Epitope-paratope Interaction Research Articles

Structural insights into epitope-paratope interactions of a monoclonal antibody targeting CEACAM5-expressing tumors

Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) are overexpressed in some tumor types. The antibody-drug conjugate tusamitamab ravtansine specifically recognizes the A3-B3 domains of human CEACAM5 (hCEACAM5). To understand this specificity, here we map the epitope-paratope interface between the A3-B3 domains of hCEACAM5 (hCEACAM5A3-B3) and the antigen-binding fragment of tusamitamab (tusa Fab). We use hydrogen/deuterium exchange mass spectrometry to identify the tusa Fab paratope, which involves heavy chain (HC) residues 101–109 and light chain residues 48–54 and 88–104. Using surface plasmon resonance, we demonstrate that alanine variants of HC residues 96–108 abolish binding to hCEACAM5, suggesting that these residues are critical for tusa-Fab–antigen complex formation. The cryogenic electron microscopy structure of the hCEACAM5A3-B3- tusa Fab complex (3.11 Å overall resolution) reveals a discontinuous epitope involving residues in the A3-B3 domains and an N-linked mannose at residue Asn612. Conformational constraints on the epitope-paratope interface enable tusamitamab to target hCEACAM5A3-B3 and distinguish CEACAM5 from other CEACAMs.

Light chain of a public SARS-CoV-2 class-3 antibody modulates neutralization against Omicron

The pairing of antibody genes IGHV2-5/IGLV2-14 is established as a public immune response that potently cross-neutralizes SARS-CoV-2 variants, including Omicron, by targeting class-3/RBD-5 epitopes in the receptor binding domain (RBD). LY-CoV1404 (bebtelovimab) exemplifies this, displaying exceptional potency against Omicron sub-variants up to BA.5. Here, we report a human antibody, 002-S21B10, encoded by the public clonotype IGHV2-5/IGLV2-14. While 002-S21B10 neutralized key SARS-CoV-2 variants, it did not neutralize Omicron, despite sharing >92% sequence similarity with LY-CoV1404. The structure of 002-S21B10 in complex with spike trimer plus structural and sequence comparisons with LY-CoV1404 and other IGHV2-5/IGLV2-14 antibodies revealed significant variations in light-chain orientation, paratope residues, and epitope-paratope interactions that enable some antibodies to neutralize Omicron but not others. Confirming this, replacing the light chain of 002-S21B10 with the light chain of LY-CoV1404 restored 002-S21B10's binding to Omicron. Understanding such Omicron evasion from public response is vital for guiding therapeutics and vaccine design.

Determination of HER2 binding domain in antigen-antibody complexes based on chemical crosslinking mass spectrometry

Chemical crosslinking (XL) of non-covalent antigen-antibody complexes followed by mass spectrometric identification (MS) of inter-protein crosslinks can provide spatial constraints between relevant residues, which are valuable structural information associated with the molecular binding interface. To highlight the potential of XL/MS in the biopharmaceutical industry, we herein developed and validated an XL/MS workflow that employed a zero-length linker, 1,1′‑carbonyldiimidazole (CDI), and a widely used medium-length linker, disuccinimidyl sulfoxide (DSSO), for fast, accurate determination of antigen domains targeted by therapeutic antibodies. To avoid false identification, system suitability samples and negative samples were designed for all experiments, and all tandem mass spectra were manually examined. To validate the proposed XL/MS workflow, two complexes involving human epidermal growth factor receptor 2 Fc fusion protein (HER2Fc) with known crystal structures, including HER2Fc-pertuzumab and HER2Fc-trastuzumab, have been subjected to CDI and DSSO crosslinking. Crosslinks established by CDI and DSSO between HER2Fc and pertuzumab accurately revealed their interaction interface. CDI crosslinking contributes more than DSSO because of its short spacer arm and high reactivity towards hydroxyl groups, demonstrating its capacity in protein interaction analysis. The correct binding domain cannot be revealed solely based on DSSO in the HER2Fc-trastuzumab complex, because domain proximity revealed by this 7-atom spacer linker cannot be directly translated as binding interfaces. As the first successful XL/MS application in early-stage therapeutic antibody discovery, we analyzed the molecular binding interface between HER2Fc and H-mab, an innovant drug candidate whose paratopes have not been studied yet. We predict that H-mab probably targets HER2 Domain I. The proposed XL/MS workflow can serve as an accurate, fast, and low-cost method to study the interaction between antibodies and large multi-domain antigens. SignificanceThis article described a fast, low-consumption approach based on chemical crosslinking mass spectrometry (XL/MS) using two linkers for binding domain determination in multidomain antigen-antibody complexes. Our results highlighted the higher importance of zero-length crosslinks established by CDI than 7-atom DSSO crosslinks, as residue proximity revealed by zero-length crosslinks is closely related to epitope-paratope interaction surfaces. Furthermore, the higher reactivity of CDI towards hydroxyl groups broadens the ranges of possible crosslinks, despite the necessity of delicate operation in CDI crosslinking. We suggest that all established CDI and DSSO crosslinks should be comprehensively considered for correct binding domain analysis because predictions solely based on DSSO might be ambiguous. We have determined the binding interface in the HER2-H-mab using CDI and DSSO, which is the first successful application of XL/MS in real-world early-stage biopharmaceutical development.

Identifying Epitope Sites on the Receptor Binding Domain (RBD) of SARS-CoV-2

Abstract COVID-19 and SARS-CoV-2 variants continue to threaten human health and life worldwide. Thousands of structures related to SARS-CoV-2 have been rapidly determined, either by X-ray crystallography or CryoEM and deposited in the Protein Data Bank (PDB) since 2020. Here, we systematically investigated the structures of 302 antibodies and 78 nanobodies in complex with spike protein or the receptor binding domain (RBD) of SARS-CoV-2. We identified 23 common epitopic sites (ES) on the RBD surface and revealed the vital role of the complementarity-determining Region (CDR) loops in recognizing epitopes. The 23 ES are characterized according to the secondary structure feature and accessible surface area. About 75% of the total surface of RBD areas could access by the elicited antibodies, while the CDR3 loops occupied 50% of the contact surface. The analysis of paratope-epitope (antibody-antigen) interaction based on these epitope sites revealed the features of potent neutralizing the virus and the unique usage of amino acids of antibody. The clustering analysis of the antibody biophysical properties and surface area availabilities on the RBD found many binding motifs. Remarkably, most variants of concern (VOC) escape mutations, including Omicron occur within these 23 ES. This analysis not only explains the differential ability of antibodies to recognize epitope sites on the RBD surface but also offers predictive guidelines for understanding the role that accumulated SARS mutations might play in escape from antibodies elicited by immunogens. This structural characterization of the epitope-paratope interactions provides insights for structure-based vaccine design and therapeutic strategies and drugs against the future virus.

Ara h 2-specific IgE epitope-like peptides inhibit the binding of IgE to Ara h 2 and suppress lgE-dependent effector cell activation.

Clinical and experimental analyses indicate a pathognomonic role for allergen IgE crosslinking through epitope-paratope interactions as a major initial step in the cascade leading to effector cell activation and clinical manifestations of lgE-mediated food allergies. We aimed to undertake the initial development and assessment of Ara h 2-specific IgE epitope-like peptides that can bind to allergen-specific IgE paratopes and suppress effector cell activation. We performed biopanning, screening, IgE binding, selection and mapping of peptides. We generated synthetic peptides for use in all functional experiments. ImmunoCAP inhibition, basophil and mast cell activation tests, with LAD2 cells, a human mast cell line were performed. Twenty-six children or young adults who had peanut allergy were studied. We identified and selected three linear peptides (DHPRFNRDNDVA, DHPRYGP and DHPRFST), and immunoblot analyses revealed binding to lgE from peanut-allergic individuals. The peptide sequences were aligned to the disordered region corresponding to the loop between helices 2 and 3 of Ara h 2, and conformational mapping showed that the peptides match the surface of Ara h 2 and h 6 but not other peanut allergens. In ImmunoCAP inhibition experiments, the peptides significantly inhibit the binding of IgE to Ara h 2 (p < .001). In basophil and mast cell activation tests, the peptides significantly suppressed Ara h 2-induced effector cell activation (p < .05) and increased the half-maximal Ara h 2 effective concentration (p < .05). Binding of the peptides to specific IgEs did not induce activation of basophils or mast cells. These studies show that the indicated peptides reduce the allergenic activity of Ara h 2 and suppress lgE-dependent basophil and mast cell activation. These observations may suggest a novel therapeutic strategy for food allergy based on epitope-paratop blocking.

Structural constraints link differences in neutralization potency of human anti-Eastern equine encephalitis virus monoclonal antibodies

Selection and development of monoclonal antibody (mAb) therapeutics against pathogenic viruses depends on certain functional characteristics. Neutralization potency, or the half-maximal inhibitory concentration (IC50) values, is an important characteristic of candidate therapeutic antibodies. Structural insights into the bases of neutralization potency differences between antiviral neutralizing mAbs are lacking. In this report, we present cryo-electron microscopy (EM) reconstructions of three anti-Eastern equine encephalitis virus (EEEV) neutralizing human mAbs targeting overlapping epitopes on the E2 protein, with greater than 20-fold differences in their respective IC50 values. From our structural and biophysical analyses, we identify several constraints that contribute to the observed differences in the neutralization potencies. Cryo-EM reconstructions of EEEV in complex with these Fab fragments reveal structural constraints that dictate intravirion or intervirion cross-linking of glycoprotein spikes by their IgG counterparts as a mechanism of neutralization. Additionally, we describe critical features for the recognition of EEEV by these mAbs including the epitope-paratope interaction surface, occupancy, and kinetic differences in on-rate for binding to the E2 protein. Each constraint contributes to the extent of EEEV inhibition for blockade of virus entry, fusion, and/or egress. These findings provide structural and biophysical insights into the differences in mechanism and neutralization potencies of these antibodies, which help inform rational design principles for candidate vaccines and therapeutic antibodies for all icosahedral viruses.

Designing and Computational Analysis of Chimeric Avian Influenza Antigen: A Yeast-Displayed Universal and Cross-Protective Vaccine Candidate

This study describes the development of a cross-protective vaccine candidate against avian influenza virus, which was designed using M2e, a highly preserved antigen. The consensus sequence of M2e was obtained using 31 sequences of avian influenza virus subtypes (H5N8, H5N1, H9N2, and H7N9) isolated from seven avian species in five Asian countries. An adjuvant, a partial sequence of flagellin, was also considered. Two chimeric antigens were designed and virtually cloned and expressed using the PYD1 vector and EBY100 yeast strain. Molecular dynamic simulations were used to assess the stability and conformational features of these antigens. The likelihood of detection by a specific monoclonal antibody, MAb148, was estimated for the designed peptides using docking studies. The second chimeric antigen was more compact and stable than the first design, but it was less detectable by MAb148. In the first design, two of the four desired epitopes ("SLLTEVETP") were exposed, while only a partial sequence of this epitope was detectable in the second design. In contrast to the second chimeric antigen, electrostatic, and binding energies related to the interaction of the first antigen and MAb148 were significantly closer to the positive control. This suggests that epitopes of the first chimeric antigen could be correctly located in the specific paratope of MAb148. In conclusion, the first chimeric antigen exhibits favorable conformational features and epitope-paratope interactions, highlighting its potential as a promising cross-protective vaccine candidate against a range of avian influenza virus subtypes.

Development and characterization of a panel of anti-idiotype antibodies to 1C10 that cross-neutralize HIV-1 subtype B viruses

The V3 loop of the human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) is one of the conserved immunogenic regions targeted by neutralizing antibodies (nAb). Two different binding modes of anti-V3 abs have been reported in studies using two V3 mimotopes: the ladle-type and cradle-type. We previously isolated a ladle-type nAb, 1C10, that potently and broadly neutralized clade B viruses. Despite its potent neutralization activity, 1C10 possesses no unique features in its amino acid sequence. We hypothesized that the neutralization potency of 1C10 is derived from its antigen-binding characteristics, which are not a consequence of the two previously reported binding modes of anti-V3 nAbs. To analyze epitope-paratope interactions between 1C10 and the V3 loop, we produced five anti-idiotypic antibodies (anti-Id abs) from mice immunized with 1C10 nAb. The idiotopes of the anti-Id Abs on the 1C10 heavy chain were estimated by alanine scanning, germline reversion mutagenesis, and a 1C10 sibling clone. Next-generation sequencing combined with homology modeling revealed contact between R315 at the tip of the V3 loop and 1C10 by D53 of CDRH2 and Phe/Asp of CDRH3. These amino acids were enriched in the anti-Id-ab-reactive B cell receptors encoded by the IGHV3-30 gene. We also found that 20% of HIV-infected individuals had abs specific to the anti-Id abs, as well as both of the V3 mimotopes, that did not respond to the linear V3 peptide. Our findings showed that the anti-Id abs induced by 1C10 recognized a key amino acid formation essential for steric interactions between the ladle-type nAb and the V3 loop. We also revealed the coexistence of anti-V3 ab reactivity to V3 loop mimotopes and to the anti-Id abs in HIV-positive individuals.

Square wave voltammetric approach to leptin immunosensing and optimization of driving parameters with chemometrics

Square wave voltammetry serves as an effective analytical means to evaluate antigen-antibody coupling at the solid-liquid interface. Herein, we describe 3-aminopropyltrimethoxysilane (APTMS) induced irreversible immobilization of anti-leptin to micellar gold nanoparticles (AuNPs). Antibodies (Abs) were orthogonally loaded on micellized AuNP assemblies via amino residual groups. The ratio of bound Ab molecules was determined by the Bradford assay. The AuNP/Ab layer modified electrodes with variable antibody surface coverage (∼400 ± 55–200 ± 30 Ab/NP) were analyzed in terms of change in backward, net current (Ip) components. The rate of antigen coupling was found to be consistent with the variation in antibody density as well as the binding affinity. The lowest detection limit was observed at the femtomolar level (0.25 fM/mL) over a wide range of antigen concentration (6.2 ng/mL to 0.12 fg/mL). The variables affecting the epitope-paratope interaction were further optimized using a chemometric approach and a response surface methodology (RSM).

Complexity of Viral Epitope Surfaces as Evasive Targets for Vaccines and Therapeutic Antibodies.

The dynamic interplay between virus and host plays out across many interacting surfaces as virus and host evolve continually in response to one another. In particular, epitope-paratope interactions (EPIs) between viral antigen and host antibodies drive much of this evolutionary race. In this review, we describe a series of recent studies examining aspects of epitope complexity that go beyond two interacting protein surfaces as EPIs are typically understood. To structure our discussion, we present a framework for understanding epitope complexity as a spectrum along a series of axes, focusing primarily on 1) epitope biochemical complexity (e.g., epitopes involving N-glycans) and 2) antigen conformational/dynamic complexity (e.g., epitopes with differential properties depending on antigen state or fold-axis). We highlight additional epitope complexity factors including epitope tertiary/quaternary structure, which contribute to epistatic relationships between epitope residues within- or adjacent-to a given epitope, as well as epitope overlap resulting from polyclonal antibody responses, which is relevant when assessing antigenic pressure against a given epitope. Finally, we discuss how these different forms of epitope complexity can limit EPI analyses and therapeutic antibody development, as well as recent efforts to overcome these limitations.

Noninvasive Point-of-Care Nanobiosensing of Cervical Cancer as an Auxiliary to Pap-Smear Test.

A potential cancer antigen (Ag), protein-phosphatase-1-gamma-2 (PP1γ2), with a restricted expression in testis and sperms has been identified as a biomarker specific to cervical cancer (CaCx). Detection of this cancer biomarker antigen (NCB-Ag) in human urine opens up the possibility of noninvasive detection of CaCx to supplement the dreaded and invasive Pap-smear test. A colorimetric response of an assembly of gold nanoparticles (Au NPs) has been employed for the quantitative, noninvasive, and point-of-care-testing of CaCx in the urine. In order to fabricate the immunosensor, Au NPs of sizes ∼5-20 nm have been chemically modified with a linker, 3,3'-di-thio-di-propionic-acid-di(n-hydroxy-succinimide-ester) (DTSP) to attach the antibody (Ab) specific to the NCB-Ag. Interestingly, the addition of Ag to the composite of Ab-DTSP-Au NPs leads to a significant hypsochromic shift due to a localized surface plasmon resonance phenomenon, which originates from the specific epitope-paratope interaction between the NCB-Ag and Ab-DTSP-Au NPs. The variations in the absorbance and wavelength shift during such attachments of different concentrations of NCB-Ag on the Ab-DTSP-Au NPs composite have been employed as a calibration to identify NCB-Ag in human urine. An in-house prototype has been assembled by integrating a light-emitting diode of a narrow range wavelength in one side of a cuvette in which the reaction has been performed while a sensitive photodetector to the other side to transduce the transmitted signal associated with the loading of NCB-Ag in the Ab-DTSP-Au NPs composite. The proposed immunosensing platform has been tested against other standard proteins to ensure noninterference alongside proving the proof-for-specificity of the NCB detection.

Structural basis for differential recognition of phosphohistidine-containing peptides by 1-pHis and 3-pHis monoclonal antibodies

In 2015, monoclonal antibodies (mAbs) that selectively recognize the 1-pHis or 3-pHis isoforms of phosphohistidine were developed by immunizing rabbits with degenerate Ala/Gly peptides containing the nonhydrolyzable phosphohistidine (pHis) analog- phosphotriazolylalanine (pTza). Here, we report structures of five rabbit mAbs bound to cognate pTza peptides: SC1-1 and SC50-3 that recognize 1-pHis, and their 3-pHis-specific counterparts, SC39-4, SC44-8, and SC56-2. These cocrystal structures provide insights into the binding modes of the pTza phosphate group that are distinct for the 1- and 3-pHis mAbs with the selectivity arising from specific contacts with the phosphate group and triazolyl ring. The mode of phosphate recognition in the 3-pHis mAbs recapitulates the Walker A motif, as present in kinases. The complementarity-determining regions (CDRs) of four of the Fabs interact with the peptide backbone rather than peptide side chains, thus conferring sequence independence, whereas SC44-8 shows a proclivity for binding a GpHAGA motif mediated by a sterically complementary CDRL3 loop. Specific hydrogen bonding with the triazolyl ring precludes recognition of pTyr and other phosphoamino acids by these mAbs. Kinetic binding experiments reveal that the affinity of pHis mAbs for pHis and pTza peptides is submicromolar. Bound pHis mAbs also shield the pHis peptides from rapid dephosphorylation. The epitope-paratope interactions illustrate how these anti-pHis antibodies are useful for a wide range of research techniques and this structural information can be utilized to improve the specificity and affinity of these antibodies toward a variety of pHis substrates to understand the role of histidine phosphorylation in healthy and diseased states.

Dissecting the two models of TCR structure–function relationships

There are only two comprehensive models attempting to account for the TCR structure-function relationships, referred to as the Standard or Centric model (Model I) and the Tritope model (Model II). This essay is written to analyze comparatively the two formulations of restrictive reactivity, stressing in particular the logic of each. Model I is essentially built on an analogy between the TCR and the BCR. Given a TCR with only one combining site (paratope), restrictive recognition requires that its ligand be viewed as a composite structure between the peptide and restricting element. It is this relationship that entrains a set of correlates that makes Model I untenable. Model II is predicated on the postulate that the recognition of the allele-specific determinants expressed by MHC-encoded restricting elements (R) is germline encoded and selected, whereas the recognition of peptide (P) is somatically encoded and selected. These selective pressures must operate on definable structures and this, in turn, necessitates a multiply recognitive T cell antigen receptor (TCR) with independent anti-R and anti-P paratopes that function coherently to signal restrictive reactivity. The consequences of this "two repertoire" postulate give us a concept of TCR structure quite distinct from that at present generally accepted, as well as a surprising relationship between numbers of functional TCR V gene segments and allele-specific determinants in the species. In the end, both models must deal with the relationship between the epitope-paratope interaction(s) and the signals to the T cell necessary for its differentiation and function.

EPI-peptide designer: a tool for designing peptide ligand libraries based on epitope-paratope interactions.

Antibodies are an important class of biological drugs, but with limitations, such as inadequate pharmacokinetics, adverse immunogenicity and high production costs. Synthetic peptides for the desired target represent an important alternative to antibodies. However, no computational tool exists to guide the design of these peptides. To identify the interacting residues in a given antibody-antigen (Ab-Ag) interface we used Interface Interacting Residue (I2R), a selection method based on computed molecular interactions. The aggregation of all the molecular interactions between epitope and paratope residues allowed us to transform the 3D Ab-Ag complex structures into interface graphs. Based on these data and the probability of molecular interaction we developed EPI-Peptide Designer tool that uses predicted paratope residues for an epitope of interest to generate targeted peptide ligand libraries. EPI-Peptide Designer successfully predicted 301 peptides able to bind to LiD1 target protein (65% of the experimentally tested peptides), an enrichment of 22% compared to randomly generated peptides. This tool should enable the development of a new generation of synthetic interacting peptides that could be very useful in the biosensor, diagnostic and therapeutic fields. All software developed in this work are available at http://www.biocomp.icb.ufmg.br/biocomp/ liza@icb.ufmg.br Supplementary data are available at Bioinformatics online.

Proteochemometric modeling of the antigen-antibody interaction: new fingerprints for antigen, antibody and epitope-paratope interaction.

Despite the high specificity between antigen and antibody binding, similar epitopes can be recognized or cross-neutralized by paratopes of antibody with different binding affinities. How to accurately characterize this slight variation which may or may not change the antigen-antibody binding affinity is a key issue in this area. In this report, by combining cylinder model with shell structure model, a new fingerprint was introduced to describe both the structural and physical-chemical features of the antigen and antibody protein. Furthermore, beside the description of individual protein, the specific epitope-paratope interaction fingerprint (EPIF) was developed to reflect the bond and the environment of the antigen-antibody interface. Finally, Proteochemometric Modeling of the antigen-antibody interaction was established and evaluated on 429 antigen-antibody complexes. By using only protein descriptors, our model achieved the best performance () among peers. Further, together with EPIF as a new cross-term, our model () can significantly outperform peers with multiplication of ligand and protein descriptors as a cross-term (). Results illustrated that: 1) our newly designed protein fingerprints and EPIF can better describe the antigen-antibody interaction; 2) EPIF is a better and specific cross-term in Proteochemometric Modeling for antigen-antibody interaction. The fingerprints designed in this study will provide assistance to the description of antigen-antibody binding, and in future, it may be valuable help for the high-throughput antibody screening. The algorithm is freely available on request.

Adjustable locks and flexible keys: plasticity of epitope-paratope interactions in germline antibodies.

Ag recognition by independent primary Abs against a small flexible Ag with overlapping epitopes was analyzed to address the determinants of Ag specificity during the initial encounter. Crystal structures of two distinct dodecapeptide Ags, GDPRPSYISHLL and PPYPAWHAPGNI, in complex with the germline mAb 36-65 were determined and compared with the structures of the same Ags bound to another independent germline mAb, BBE6.12H3. For each peptide Ag, the two germline mAbs recognized overlapping epitopes, but in different topologies. The peptide structures differed, and the two paratopes attained discrete conformations, leading to different surface topologies, in a mode that can be described as adjustable locks and flexible keys. This is in contrast to mature mAbs, in which conformational convergence of different paratopes while binding to a common epitope in a similar conformation has been reported. These results suggest that the primary immune receptor repertoire is highly versatile as compared with its mature counterpart. Germline and mature mAbs adopt distinct mechanisms for recognizing a flexible epitope. Whereas conservation of conformational repertoire is a key characteristic of mature mAbs achieved through affinity maturation, the germline mAbs, at the initial stages of Ag encounter, maintain substantial plasticity, accommodating a broad specificity repertoire.

Clonality characterization of natural epitope-specific antibodies against the tumor-related antigen topoisomerase IIa by peptide chip and proteome analysis: a pilot study with colorectal carcinoma patient samples

Patient-specific sequential epitopes were identified by peptide chip analysis using 15mer peptides immobilized on glass slides that covered the topoisomerase IIa protein with a frameshift of five amino acids. Binding specificities of serum antibodies against sequential epitopes were confirmed as being mono-specific by peptide chip re-analysis of epitope-affinity-purified antibody pools. These results demonstrate that serum samples from colon carcinoma patients contain antibodies against sequential epitopes from the topoisomerase IIa antigen. Interactions of patients' antibodies with sequential epitopes displayed by peptides on glass surfaces may thus mirror disease-specific immune situations. Consequently, these data suggest epitope-antibody reactivities on peptide chips as potential diagnostic readouts of individual immune response characteristics, especially because monospecific antibodies can be interrogated. Subsequently, the clonality of the antibodies present in the mono-specific antibody pools was characterized by 2D gel electrophoresis. This analysis suggested that the affinity-purified antibodies were oligoclonal. Similarly to large-scale screening approaches for specific antigen-antibody interactions in order to improve disease diagnostic, we suggest that "protein-wide" screening for specific epitope-paratope interactions may help to develop novel assays for monitoring of personalized therapies, since individual properties of antigen-antibody interactions remain distinguishable.

Kinetic analysis of a human chorionic gonadotropin-β epitope–paratope interaction

Kinetics of protein–protein or ligand–ligate interaction has predominantly been studied by optical spectroscopy (particularly fluorescence) and surface plasmon resonance biosensors. Almost all such studies are based on association kinetics between ligand–ligate and suffer from certain methodological and interpretational limitations. Therefore, kinetic analyses of dissociation data of such interactions become indispensable. In the present investigation, the radiolabeled human chorionic gonadotropin-β (125IhCGβ) was employed as a probe and nitrocellulose (NC) as a solid support to immobilize monoclonal antibody (MAb) G1G10.1. The NC–G1G10.1–125IhCGβ complex (NCcom) was prepared and the dissociation of radiolabeled hCGβ was carried out in the presence of excess unlabeled ligate. From the experimental dissociation data under varying ionic strength, dissociation constants (k− 1), association constants (k+1) and affinity constants (ka) were calculated. The values obtained were utilized in exploring the amino acid residues constituting an epitopic region of hCGβ involved in interaction with the complementary paratope on MAb G1G10.1. Kinetic data of the present study supported our recently published findings [using single step-solid phase radioimmunoassay (SS-SPRIA)] that the core region of hCGβ epitope consists of Arg (94,95) and Asp (99) while a Lys (104) and a His (106) are in proximity to the core epitopic region. Based on the results of present investigation, we conclude that dissociation kinetics coupled with SS-SPRIA unequivocally provides considerable insight into the study of ligand–ligate interactions and epitope analysis.

Molecular dissection of an hCG-β epitope using single-step solid phase radioimmunoassay

BackgroundPeptides and proteins have both sequence-specific (contiguous) and conformation-specific (discontiguous) epitopes. Sequence-specific epitopes are delineated by peptide approach and other robust methods like competition assays, gene expression assays, synthetic peptide library based assays etc. Available methods for delineation of conformation-specific epitopes are cumbersome (X-ray crystallography etc.), time-consuming and require costly sophisticated equipments. Hence, there is a need to develop a simple method for identification and mapping of conformation-specific epitopes. MethodIn the single-step solid phase radioimmunoassay (SS-SPRIA), an immunochemical bridge of ‘mouse IgG-anti-mouse IgG’ was prepared in the polypropylene wells followed by adsorption with hCG specific monoclonal antibody (MAb) G1G10.1. The extent of competitive inhibition in binding ability of 125IhCG-β with chemically or enzymatically modified hCG-β to immobilized MAb G1G10.1 in comparison to hCG-β standards was utilized to identify the epitopic amino acid involved in epitope–paratope interaction. ResultsData clearly suggest that the epitope under investigation consisted of Arg (94, 95) and Asp (99) at the core region with a Lys (104) and a His (106) in the proximity and absence of chymotrypsin susceptible Phe or Tyr in this region. ConclusionThe data of SS-SPRIA revealed the 93–100 loop of amino acid sequence, as the core region of conformation-specific epitope of hCG-β at or near the receptor-binding region. Hence, SS-SPRIA seems to be a simple method for identification and mapping of conformation-specific epitopes.

Epitope mapping from real time kinetic studies — Role of cross-linked disulphides and incidental interacting regions in affinity measurements: Study with human chorionic gonadotropin and monoclonal antibodies

Real time kinetic studies were used to map conformational epitopes in human chorionic gonadotropin (hCG) for two monoclonal antibodies (MAbs). The epitopes were identified in the regions (alpha 5--14 and alpha 55--62). The association rate constant (k+1) was found to be altered by chemical modification of hCG, and the ionic strength of the reaction medium. Based on these changes, we propose the presence of additional interactions away from the epitope- paratope region in the hCG-MAb reaction. We have identified such incidental interacting regions (IIRs) in hCG to be the loop region alpha 35--47 and alpha 60--84. The IIRs contribute significantly towards the KA of the interaction. Therefore, in a macromolecular interaction of hCG and its MAb, KA is determined not only by epitopeparatope interaction but also by the interaction of the nonepitopic-nonparatopic IIRs. However, the specificity of the interaction resides exclusively with the epitope-paratope pair.