Antibody Binding Affinity Research Articles

SARS-CoV-2 Variants, RBD Mutations, Binding Affinity, and Antibody Escape.

Since 2020, the receptor-binding domain (RBD) of the spike protein of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been constantly mutating, producing most of the notable missense mutations in the context of “variants of concern”, probably in response to the vaccine-driven alteration of immune profiles of the human population. The Delta variant, in particular, has become the most prevalent variant of the epidemic, and it is spreading in countries with the highest vaccination rates, causing the world to face the risk of a new wave of the contagion. Understanding the physical mechanism responsible for the mutation-induced changes in the RBD’s binding affinity, its transmissibility, and its capacity to escape vaccine-induced immunity is the “urgent challenge” in the development of preventive measures, vaccines, and therapeutic antibodies against the coronavirus disease 2019 (COVID-19) pandemic. In this study, entropy–enthalpy compensation and the Gibbs free energy change were used to analyze the impact of the RBD mutations on the binding affinity of SARS-CoV-2 variants with the receptor angiotensin converting enzyme 2 (ACE2) and existing antibodies. Through the analysis, we found that the existing mutations have already covered almost all possible detrimental mutations that could result in an increase of transmissibility, and that a possible mutation in amino-acid position 498 of the RBD can potentially enhance its binding affinity. A new calculation method for the binding energies of protein–protein complexes is proposed based on the entropy–enthalpy compensation rule. All known structures of RBD–antibody complexes and the RBD–ACE2 complex comply with the entropy–enthalpy compensation rule in providing the driving force behind the spontaneous protein–protein docking. The variant-induced risk of breakthrough infections in vaccinated people is attributed to the L452R mutation’s reduction of the binding affinity of many antibodies. Mutations reversing the hydrophobic or hydrophilic performance of residues in the spike RBD potentially cause breakthrough infections of coronaviruses due to the changes in geometric complementarity in the entropy–enthalpy compensations between antibodies and the virus at the binding sites.

Enhancement of Binding Affinity of Anti-Hapten Polyclonal IgG Recognizing Mitragynine using Affinity Purification

Antibodies are glycoproteins found in peritoneal fluid, serum, and blood. The antibody-based assay has been used for broad applications such as immunodiagnostic and other biomedical applications. Depending on the intended application, a highly purified polyclonal antibody could be used as an alternative. Purification of antibodies from anti-sera has been proven as one of the methods to enhance the binding affinity of antibodies towards its antigen. We report herein the enhancement of the binding affinity of anti-hapten polyclonal IgG recognizing mitragynine using affinity purification. Serum from the terminal bleed of New Zealand White (NZW) rabbits immunized with mitragynine conjugated with cationized– bovine serum albumin at methyl ester (C22-MG-cBSA), or aromatic ether modification (C9-MG-cBSA) were subjected to HiTrap Protein G affinity purification using fast protein liquid chromatography (FPLC). The elution peak from chromatography fractions was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Here, we report the binding of polyclonal antibodies produced from inoculation of either C22-MG-cBSA or C9-MG-cBSA immunogens of which mitragynine-ovalbumin (MG-OVA) was used as coating antigen in the ELISA assay. Non purified anti-sera from C22-MG-cBSA-inoculated rabbits showed higher titer than C9-MG-cBSA at 1/128 000 and 1/32 000 dilutions, respectively. The affinity of purified poly-IgGs from rabbits immunized with C22-MG-cBSA showed a mean Kd value of 7.965 × 10-6 μM, which was lower than those immunized with C9-MG-cBSA at mean Kd of 1.390 × 10-4 μM. In addition, the purified poly- IgGs showed higher binding towards MG-OVA than non-purified anti-sera at comparable protein concentrations. These results indicated that the higher binding affinity of purified polyclonal IgG is due to the reduced competition among polyclonal antibodies with non- IgG proteins that co-existed in the non-purified anti-sera after the affinity purification.

Neutralizing antibody 5-7 defines a distinct site of vulnerability in SARS-CoV-2 spike N-terminal domain.

Antibodies that potently neutralize SARS-CoV-2 target mainly the receptor-binding domain or the N-terminal domain (NTD). Over a dozen potently neutralizing NTD-directed antibodies have been studied structurally, and all target a single antigenic supersite in NTD (site 1). Here, we report the cryo-EM structure of a potent NTD-directed neutralizing antibody 5-7, which recognizes a site distinct from other potently neutralizing antibodies, inserting a binding loop into an exposed hydrophobic pocket between the two sheets of the NTD β sandwich. Interestingly, this pocket was previously identified as the binding site for hydrophobic molecules, including heme metabolites, but we observe that their presence does not substantially impede 5-7 recognition. Mirroring its distinctive binding, antibody 5-7 retains neutralization potency with many variants of concern (VOCs). Overall, we reveal that a hydrophobic pocket in NTD proposed for immune evasion can be used by the immune system for recognition.

Ultrasensitive capacitance sensor to detect amyloid-beta 1-40 in human serum using supramolecular recognition of β-CD/RGO/ITO micro-disk electrode

In this paper, we developed a new ultrasensitive capacitance sensor for detection of amyloid beta 1-40 (aβ40) protein (one of Alzheimer's disease core biomarkers) in human serum based on the high supramolecular recognition of the β-cyclodextrin/reduced graphene oxide (β-CD/RGO) nanohybrid toward the anti-aβ40 antibody molecule. The sensor was established by immobilizing specific anti-aβ40 antibody onto the β-CD/RGO nanohybrid functionalized on indium tin oxide micro-disk electrode (anti-aβ40/β-CD/RGO/ITO). Detection of aβ40 in the human serum (HS) using the sensor anti-aβ40/β-CD/RGO/ITO is carried out by capacitance measurement without a redox probe to prevent protein denaturation, serving as a convenient strategy for point-of-care diagnosis. In comparison with other studies, the sensor shows a very low limit of detection of 0.69 fg mL−1 in HS, demonstrating its ability for the ultrasensitive detection of aβ40. Using this sensor, the dissociation constant KD of the binding interaction between anti-aβ40 and aβ40 in HS is found to be 2.9 × 10−7 nM, indicating the high binding affinity of antibody–antigen and the suitability of the anti-aβ40/β-CD/RGO/ITO sensor for aβ40 protein detection. The good selectivity of the anti-aβ40/β-CD/RGO/ITO sensor in the presence of differential analytes was also performed in this paper.

Exploring Drug and Antibody-Based Treatment Options for Creutzfeldt-Jakob Disease

Creutzfeldt-Jakob Disease (CJD) is a neurodegenerative disease characterized by mutant PrP prion proteins, which accumulates and impairs the function of wild-type PrPc proteins. The interaction of prion proteins with wild-type proteins converts the PrPc proteins to mutant PrP proteins. These mutant prion proteins lead to neural tissue degradation and other nervous system problems that can eventually lead to death. The use of antibodies to target and destroy prion proteins can be used to decrease PrP levels that can stop CJD progression. The binding affinities of different anti-PrP Fab antibodies are analyzed to determine which antibody best binds to PrP proteins and targets them for destruction. Through antibody-based targeting of prion proteins, potential treatment methods could be developed for CJD. In addition, the use of drugs, such as quinacrine and doxycycline, also show short-term effects in decreasing the progression of CJD. These drugs extend the average lifespan of tested subjects with CJD but also lead to the development of drug-resistant prion proteins that eventually cause the death of the subject affected by CJD.

Electrostatic Interactions Explain the Higher Binding Affinity of the CR3022 Antibody for SARS-CoV-2 than the 4A8 Antibody.

A structural understanding of the mechanism by which antibodies bind SARS-CoV-2 at the atomic level is highly desirable as it can tell the development of more effective antibodies to treat Covid-19. Here, we use steered molecular dynamics (SMD) and coarse-grained simulations to estimate the binding affinity of the monoclonal antibodies CR3022 and 4A8 to the SARS-CoV-2 receptor-binding domain (RBD) and SARS-CoV-2 N-terminal domain (NTD). Consistent with experiments, our SMD and coarse-grained simulations both indicate that CR3022 has a higher affinity for SARS-CoV-2 RBD than 4A8 for the NTD, and the coarse-grained simulations indicate the former binds three times stronger to its respective epitope. This finding shows that CR3022 is a candidate for Covid-19 therapy and is likely a better choice than 4A8. Energetic decomposition of the interaction energies between these two complexes reveals that electrostatic interactions explain the difference in the observed binding affinity between the two complexes. This result could lead to a new approach for developing anti-Covid-19 antibodies in which good candidates must contain charged amino acids in the area of contact with the virus.

Computational-Driven Epitope Verification and Affinity Maturation of TLR4-Targeting Antibodies.

Toll-like receptor (TLR) signaling plays a critical role in the induction and progression of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematous, experimental autoimmune encephalitis, type 1 diabetes mellitus and neurodegenerative diseases. Deciphering antigen recognition by antibodies provides insights and defines the mechanism of action into the progression of immune responses. Multiple strategies, including phage display and hybridoma technologies, have been used to enhance the affinity of antibodies for their respective epitopes. Here, we investigate the TLR4 antibody-binding epitope by computational-driven approach. We demonstrate that three important residues, i.e., Y328, N329, and K349 of TLR4 antibody binding epitope identified upon in silico mutagenesis, affect not only the interaction and binding affinity of antibody but also influence the structural integrity of TLR4. Furthermore, we predict a novel epitope at the TLR4-MD2 interface which can be targeted and explored for therapeutic antibodies and small molecules. This technique provides an in-depth insight into antibody–antigen interactions at the resolution and will be beneficial for the development of new monoclonal antibodies. Computational techniques, if coupled with experimental methods, will shorten the duration of rational design and development of antibody therapeutics.

Covalent Stabilization of Antibody Recruitment Enhances Immune Recognition of Cancer Targets.

Antibody recruiting molecules (ARMs) represent an important class of "proximity-inducing" chemical tools with therapeutic potential. ARMs function by simultaneously binding to a hapten-specific serum antibody (Ab) (e.g., anti-dinitrophenyl (DNP)) and a cancer cell surface protein, enforcing their proximity. ARM anticancer efficacy depends on the formation of ARM:Ab complexes on the cancer cell surface, which activate immune cell recognition and elimination of the cancer cell. Problematically, ARM function in human patients may be limited by conditions that drive the dissociation of ARM:Ab complexes, namely, intrinsically low binding affinity and/or low concentrations of anti-hapten antibodies in human serum. To address this potential limitation, we previously developed a covalent ARM (cARM) chemical tool that eliminates the ARM:antibody equilibrium through a covalent linkage. In the current study, we set out to determine to what extent maximizing the stability of ARM:antibody complexes via cARMs enhances target immune recognition. We observe cARMs significantly increase target immune recognition relative to ARMs across a range of therapeutically relevant antibody concentrations. These results demonstrate that ARM therapeutic function can be dramatically enhanced by increasing the kinetic stability of ARM:antibody complexes localized on cancer cells. Our findings suggest that a) high titres/concentrations of target antibody in human serum are not neccessary and b) saturative antibody recruitment to cancer cells not sufficient, to achieve maximal ARM therapeutic function.

The Masking Game: Design of Activatable Antibodies and Mimetics for Selective Therapeutics and Cell Control.

The high selectivity and affinity of antibody binding have made antibodies all-pervasive tools in therapy, diagnosis, and basic science. A plethora of chemogenetic approaches has been devised to make antibodies responsive to stimuli ranging from light to enzymatic activity, temperature, pH, ions, and effector molecules. Within a single decade, the field of activatable antibodies has yielded marketed therapeutics capable of engaging antigens that could not be targeted with traditional antibodies, as well as new tools to control intracellular protein location and investigate biological processes. Many opportunities remain untapped, waiting for more efficient and generally applicable masking strategies to be developed at the interface between chemistry and biotechnology.

Genotyping and Molecular Characterization of Classical Swine Fever Virus Isolated in China during 2016–2018

Classical swine fever (CSF) is a highly contagious disease of swine caused by classical swine fever virus (CSFV). For decades the disease has been controlled in China by a modified live vaccine (C-strain) of genotype 1. The emergent genotype 2 strains have become predominant in China in the past years that are genetically distant from the vaccine strain. Here, we aimed to evaluate the current infectious status of CSF, and for this purpose 24 isolates of CSFV were identified from different areas of China during 2016–2018. Phylogenetic analysis of NS5B, E2 and full genome revealed that the new isolates were clustered into subgenotype 2.1d and 2.1b, while subgenotype 2.1d was predominant. Moreover, E2 and Erns displayed multiple variations in neutralizing epitope regions. Furthermore, the new isolates exhibited capacity to escape C-strain-derived antibody neutralization compared with the Shimen strain (genotype 1). Potential positive selection sites were identified in antigenic regions of E2 and Erns, which are related with antibody binding affinity. Recombination events were predicted in the new isolates with vaccine strains in the E2 gene region. In conclusion, the new isolates showed molecular variations and antigenic alterations, which provide evidence for the emergence of vaccine-escaping mutants and emphasize the need of updated strategies for CSF control.

Immunoinformatic analysis of structural and epitope variations in the spike and Orf8 proteins of SARS-CoV-2/B.1.1.7.

A newly emerged strain of SARS‐CoV‐2 of B.1.1.7 lineage has caused a significant surge in the SARS‐CoV‐2 infections in the UK. In this study, changes in the epitopes of spike and orf8 proteins in SARS‐CoV‐2 of B.1.1.7 lineage were investigated. Genomic alignment of the SARS‐CoV‐2/B.1.1.7 with SARS‐CoV‐2/Wuhan showed the presence of several mutations in orf1a/b, spike, orf8, and N proteins of SARS‐CoV‐2/B.1.1.7. Molecular models of spike and orf8 proteins were constructed by homology modeling. Superimposition between the spike proteins of SARS‐CoV‐2/Wuhan and SARS‐CoV‐2/B.1.1.7 showed noticeable variations in the spatial orientation in Val70‐Asn74 and Thr250‐Ser255 regions. This may have also resulted in the extension of the epitopic region at Ser244‐Gly249 in the SARS‐CoV‐2/B.1.1.7 spike protein. Superimposition of the SARS‐CoV‐2/B.1.1.7 spike protein over Fab‐spike protein complexes of SARS‐CoV‐2/Wuhan also showed subtle variations in the antibody binding affinity targeting the N‐terminal domain of the spike protein. Epitopic variations were also observed between the corresponding orf8 regions of SARS‐CoV‐2/Wuhan and SARS‐CoV‐2/B.1.1.7. Moreover, the presence of a stop codon at position 27 in orf8 connotes the emergence of two frames (orf8a and orf8b) in SARS‐CoV‐2, which further hampers its extracellular secretion, and in turn, immunogenicity. The findings of the present study could further be used to develop targeted immunotherapeutics.

Dual Antibody-Conjugated Amyloid Nanorods to Promote Selective Cell-Cell Interactions.

Grafting biomolecules on nanostructures’ surfaces is an increasingly used strategy to target pathogenic cells, with both diagnostic and therapeutic applications. However, nanomaterials monofunctionalized by conjugating a single type of ligand find limited uses in pathologies/therapies that require two or more targets/receptors to be targeted and/or activated with a single molecular entity simultaneously. Therefore, multivalent nanomaterials for dual- or multitargeting are attracting significant interest. This study provides a proof of concept of such nanostructures. We have recently developed a modular methodology that allows obtaining amyloid-based materials decorated with active globular domains. Here, this approach is exploited to generate functional amyloid fibrils displaying antibody capture moieties. A high antibody binding affinity and capacity for the resulting nanofibrils, whose size can be manipulated to obtain homogeneous nanorods with high biocompatibility, are demonstrated. These nanorods are then used for specific antibody-mediated targeting of different cell types. Simultaneous conjugation of these nanorods with different antibodies allows obtaining a mimic of a bispecific antibody that redirects T lymphocytes to tumoral cells, holding high potential for immunotherapy. Overall, the work illustrates a modular and straightforward strategy to obtain preparative quantities of multivalent antibody-functionalized nanomaterials with multitargeting properties without the need for covalent modification.

Comparison of the SARS-CoV-2 (2019-nCoV) M protein with its counterparts of SARS-CoV and MERS-CoV species

Coronaviruses M proteins are well-represented in the major protein component of the viral envelope. During the viral assembly, they play an important role by association with all other viral structural proteins. Despite their crucial functions, very little information regarding the structures and functions of M proteins is available. Here we utilize bioinformatic tools from available sequences and 3D structures of SARS-CoV, SARS-CoV2, and MERS-CoV M proteins in order to predict potential B-cell epitopes and assessing antibody binding affinity. Such study aims to aid finding more effective vaccines and recognize neutralizing antibodies. we found some rather exciting differences between SARS-COV-2, SARS-Cov and MERS-CoV M proteins. Two SARS-CoV-2 peptides with significant antigen presentation scores for human cell surface proteins have been identified. The results reveal that N-terminal domains of M proteins of SARS-CoV and SARS-CoV2 are translocated (outside) whereas it is inside (cytoplasmic side) in MERS-CoV.

Molecular Cloning, Expression and Purification of Recombinant VHH Proteins Expressed in <i>E. coli</i>

Variable Heavy-chain Homodimer (VHH) or Nanobody is a recombinant dromedary antibody fragment which is classified as the smallest antibody fragments with the highest binding affinity and specificity of the original whole antibody. In this study the Expression of Nanobodies in E. coli WK6 cell periplasm was performed. The protein expression and purity was and analyzed by Affinity Chromatography, SDS PAGE and Western Blot. Upon elution with Imidazole, the concentrations observed using the OD280 nm of the eluted fractions EI, E2 and E3 were observed to be 0.42 μg/ml, 0.13 μg/ml and −0.46 μg/ml respectively. This gives an Antilog of 7.88 kDa which showed the calculated molecular size of our band. The SDS-PAGE gel reading was confirmed using Western blot analysis and illustrated as the specific binding of the mouse Anti-His antibody to the Histidine tag of the Nanobody. The Nanobody protein expression was then analyzed further with western blotting showed a strong signal at the region corresponding to the 15 kDa marker indicating presence of the Nanobody gene. This was taken as further confirmation of the protein expression from the bacterial cells.

Covalent Stabilization of Antibody Recruitment Enhances Immune Recognition of Cancer Targets

Antibody recruiting molecules (ARMs) represent an important class of chemical tools with therapeutic potential. ARMs function by simultaneously binding to a hapten specific serum antibody (Ab) and a cancer cell surface protein, thereby forming ARM:Ab complexes on the cancer cell surface. These complexes activate immune cell recognition and elimination of the cancer cell. Problematically, ARM function in human patients may be limited by conditions that drive the dissociation of ARM:Ab complexes; namely intrinsically low binding affinity of anti-hapten antibodies in human serum. To address this potential limitation, we applied covalent ARM (cARM) chemical tools to eliminate the ARM:Ab equilibrium through a covalent linkage. In the current study we determine to what extent stabilizing ARM:Ab complexes via cARMs enhances target immune recognition. We observe cARMs significantly enhance immune function relative to ARMs and demonstrate ARM therapeutic function can be dramatically enhanced by increasing the stability of ARM:antibody complexes on cancer cells.

The SARS-CoV-2 B.1.618 variant slightly alters the spike RBD-ACE2 binding affinity and is an antibody escaping variant: a computational structural perspective.

Continuing reports of new SARS-CoV-2 variants have caused worldwide concern and created a challenging situation for clinicians. The recently reported variant B.1.618, which possesses the E484K mutation specific to the receptor-binding domain (RBD), as well as two deletions of Tyr145 and His146 at the N-terminal binding domain (NTD) of the spike protein, must be studied in depth to devise new therapeutic options. Structural variants reported in the RBD and NTD may play essential roles in the increased pathogenicity of this SARS-CoV-2 new variant. We explored the binding differences and structural-dynamic features of the B.1.618 variant using structural and biomolecular simulation approaches. Our results revealed that the E484K mutation in the RBD slightly altered the binding affinity through affecting the hydrogen bonding network. We also observed that the flexibility of three important loops in the RBD required for binding was increased, which may improve the conformational optimization and consequently binding of the new variant. Furthermore, we found that deletions of Tyr145 and His146 at the NTD reduced the binding affinity of the monoclonal antibody (mAb) 4A8, and that the hydrogen bonding network was significantly affected consequently. This data show that the new B.1.618 variant is an antibody-escaping variant with slightly altered ACE2–RBD affinity. Moreover, we provide insights into the binding and structural-dynamics changes resulting from novel mutations in the RBD and NTD. Our results suggest the need for further in vitro and in vivo studies that will facilitate the development of possible therapies for new variants such as B.1.618.

Structure, Function, and Therapeutic Use of IgM Antibodies.

Natural immunoglobulin M (IgM) antibodies are pentameric or hexameric macro-immunoglobulins and have been highly conserved during evolution. IgMs are initially expressed during B cell ontogeny and are the first antibodies secreted following exposure to foreign antigens. The IgM multimer has either 10 (pentamer) or 12 (hexamer) antigen binding domains consisting of paired µ heavy chains with four constant domains, each with a single variable domain, paired with a corresponding light chain. Although the antigen binding affinities of natural IgM antibodies are typically lower than IgG, their polyvalency allows for high avidity binding and efficient engagement of complement to induce complement-dependent cell lysis. The high avidity of IgM antibodies renders them particularly efficient at binding antigens present at low levels, and non-protein antigens, for example, carbohydrates or lipids present on microbial surfaces. Pentameric IgM antibodies also contain a joining (J) chain that stabilizes the pentameric structure and enables binding to several receptors. One such receptor, the polymeric immunoglobulin receptor (pIgR), is responsible for transcytosis from the vasculature to the mucosal surfaces of the lung and gastrointestinal tract. Several naturally occurring IgM antibodies have been explored as therapeutics in clinical trials, and a new class of molecules, engineered IgM antibodies with enhanced binding and/or additional functional properties are being evaluated in humans. Here, we review the considerable progress that has been made regarding the understanding of biology, structure, function, manufacturing, and therapeutic potential of IgM antibodies since their discovery more than 80 years ago.

Computational engineering the binding affinity of Adalimumab monoclonal antibody for designing potential biosimilar candidate

Amongst the anti-TNF-α therapy for rheumatoid arthritis and other autoimmune diseases, Adalimumab mAb is one of the best candidates. However, several risk factors are found to be associated with higher doses. Improvement of the binding properties will therefore significantly increase its therapeutic efficacy, reduce the dosage requirements, and ultimately the associated toxicity and treatment cost. Here, we proposed a systematic in silico approach of finding newer mAb variants with improved binding properties. Using various bioinformatics tools, we have identified the significant amino acid residues on Adalimumab mAb. Next, we searched for the suitability of the other residues for mutating the significant residues and from the combinations of suitable mutations, variants were designed. To find the most significant ones, binding properties of the variants were compared with the wild type Adalimumab mAb using molecular docking scrutiny and molecular dynamics simulation. Finally, structural properties between the variant and wild type were analyzed. We have identified the six most significant residues on Adalimumab mAb involved in the antigen-antibody interactions. Using the suitable mutations replacing each of these residues, we have modeled 143 variants. From several docking analyses, we have found five significant variants and after molecular dynamics simulation, one most significant variant with improved binding affinity was identified whose structural properties are similar to the wild type Adalimumab mAb. Designed variant from this study, may provide newer insights on the structure-based affinity improvements of monoclonal antibodies and likewise modifications of the Fc region will also improve the therapeutic effector functions of antibodies too.

Affinity-Restricted Memory B Cells Dominate Recall Responses to Heterologous Flaviviruses

Memory B cells (MBCs) can respond to heterologous antigens either by molding new specificities through secondary germinal centers (GCs) or by selecting preexisting clones without further affinity maturation. To distinguish these mechanisms in flavivirus infections and immunizations, we studied recall responses to envelope protein domain III (DIII). Conditional deletion of activation-induced cytidine deaminase (AID) between heterologous challenges of West Nile, Japanese encephalitis, Zika, and dengue viruses did not affect recall responses. DIII-specific MBCs were contained mostly within the plasma-cell-biased CD80+ subset, and few GCs arose following heterologous boosters, demonstrating that recall responses are confined by preexisting clonal diversity. Measurement of monoclonal antibody (mAb) binding affinity to DIII proteins, timed AID deletion, single-cell RNA sequencing, and lineage tracing experiments point to selection of relatively low-affinity MBCs as a mechanism to promote diversity. Engineering immunogens to avoid this MBC diversity may facilitate flavivirus-type-specific vaccines with minimized potential for infection enhancement.

Binding affinity determination of therapeutic antibodies to membrane protein targets: Kinetic Exclusion Assay using cellular membranes for anti-CD20 antibody

Antibody-based therapeutics targeting membrane proteins have evolved as a major modality for the treatment of cancer, inflammation and autoimmune diseases. There are numerous challenges, ranging from desired epitope expression to reliable binding/functional assays which are associated with developing antibodies for this target class. Specifically, having a robust methodology for characterizing antibody interaction with a membrane protein target is essential for providing guidance on dosing, potency and thus expected efficacy. Fluorescence-activated cell sorting (FACS) has been commonly used to characterize antibodies binding to membrane protein targets. FACS provides information about the antibody-receptor complex (antibody bound to cells) and the apparent equilibrium dissociation constant (KD′) is elucidated by fitting the antibody-receptor binding isotherm as a function of total antibody concentration to a nonlinear regression model. Conversely, Kinetic Exclusion Assay (KinExA) has been used to measure solution-based equilibrium dissociation constant (KD) of antibodies. Here, KD is determined by measuring the free antibody concentration at equilibrium in a series of solutions in which the antibody is at constant concentration and the receptor (either in the membrane or the cell) is titrated. We measured the binding affinity of the anti-CD20 antibody, Rituximab, using both FACS and KinExA. There was ~25-fold difference in the binding affinity measured by these two techniques. We have explored this discrepancy through additional experiments around the mathematical framework involved in the analysis of these two different binding assays. Finally, our study concluded that KinExA enables accurate measurement of the KD for strong protein-protein interactions (sub-nanomolar values) compared to FACS.