Evaluation of Binding Kinetics and Thermodynamics of Antibody-Antigen Interactions and Interactions Involving Complement Proteins.

The study of kinetics and thermodynamics of protein-protein interactions can contribute to assessment of the mechanism of molecular recognition process. These analyses can provide information about conformational changes and noncovalent forces that influence the initial recognition between proteins and stabilization of the complex. Studying these aspects may lead to a better comprehension of functions of proteins in biological environment and can become useful for the rational modification of some interactions by engineering of one of the implicated partners. Real-time biosensor assays based on surface plasmon resonance have been widely applied for the label-free evaluation of protein-protein interactions, allowing their characterization in term of binding affinity and kinetics. In the present chapter, we provide a protocol for the assessment of interactions involving complement proteins or antibodies, the protagonists of the immune system. We reported guidelines and indications concerning the analysis of the experimental data for the estimation of the kinetic parameters and for the evaluation of activation and equilibrium binding thermodynamics.

Immunology of carbohydrate-based vaccines.

Carbohydrates are considered as promising targets for vaccine development against infectious diseases where cell surface glycan's on many infectious agents are attributed to playing an important role in pathogenesis. Understanding the relationship between carbohydrates and immune components at a molecular level is crucial for the development of well-defined vaccines. Recently, carbohydrate immunology research has been accelerated by the development of new technologies that contribute to the design of optimum antigens, synthesis of antigens and the studies of antigen-antibody interactions, and as a result, several promising carbohydrate-based vaccine candidates have been prepared in recent years. This article briefly presents the mechanistic pathways of polysaccharide, glycoconjugate, glycolipid and zwitterionic vaccines and the interplay between carbohydrate antigen and immune response.

Long term kinetic measurements revealing precision and general performance of surface plasmon resonance biosensors.

This work presents an extensive parameter list that facilitates a survey of biosensor performance using Biacore instruments for kinetic binding studies. Six long term measurements were performed using a strongly interacting antigen-antibody (ß2 microglobulin) system. Both Single Cycle Kinetic (SCK) and Multi Cycle Kinetic (MCK) were executed each with five different analyte concentrations. The overall comparison of the long term monitored parameters, like the dissociation constant (KD with approximately 3-6% relative percental standard deviation), the association and dissociation rate constants (ka, kd), the analyte binding capacity (Rmax), chi2 and the sum of the absolute values of the residuals, revealed the delicate factors that make the system performance vulnerable. The main influential factors on kinetic performance were the regeneration conditions, the quality of the sensor surface, the usage time and alteration of the sensor surface, the dilution series and the number of run cycles (about 250-600 per chip). Moreover the direct comparison of MCK and SCK uncovered distinct differences in the accuracy of the KD values. The study of sensor chips from two manufacturers showed distinct differences in the precision of the data. Using control charts for the surveillance of these parameters contributes to an overall better system performance.

Extending the throughput of Biacore 4000 biosensor to accelerate kinetic analysis of antibody-antigen interaction.

The surface plasmon resonance (SPR) biosensors are being routinely used in different stages of drug discovery and development. However, the lack of high throughput SPR biosensors continues to be a primary bottleneck for the rapid kinetic screening of large panels of monoclonal antibodies (mAbs). To further increase the throughput of the Biacore 4000 biosensor, we have developed three kinetic screening assays to characterize mAb-antigen interactions - (i) 16-mAb capture kinetic, (ii) single cycle kinetic (SCK), and (iii) parallel kinetic (PK). The performance of all three kinetic assays was evaluated by characterizing the binding of kinetically diverse human mAbs to four antigens with molecular weights of 14kD, 29kD, 38kD, and 48kD and binding affinities ranging from 130pM to 200 nM. The binding rate constants measured using all three kinetic assays were reproducible across multiple experiments and correlated with the values generated using the conventional 8-mAb capture kinetic assay on the Biacore 4000 (R2 > 0.94). Moreover, the 16-mAb capture assay decreased experiment time and analyte consumption by 35% and 50%, respectively. This work illustrates the significance of the 16-mAb capture kinetic, SCK, and PK assays to increase the throughput of Biacore 4000 and to support rapid kinetic screening of mAbs.

Exploring sensitivity & throughput of a parallel flow SPRi biosensor for characterization of antibody-antigen interaction.

Rapid growth in the field of biotherapeutics has led to an increased demand for high-throughput, label-free biosensors exhibiting high sensitivity. To support the current needs, Sierra Sensors introduced a surface plasmon resonance imaging (SPRi) based biosensor, Molecular Affinity Screening System (MASS-1). We assessed the potential utility of MASS-1 to support Regeneron's therapeutic antibody discovery. A large panel of antibody-antigen interactions was characterized using MASS-1 and the kinetic data were compared with the Biacore 4000 biosensor. Less than 10% deviation in the binding rate constants measured across eight flow channels of MASS-1 was observed. The single injection cycle kinetic assay allowed rapid measurement of binding rate constants for antibody-antigen interactions. MASS-1 sensitivity was independent of protein immobilization level and kinetic analysis performed using ultra-low density mAb surfaces allowed characterization of picomolar affinity interactions without mass transport limitation. High-throughput characterization of a panel of 189 monoclonal antibodies to 13 different antigens with molecular weights ranging from 14kD to 105kD revealed that binding kinetic parameters measured on MASS-1 were comparable to those measured on Biacore 4000. Our data demonstrate that MASS-1 measures reliable binding kinetic parameters and has an appropriate combination of throughput and sensitivity to support discovery and development of therapeutic antibodies.

A Rough Energy Landscape to Describe Surface-Linked Antibody and Antigen Bond Formation.

Antibodies and B cell receptors often bind their antigen at cell-cell interface while both molecular species are surface-bound, which impacts bond kinetics and function. Despite the description of complex energy landscapes for dissociation kinetics which may also result in significantly different association kinetics, surface-bound molecule (2D) association kinetics usually remain described by an on-rate due to crossing of a single free energy barrier, and few experimental works have measured association kinetics under conditions implying force and two-dimensional relative ligand-receptor motion. We use a new laminar flow chamber to measure 2D bond formation with systematic variation of the distribution of encounter durations between antigen and antibody, in a range from 0.1 to 10 ms. Under physiologically relevant forces, 2D association is 100-fold slower than 3D association as studied by surface plasmon resonance assays. Supported by brownian dynamics simulations, our results show that a minimal encounter duration is required for 2D association; an energy landscape featuring a rough initial part might be a reasonable way of accounting for this. By systematically varying the temperature of our experiments, we evaluate roughness at 2kBT, in the range of previously proposed rough parts of landscapes models during dissociation.

Protein Cross-Linking Capillary Electrophoresis for Protein-Protein Interaction Analysis.

Capillary electrophoresis (CE) has been identified as a useful platform for detecting, quantifying, and screening for modulators of protein-protein interactions (PPIs). In this method, one protein binding partner is labeled with a fluorophore, the protein binding partners are mixed, and then, the complex is separated from free protein to allow direct determination of bound to free ratios. Although it possesses many advantages for PPI studies, the method is limited by the need to have separation conditions that both prevent protein adsorption to capillary and maintain protein interactions during the separation. In this work, we use protein cross-linking capillary electrophoresis (PXCE) to overcome this limitation. In PXCE, the proteins are cross-linked under binding conditions and then separated. This approach eliminates the need to maintain noncovalent interactions during electrophoresis and facilitates method development. We report PXCE methods for an antibody-antigen interaction and heterodimer and homodimer heat shock protein complexes. Complexes are cross-linked by short treatments with formaldehyde after reaching binding equilibrium. Cross-linked complexes are separated by electrophoretic mobility using free solution CE or by size using sieving electrophoresis of SDS complexes. The method gives good quantitative results; e.g., a lysozyme-antibody interaction was found to have Kd = 24 ± 3 nM by PXCE and Kd = 17 ± 2 nM using isothermal calorimetry (ITC). Heat shock protein 70 (Hsp70) in complex with bcl2 associated athanogene 3 (Bag3) was found to have Kd = 25 ± 5 nM by PXCE which agrees with Kd values reported without cross-linking. Hsp70-Bag3 binding site mutants and small molecule inhibitors of Hsp70-Bag3 were characterized by PXCE with good agreement to inhibitory constants and IC50 values obtained by a bead-based flow cytometry protein interaction assay (FCPIA). PXCE allows rapid method development for quantitative analysis of PPIs.

Maximum-Entropy Models of Sequenced Immune Repertoires Predict Antigen-Antibody Affinity.

The immune system has developed a number of distinct complex mechanisms to shape and control the antibody repertoire. One of these mechanisms, the affinity maturation process, works in an evolutionary-like fashion: after binding to a foreign molecule, the antibody-producing B-cells exhibit a high-frequency mutation rate in the genome region that codes for the antibody active site. Eventually, cells that produce antibodies with higher affinity for their cognate antigen are selected and clonally expanded. Here, we propose a new statistical approach based on maximum entropy modeling in which a scoring function related to the binding affinity of antibodies against a specific antigen is inferred from a sample of sequences of the immune repertoire of an individual. We use our inference strategy to infer a statistical model on a data set obtained by sequencing a fairly large portion of the immune repertoire of an HIV-1 infected patient. The Pearson correlation coefficient between our scoring function and the IC50 neutralization titer measured on 30 different antibodies of known sequence is as high as 0.77 (p-value 10-6), outperforming other sequence- and structure-based models.

Origins of specificity and affinity in antibody-protein interactions.

Natural antibodies are frequently elicited to recognize diverse protein surfaces, where the sequence features of the epitopes are frequently indistinguishable from those of nonepitope protein surfaces. It is not clearly understood how the paratopes are able to recognize sequence-wise featureless epitopes and how a natural antibody repertoire with limited variants can recognize seemingly unlimited protein antigens foreign to the host immune system. In this work, computational methods were used to predict the functional paratopes with the 3D antibody variable domain structure as input. The predicted functional paratopes were reasonably validated by the hot spot residues known from experimental alanine scanning measurements. The functional paratope (hot spot) predictions on a set of 111 antibody-antigen complex structures indicate that aromatic, mostly tyrosyl, side chains constitute the major part of the predicted functional paratopes, with short-chain hydrophilic residues forming the minor portion of the predicted functional paratopes. These aromatic side chains interact mostly with the epitope main chain atoms and side-chain carbons. The functional paratopes are surrounded by favorable polar atomistic contacts in the structural paratope-epitope interfaces; more that 80% these polar contacts are electrostatically favorable and about 40% of these polar contacts form direct hydrogen bonds across the interfaces. These results indicate that a limited repertoire of antibodies bearing paratopes with diverse structural contours enriched with aromatic side chains among short-chain hydrophilic residues can recognize all sorts of protein surfaces, because the determinants for antibody recognition are common physicochemical features ubiquitously distributed over all protein surfaces.

Structure of a human IgA1 Fab fragment at 1.55†Šresolution: potential effect of the constant domains on antigen-affinity modulation.

Despite being the most abundant class of immunoglobulins in humans and playing central roles in the adaptive immune response, high-resolution structural data are still lacking for the antigen-binding region of human isotype A antibodies (IgAs). The crystal structures of a human Fab fragment of IgA1 in three different crystal forms are now reported. The three-dimensional organization is similar to those of other Fab classes, but FabA1 seems to be more rigid, being constrained by a hydrophobic core in the interface between the variable and constant domains of the heavy chain (VH-CH1) as well as by a disulfide bridge that connects the light and heavy chains, influencing the relative heavy/light-chain orientation. The crystal structure of the same antibody but with a G-isotype CH1 which is reported to display different antigen affinity has also been solved. The differential structural features reveal plausible mechanisms for constant/variable-domain long-distance effects whereby antibody class switching could alter antigen affinity.

Dynamic antibody-binding properties in the pathogenesis of HIT.

Rapid laboratory assessment of heparin-induced thrombocytopenia (HIT) is important for disease recognition and management. The utility of contemporary immunoassays to detect antiplatelet factor 4 (PF4)/heparin antibodies is hindered by detection of antibodies unassociated with disease. To begin to distinguish properties of pathogenic anti-PF4/heparin antibodies, we compared isotype-matched monoclonal antibodies that bind to different epitopes: KKO causes thrombocytopenia in an in vivo model of HIT, whereas RTO does not. KKO binding to PF4 and heparin is specifically inhibited by human HIT antibodies that activate platelets, whereas inhibition of RTO binding is not differentially affected. Heparin increased the avidity of KKO binding to PF4 without affecting RTO, but it did not increase total binding or binding to nontetrameric PF4(K50E). Single-molecule forced unbinding demonstrated KKO was 8-fold more reactive toward PF4 tetramers and formed stronger complexes than RTO, but not to PF4(K50E) dimers. KKO, but not RTO, promoted oligomerization of PF4 but not PF4(K50E). This study reveals differences in the properties of anti-PF4 antibodies that cause thrombocytopenia not revealed by ELISA that correlate with oligomerization of PF4 and sustained high-avidity interactions that may simulate transient antibody-antigen interactions in vivo. These differences suggest the potential importance of epitope specificity in the pathogenesis of HIT.

The clinical relevance of persistent recombinant immunoblot assay-indeterminate reactions: insights into the natural history of hepatitis C virus infection and implications for donor counseling.

BACKGROUND: Recombinant immunoblot assay (RIBA) is used to determine the specificity of antibody to hepatitis C virus (anti-HCV). The RIBA result is recorded as positive, negative, or indeterminate. The interpretation and significance of RIBA-indeterminate reactions are unclear. We addressed the clinical relevance of these reactions in the context of the natural history of HCV infection in a prospectively followed cohort of anti-HCV-positive blood donors. STUDY DESIGN AND METHODS: Donor demographics, exposure history, and humoral and cell-mediated immunity (CMI) were compared in 15 RIBA-indeterminate subjects, nine chronic HCV carriers, and eight spontaneously recovered subjects. Serum samples were tested for anti-HCV by a quantitative, liquid luciferase immunoprecipitation system (LIPS). CMI was assessed by interferon-γ enzyme-linked immunosorbent spot assay. RESULTS: In the LIPS assay, the sum of antibody responses to six HCV antigens showed significant (p < 0.001) stepwise diminution progressing from chronic carriers to spontaneously recovered to RIBA-indeterminate subjects. CMI responses in RIBA-indeterminate subjects were similar to spontaneously recovered subjects and greater than chronic carriers and controls (p < 0.008). A parenteral risk factor was identified in only 13% of RIBA-indeterminate subjects compared to 89% of chronic carriers and 87% of spontaneously recovered subjects. RIBA-indeterminate donors were older than the other groups. CONCLUSION: The CMI and LIPS results suggest that persistent RIBA-indeterminate reactions represent waning anti-HCV responses in persons who have recovered from a remote HCV infection. In such cases, detectable antibody may ultimately disappear leaving no residual serologic evidence of prior HCV infection, as reported in a minority of long-term HCV-recovered subjects.

Epitope binning of murine monoclonal antibodies by a multiplexed pairing assay.

We describe an assay and data evaluation technique for sorting a panel of murine monoclonal antibodies according to epitope specificities. The assay analyzes the simultaneous binding (pairing) of antibodies to an antigen and groups together antibodies with similar pairing profiles. Similar profiles indicate that the antibodies bind to the same or closely related epitopes. The assay works well with crude hybridoma supernatants and can be multiplexed. These features make the assay particularly suitable for the early phase of hybridoma/antibody screening when antibodies are available only as low volume culture harvest samples.

Evaluation of three Immunochromatographic Assays for Detection of Legionella pneumophila serogroup 1 Antigen in Urine Samples

Los métodos inmunocromatográficos Uni-Gold, SAS y BinaxNOW para la detección cualitativa del antígeno de Legionellapneumophila serogrupo 1 en orina fueron comparadosempleando 39 muestras de orina, sin congelar y sin concentrar,de pacientes con Enfermedad del Legionario. La prueba Uni-Gold detectó el antigen en el 41% de los casos (16/39), SAS enel 61,5% (24/39) y Binax NOW en el 74,3% (29/39). La pruebaBinax NOW mostró los mejores resultados en la detección delantígeno de L. pneumophila serogrupo 1 en muestras de orina(AU)

The Uni-Gold, the SAS and the Binax NOW immunochromatographictest (ICT) urinary antigen assays for the qualitativedetection of Legionella pneumophila serogroup 1 were comparedusing 39 unfrozen and nonconcentrated urine samplesfrom patients with Legionnaires´ disease (LD). The Uni-Gold antigentest detected the urinary antigen in 41% (16/39), the SASantigen test in 61.5% (24/39), and the Binax NOW antigen test in74.3% (29/39). The Binax NOW ICT assay showed the best resultswhen detecting L. pneumophila urinary antigen(AU)

Antigen loss from antibody-coated red blood cells.

Clinically significant signs and symptoms of hemolysis can result from the transfusion of crossmatch incompatible blood, the development of anti-red blood cell (RBC) alloantibodies posttransfusion, or the development of autoantibodies to RBCs (ie, autoimmune hemolytic anemia). However, a less understood and poorly appreciated phenomenon is when an antibody induces the loss of its target antigen without significant damage to the cells themselves. This has been referred to as "depressed antigen," "antigen suppression," "weakened antigenicity," and "antigen loss." This phenomenon has been observed for multiple blood group antigens on human RBCs, in addition to antigens on leukocytes, platelets, neurons, and neoplastic cells. This review discusses the published human case reports of antigen loss in each of the contexts described above and describes several experimental models, including whole animal models and in vitro culture systems. Our current understanding of the cellular and molecular mechanisms is discussed, and the role of antigen loss phenomena in both normal immune function and in disease states is reviewed.

The immunoglobulin-binding Eib proteins from Escherichia coli are receptors for IgG Fc.

The immunoglobulin-binding proteins from Escherichia coli (Eibs) comprise a family of six proteins homologous to the Yersinia adhesin YadA. These proteins are postulated to bind to the Fc portion of immunoglobulin G (IgG) in a non-immune manner. However, a recent study [Ghumra, A., Pleass, R.J., 2007. Escherichia coli do not express Fc-receptors for human immunoglobulin G (IgG). Mol. Immunol. 44, 2144-2146] appeared to show that these proteins do not bind Fc and suggested that the binding seen in earlier studies is due to the polyclonal preparations used in the assays containing antibodies specific to epitopes in the Eib proteins. To resolve this matter, we produced purified, recombinant Eibs for the first time and investigated their binding to intact antibodies and Fc fragments by immunoblot and ELISA techniques. We were able to purify four members of the family, EibA, -C, -D and -F, and show conclusively that these bind IgG Fc. We were also able to block the binding of full-length antibody with IgG Fc, but not with IgG Fab. Binding to IgG Fab was not detectable by surface plasmon resonance, whereas the affinities of Eibs to IgG and IgG Fc were in the range of 50-200 nM. We further demonstrate that deglycosylating IgG Fc does not affect Eib binding. Our results show that the Eib proteins do indeed bind human IgG Fc and that IgG Fc receptors are present in E. coli.

Some antiphospholipid antibodies bind to hemostasis and fibrinolysis proteases and promote thrombosis.

It is generally accepted that the major autoantigen for antiphospholipid antibodies (aPL) is beta(2)glycoprotein I (beta(2)GPI). Interestingly, some aPL bind to beta(2)GPI and the homologous enzymatic domains of several proteases involved in hemostasis and fibrinolysis, and correspondingly hinder anticoagulant regulation and resolution of clots. These findings are consistent with several early findings of aPL and provide a new perspective about some aPL in terms of their binding specificities and related functional properties in promoting thrombosis. In addition, homologous enzymatic domains of the involved proteases share conformation epitope(s) with beta(2)GPI, thus providing a possible structural basis for some non-mutually exclusive mechanisms of aPL-mediated thrombosis.

Tissue factor in antiphospholipid antibody-induced pregnancy loss: a pro-inflammatory molecule.

Fetal loss in patients with antiphospholipid antibodies (aPL) has been ascribed to thrombosis of placental vessels. However, we have shown that inflammation, specifically complement activation with generation of the anaphylotoxin C5a, is an essential mediator of fetal injury. We have analysed the role of tissue factor (TF) in a mouse model of aPL-induced pregnancy loss. TF is the major cellular activator of the coagulation cascade but also has cell signaling activity. Mice that received aPL-IgG showed strong TF staining throughout the decidua and on embryonic debris. This TF staining was not associated with either fibrin staining or thrombi in deciduas. The absence of fibrin deposition and thrombi suggests that TF-dependent activation of coagulation does not mediate aPL-induced pregnancy loss.We found that either blockade of TF with a monoclonal antibody in wild type mice or a genetic reduction of TF prevented aPL-induced inflammation and pregnancy loss indicated a pathogenic role for TF in aPL-induced pregnancy complications. In response to aPL-generated C5a, neutrophils express TF potentiating inflammation in the deciduas and leading to miscarriages. Importantly, we showed that TF in myeloid cells, but not fetal-derived cells (trophoblasts), was associated with fetal injury, suggesting that the site for pathologic TF expression is neutrophils. We found that TF expression in neutrophils contributes to respiratory burst and subsequent trophoblast injury and pregnancy loss induced by aPL. The identification of TF, acting as an important pro-inflammatory mediator in aPL-induced fetal injury, provides a new target for therapy to prevent pregnancy loss in the aPL syndrome.

Tissue factor in the antiphospholipid syndrome.

Antiphospholipid (aPL) antibodies are clinically important acquired risk factors for thrombosis and pregnancy loss and are thought to have a direct prothrombotic effect in vivo. Data suggest that a major mechanism by which aPL antibodies contribute to thrombophilia is the upregulation of tissue factor (TF) (CD142) on blood cells and vascular endothelium. TF is the physiological trigger of normal blood coagulation and thrombosis in many hypercoagulable conditions. This article reviews the physiology of TF, the molecular regulation of TF expression and the effects of aPL antibodies on intravascular TF regulation and expression. Inhibition of TF and the pathways by which aPL antibodies induce TF expression are potentially attractive therapeutic targets in the antiphospholipid syndrome.