handle of the ladle). The antigen binding side β strand of the CDRH3, decorated with 5 consecutive tyrosines (residues 100G–100K), makes main chain interactions with the N-terminal strand of the V3 crown. There is a shallow pocket at the base of CDRH3 that can be considered as the bowl of the ladle. On one side of the bowl, two tryptophans, residue 91 of the light chain (TrpL91) and TrpL96, form a wall that packs against the GPG turn of the V3 arch. On the other side of the bowl, TyrH100J and TrpH33 of the heavy chain form perfect π–cation stacking geometry that sandwiches the guanidinium group of Arg315 of V3. In addition, the side chain of Arg315 of V3 forms a salt bridge with the side chain of AspH95. This highly specific binding of the Arg315 side chain with a π–cation stacking and a salt bridge has led to speculation that Arg315 is the signature motif of the epitope of 447–52D (11). This might be the cause of 447–52D’s preference for neutralizing clade B viruses over nonclade B viruses (12). Although investigated by us and other groups, analysis of the structure-function relationship of the antigen binding site of 447–52D still presents unanswered questions. For example, although the crystal structures of the antigen binding fragments (Fabs) of 447–52D in complex with V3 peptides of clade B and nonclade B sequences have been obtained (9, 10), the role of the C-terminal region of V3 crown hairpin in the antigen antibody interaction was not clear because the residues after 316 had poor electron densities. The C-terminus was later extended to residue 319 in another crystal structure (13), but the diffraction data set was epitaxially twinned and the structure was not sufficiently refined. There are also some inaccuracies of the placement of key residue Arg315 of the V3 loop and water molecules within the antigen binding site (9, 13). Most importantly, the contribution to antigen binding of the individual residues cannot be determined from the structure alone because residues at the antigen–antibody interface do not contribute equally to the affinity and neutralization function of the antibody. Instead, studies have indicated that the residue’s contribution to the antibody function is not uniformly distributed along the binding interface; rather, the majority of the contribution is often localized to a subset of residues (14–16). To study the contribution of individual residues, a structure-guided computational approach has been used (17). However, it cannot accurately predict the affinity (kD) and Gibbs free binding energy (ΔG) from atomic structures. Purely functional studies are also limited in that they lack the precision at the single residue level (12, 18). Therefore, the best approach is to interrogate the contribution of individual residues at the molecular level by sensitive binding assays guided by computational and structural studies. SPR (19), ELISA (20), and FRET(21) assays have also been used to measure affinity with high sensitivity, but these techniques cannot determine a complete thermodynamic signature. Isothermal titration calorimetry (ITC) is a unique technique that can not only provide the accurate measurement of the binding affinities but also sensitively quantitate the thermodynamic signatures of individual residues at a specific temperature (22, 23). Here we present a detailed interrogation of the antigen binding site of 447–52D using a combination of mutagenesis, neutralization assays using pseudoviruses, and determination of affinity by ITC.

For elucidation of the role of salt bridge formation in the antigen-antibody complex, the interaction between hen egg white lysozyme (HEL) and its monoclonal antibody HyHEL10, the structure of which has been well characterized and forms one salt bridge (Lys97 of HEL and Asp32 of HyHEL10 heavy chain variable region (VH)), was investigated. Asp32 of VH was substituted with Ala, Asn, or Glu by site-directed mutagenesis, and the interaction between HEL and the mutant fragments of the variable region of light chain was investigated by inhibition of the enzymatic activity of HEL and isothermal titration calorimetry. Inhibition assay indicated that these mutations lowered the inhibition only slightly. Thermodynamic study indicated that the negative enthalpic change in the interaction between each of the mutant variable regions of light chain and HEL was significantly increased, although the association constant was slightly decreased, suggesting that these mutations increased the entropy change upon antigen-antibody binding. These results indicate that the role of salt bridge formation in the HyHEL10-HEL interaction is to lower the entropic loss due to binding. In the mutant proteins, the numbers of residues that were perturbed structurally on binding increased, suggesting that the salt bridge suppresses excess structural movement of the antibody upon binding.

Gel filtration on HPLC columns can be a convenient means for the separation of soluble immune complexes from the mixture of human serum albumin (HSA)—monoclonal serum albumin antibody (Mab anti HSA). Owing to the rapidity of the method, the equilibrium is not modified, as it may be during the salting-out separation. This method has been applied to the study of the interaction between HSA and a monoclonal antibody against human immunodeficiency virus envelope protein (Mab anti HIV gp 120). Supported by the fact that HIV proteins share numerous epitopes with human proteins, a treatment to improve the specific immune response against this pathogen is proposed: it is based on the blocking of the epitopes of the self antigens, expressed in the thymus, which are in common with the virus. Repeated injections within the thymus, of neutralizing antibodies against the pathogen, obtained from a sufficiently distant animal species and purified by affinity chromatography, would prevent the T cells of the host...

To understand the mechanism of affinity maturation, we examined the antigen-antibody interactions between 4-hydroxy-3-nitrophenylacetyl (NP) caproic acid and the Fab fragments of three anti-NP antibodies, N1G9, 3B44, and 3B62, by isothermal titration calorimetry. The analyses have revealed that all of these interactions are mainly driven by negative changes in enthalpy. The enthalpy changes decreased linearly with temperature in the range of 25-45°C, producing negative changes in heat capacity. On the basis of the dependence of binding constants on the sodium chloride concentration, we have shown that, during the affinity maturation of the anti-NP antibody, the electrostatic effect does not significantly contribute to the increase in the binding affinity. We have found that, as the logarithm of the binding constants increases during the affinity maturation of the anti-NP antibody, the magnitudes of the corresponding enthalpy, heat capacity, and unitary entropy changes increase almost linearly. On the basis of this correlation, we have concluded that, during the affinity maturation of the anti-NP antibody, a better surface complementarity is attained in the specific complex in order to obtain a higher binding affinity.

Background The immunogenic third variable region (V3) of HIV-1 gp120 is a target for AIDS vaccines. V3 is recognized by mAb 447-52D, known for its ability to neutralize viruses with a GPGR beta turn motif at the apex of V3, which is characteristic of clade B viruses. Interestingly, 447-52D can also bind non-clade B V3 peptides containing a GPGQ motif. A detailed biochemical and biophysical dissection of the antigen-antibody interaction of 447-52D was undertaken to understand this disparity.

A simple and reliable method based upon law of mass action for calculating affinity of a monoclonal antibody using non-competitive ELISA, is described. In this method, the binding of an antibody (Ab) with an antigen (Ag) is measured by ELISA using serial dilutions of both antigen (coated on the plate) as well as antibody. When the OD measured after the antigen antibody interaction was plotted against the concentration of Ab, added to the wells, a hyperbolic curve was obtained. The OD, at any point of the curve, was considered as a direct reflection of the amount of antibody bound to the antigen. The OD-100 denotes the occupancy of maximum no. of epitopes available on the antigen molecules, accessible to the antigen. The concentration of antibody (Ab, Ab') at corresponding levels of antigen concentration (Ag, Ag'), presents the value obtained at OD-50. The [Ag] and [Ag'] are not the true antigen concentrations but are the measurement of antigen density on the plate. The affinity constant K(aff) was calculated by using the formula K(aff) = (n - 1)/2(n[Ab'] - [Ab]), derived from law of mass action, where n = [Ag]/[Ag']. A computer program to calculate the affinity of antibody to the antigen using method described in this manuscript has been developed and discussed.

An indirect competitive enzyme-linked immunosorbent assay (icELISA) for 12 phenylurea herbicides (PUHs) was established with the half-maximum inhibition concentration (IC(50)) of 1.7-920.7 μg L(-1). A method of computer-aided molecular modeling was established in quantitative structure-activity relationship (QSAR) studies to obtain a deeper insight into the PUHs' antibody interactions on how and which molecular properties of the analytes quantitatively affect the antibody recognition. A two-dimensional (2D)-QSAR model based on the Hansch equation and a hologram QSAR (HQSAR) model were constructed, and both showed highly predictive abilities with cross-validation q(2) values of 0.820 and 0.752, respectively. It was revealed that the most important impact factor of the antibody recognition was the PUHs' hydrophobicity (log P), which provided a quadratic correlation to the antibody recognition. Hapten-carrier linking groups were less exposed to antibodies during immunization; thus, groups of the analytes in the same position were generally considered to be less contributive to antibody recognition during immunoassay. But the results of substructure-level analysis showed that these groups played an important role in the antigen-antibody interaction. In addition, the frontier-orbital energy parameter E(LUMO) was also demonstrated as a related determinant for this reaction. In short, the result demonstrated that the hydrophobicity and the lowest unoccupied molecular orbital energy (E(LUMO)) of PUH molecules were mainly responsible for antibody recognition.

Selective separation of chymotrypsinogen using anti-chymotrypsinogen-antibodies as affinity ligands was realized in reverse micellar system composed of a nonionic surfactant tetra-oxyethylenemonodecylether. Antibodies as affinity ligands were immobilized in reverse micelles by combining cholesteryl groups covalently. Selective separation of proteins using bioaffinity ligands was extended to antigen-antibody reaction system, which enables us to choose any kind of target proteins.

To evaluate the conformation of reduced HEL, the monoclonal antibodies HyC1 and HyC2, which recognize different conformational epitopes on native hen egg lysozyme (HEL), were used, and the kinetics of their interactions with native HEL, S-1,2-dicarboxyethylated HEL (DCE-HEL), and carboxymethylated Cys6 and Cys127 HEL (CM(6,127)-HEL) were assessed using surface plasmon resonance. Although their association rate constants differed 10(5)-fold, their dissociation rate constants were essentially the same, suggesting that DCE-HEL and CM(6,127)-HEL possess conformations similar to that of native HEL when they bind antibodies. We considered that the ratio of the association rate constant of reduced HEL to native HEL represents the proportion of the native format determinant in equilibrium. Reduction of the Cys6-Cys127 disulfide bond would transform the epitope recognized by HyC1 into a non-native conformation similar to that of DCE-HEL. We show that monoclonal antibodies provide a sensitive tool for evaluation of the structural and hydrodynamic changes of proteins.

This chapter examines the aspects of how the interaction between antigen and antibody can be exploited in research and clinical laboratories. The specificity and sensitivity of antibodies make them useful biochemical tools. Because immunology evolved from the sciences of biochemistry, genetics, histology, and pathology, many of the techniques used to characterize features of the immune system are derived from the methodologies of these fields. General methodologies appropriate for examining cellular, enzymatic, and genetic components are frequently used. However, immunologists can also take advantage of a unique feature of their science, the antigen–antibody bond, which has the specificity of an enzymatic reaction without its sometimes inconvenient permanent alteration of the bound substrate. The development of hybridoma technology and monoclonal antibodies has resulted in an explosion of technical applications. Antibodies are used in two main categories of assays—those based on immune complex formation, and those based on unitary antigen–antibody pair formation. Direct tag assays involve the antigen and the primary antibody, one of which is tagged. Indirect tag assays involve the antigen, the primary antibody, and a third component that is labeled and used to detect the antigen–antibody pair. This chapter closes with a discussion on antibodies as effectors and antibodies as laboratory tools.

Serology is the study of the in vitro reaction of antibodies in blood serum with antigens, i.e., usually those of microorganisms inducing infectious disease. Precipitation, agglutination, and complement fixation are serological methods used in diagnosis and research.

Two antibody preparations against cardiotoxins were raised by immunizing rabbits with cardiotoxin 1 and cardiotoxin 3, respectively. The two antibody preparations showed precipitin reactions with cardiotoxins 1, 2, 3 and 5, respectively. However, the results of competitive enzyme-linked immunoassay revealed that the respective cardiotoxin molecules exhibited different reactivity toward anticardiotoxin antibodies. Moreover, the order of reactivity with antibodies was not in line with the degree of their sequence identity. This suggest that the anticardiotoxin antibodies may recognize conformational epitopes rather than sequential ones in the toxin molecules. Alternatively, the four cardiotoxins reacted well with the antibodies in the absence of competitor, suggesting that sequence variations with cardiotoxin molecules may not exclusively influence the potential use of the anticardiotoxin antibodies for the neutralization of the activity of cardiotoxin variants.

The dissociation constant (Kd) of a monoclonal antibody with fluorescein isothiocyanate (FITC)-labeled insulin and unlabeled insulins from several species were measured using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Kd determinations were made by separating free FITC-insulin and its complex with the antibody in equilibrated solutions in 6 s or less. The use of LIF detection allowed quantification of free and bound FITC-insulin in the picomolar range, as is required to measure Kd's below 1 nM. The Kd of FITC-insulin with the antibody was determined to be 0.25 nM by Scatchard analysis. The Kd's of the antibody with unlabeled insulins from several species were obtained by fitting bound over free FITC-insulin as a function of unlabeled insulin concentration data from a series of solutions containing a fixed concentration of FITC-insulin and antibody and variable concentrations of insulin to the expected curve derived from the equilibria and mass balance of the solutions. Kd's for the different insulins were between 0.34 and 0.64 nM.

Effects of mutations on antigen-antibody interactions were energetically examined using several kinds of Fv fragments, derivatives of a monoclonal antibody, D1.3, that is specific for hen egg-white lysozyme. According to the transition-state theory, the thermodynamic parameters involved in pre and post-processes of formation of the activated complex were estimated separately. In the pre-process, there is a strong compensation between changes in entropy and changes in enthalpy caused by substitutions of amino acid residues. Thus, the differences in the association constant (KA) caused by mutations are not related to the pre-process but to the post-process.

Plasma and IgG obtained from 10 Lambert-Eaton myasthenic syndrome (LES) patients (5 with carcinoma, 5 without associated cancer), 6 healthy subjects, and 1 patient with small-cell lung cancer (SCLC) were examined in their ability to recognize chromaffin cell antigens on Western blots. The pattern of antigen recognition was compared with the magnitude of inhibition of voltage-dependent calcium and sodium currents recorded with the patch-clamp technique from chromaffin cells. Eight of the 11 patients with LES and/or SCLC recognized plasma membrane proteins and 9 of the patients' IgG interacted with cytoplasmic antigens with no apparent pattern of antigen recognition between patients. Also, there was no obvious band pattern distinguishing patients with LES from those with LES and concurrent SCLC. Eighty percent of the LES patients' antibodies were capable of reducing the calcium current (ICa) in chromaffin cells. One of the novel findings of this study is that 30% of the patients had produced antibodies which were able to inhibit both calcium and sodium currents (INa). The heterogeneous response of the IgG on the Western blots does not appear to correlate with the efficacy of reducing the inward currents.

The effect of serum. methanol/water, and detergents on the dissociation of the antigens thyroxin (T4) and triiodothyronine (T3) from immobilized antibody was investigated by an antigen binding technique and scanning electron microscopy. The antibody coatings immobilized on the different polymeric substrates showed varying degrees of denaturation and desorption due to detergents but were unaffected by a 60 40 (by volume) mixture of methanol/water after various washington procedures. The factors influencing the dissociation and its relevance to recycling immunoassay are discussed. It was found that nonspecific binding of serum proteins to the antibody coatings wa a major caused of deterioration in antibody binding capacity.

Specific penicillin-carrier receptor proteins (CRP) have been isolated from the sera of penicillin allergic rabbits and human subjects in the unconjugated native state in electrophoretically homogeneous form by employing a synthetic polymeric affinity template containing the 7-deoxy analogue of penicillin G. The synthesis of the 7-deoxy analogue has been described. In this affinity system the antipenicillin-antibody is desorbed by 0·9M thiourea and the CRP in 8M urea. The CRP after incubation with penicillin is converted into the full-fledged antigen. Studies on the origin of CRP and the nature of antibody as well as comparative studies on the properties of the rabbit antibody and those of antibodies elicited by a BSA-BPO conjugate are reported.

The availability of an electrophoretically homogeneous rabbit penicillin carrier receptor protein (CRP) and rabbit antipenicillin antibody afforded an idealin vitro system to calculate the thermodynamic parameters of the binding of14C benzyl penicillin CRP conjugate (antigen) to the purified rabbit antipenicillin antibody. The thermodynamic parameters of this antigen-antibody reaction has been studied by radio-active assay method by using millipore filter. Equilibrium constant (K) of this reaction has been found to be 2·853×109M−2 and corresponding free energy (ΔG) at 4°C and 37°C has been calculated to be −12·02 and −13·5 kcal/mole, enthalpy (ΔH) and entropy (ΔS) has been found to be 361 kcal/mole and +30 eu/mole respectively. Competitive binding studies of CRP-analogue conjugates with the divalent rabbit antibody has been carried out in the presence of14C-penicilloyl CRP. It was found that 7-deoxy penicillin-CRP complex and 6-amino penicilloyl CRP conjugate binds to the antibody with energies stronger than that with the14C-penicilloyl CRP. All the other analogue conjugates are much weaker in interfering with the binding of the penicilloyl CRP with the antibody. The conjugate of methicillin,o-nitro benzyl penicillin and ticarcillin with CRP do not materially interfere in the process.

Binding of Epstein-Barr-virus-determined nuclear antigen (EBNA) to acid-fixed nuclei could be prevented by exposing EBNA to anti-EBNA antibodies. While EBNA is eluted from acid-fixed nuclei by 0.4 M NaCl, it was not eluted even by 1.5 M NaCl if EBNA bound to acid-fixed nuclei had been exposed to anti-EBNA antibody. In this case the reaction was abolished, and EBNA was at least partially eluted by 0.2 M glycine buffer, pH 2.8, or by 3 M NaSCN. EBNA not exposed to antibody was completely eluted from acid-fixed nuclei by treatment with a solution containing 200 microgram of DNA per 1 ml of isotonic buffer, pH 6.5; however, after treatment with antibody, EBNA was not eluted by DNA.