Generation Of mAbs Research Articles

Development of Dengue Virus Serotype-Specific NS1 Capture Assays for the Rapid and Highly Sensitive Identification of the Infecting Serotype in Human Sera.

Dengue fever can be caused by one of four distinct dengue virus (DENV) serotypes that cocirculate in many parts of the world. Point of care serotype-specific nonstructural protein-1 (NS1) capture assays for the rapid serotyping of DENV in human sera would greatly support epidemiological surveillance and potentially also prognosis in individual patients. To ensure both serotype specificity and broad coverage of variants within serotypes, we have applied an innovative approach for the generation and selection of serotype-specific anti-NS1 mAbs. To elicit mAbs against conformational epitopes, NMRI mice were immunized with living HEK 293 transfectants expressing the native target Ags in multiple display on the cell surface. For each serotype, three different NS1 sequence variants were sequentially used for immunization of mice, hybridoma selection, and capture assay development, respectively. Selection of optimal combinations of capturing and detecting mAbs yielded highly sensitive and specific NS1 serotyping ELISAs (st-ELISAs) for the four serotypes. st-ELISA testing of 41 dengue patient sera showed a 100% concordance with the serotype determined by serotype-specific reverse transcriptase real-time quantitative PCR. The respective NS1 variants could be detected for ∼10 d after the onset of illness. Ab-dependent enhancement of DENV infections may be associated with a specific range of pre-existing anti-DENV serological Ab titers. Testing of patient sera with the developed st-ELISAs will not only be useful for epidemiological studies and surveillance, but it may also help to develop and validate assays that can distinguish protective versus enhancing Ab responses for risk assessment for the development of severe dengue disease in individual patients.

Analysis of the effect of promoter type and skin pretreatment on antigen expression and antibody response after gene gun-based immunization.

Monoclonal antibodies (mAbs) have enabled numerous basic research discoveries and therapeutic approaches for many protein classes. However, there still exist a number of target classes, such as multi-pass membrane proteins, for which antibody discovery is difficult, due in part to lack of high quality, recombinant protein. Here we describe the impact of several parameters on antigen expression and the development of mAbs against human claudin 4 (CLDN4), a potential multi-indication cancer target. Using gene gun-based DNA delivery and bioluminescence imaging, we optimize promoter type by comparing expression profiles of four robust in vivo promoters. In addition, we observe that most vectors rapidly lose expression, ultimately reaching almost background levels by three days post-delivery. Recognizing this limitation, we next explored skin pretreatment strategies as an orthogonal method to further boost the efficiency of mAb generation. We show that SDS pretreatment can boost antigen expression, but fails to significantly increase mAb discovery efficiency. In contrast, we find that sandpaper pretreatment yields 5-fold more FACS+ anti-CLDN4 hybridomas, without impacting antigen expression. Our findings coupled with other strategies to improve DNA immunizations should improve the success of mAb discovery against other challenging targets and enable the generation of critical research tools and therapeutic candidates.

Frontiers of monoclonal antibodies: Applications in medical practices.

With the flourishing of innovation in drug discovery into a new era of personalized therapy, the use of monoclonal antibodies (mAbs) in the treatment of various ailments lies at the forefront. Major improvements in genetic sequencing and biomedical techniques as well as research into mAbs emphasize on determining new targets for advanced therapy while maximizing efficacy for clinical application. However, a balance has to be achieved concerning developing a target with low toxicity combined with high specificity and versatility, to allow a specific antibody to facilitate several biotic effects, ranging from neutralization of virus mechanisms to modulation of immune response and maintaining low global economic cost. Presently, there are approximately 30 mAbs' permitted for therapeutic use with many more being tested in clinical trials. Nevertheless, the heavy cost of mAbs' production, stowage and management as well as the subsequent hindrances to their development are outweighed by mAbs' clinical advantages. Compared to conventional drugs, since mAbs use as pharmacologic iotas have specific physical features and modes of action, they should be considered as a discrete therapeutic category. In this review, the history of mAb generation and the innovative technological applications of mAbs that has advanced in clinical practices is reviewed.

A toolbox of immunoprecipitation-grade monoclonal antibodies to human transcription factors.

A key component of efforts to address the reproducibility crisis in biomedical research is the development of rigorously validated and renewable protein-affinity reagents. As part of the US National Institutes of Health (NIH) Protein Capture Reagents Program (PCRP), we have generated a collection of 1,406 highly validated immunoprecipitation- and/or immunoblotting-grade mouse monoclonal antibodies (mAbs) to 737 human transcription factors, using an integrated production and validation pipeline. We used HuProt human protein microarrays as a primary validation tool to identify mAbs with high specificity for their cognate targets. We further validated PCRP mAbs by means of multiple experimental applications, including immunoprecipitation, immunoblotting, chromatin immunoprecipitation followed by sequencing (ChIP-seq), and immunohistochemistry. We also conducted a meta-analysis that identified critical variables that contribute to the generation of high-quality mAbs. All validation data, protocols, and links to PCRP mAb suppliers are available at http://proteincapture.org.

Generation of a monoclonal antibody recognizing the heavily glycosylated CD45 protein and its application on identifying circulating tumor cells.

Here, we provide direct evidence that using recombinant proteins expressed in eukaryotic cells as antigen is a practical way to generate monoclonal antibodies (mAbs) against heavily glycosylated proteins. Heavily glycosylated proteins are typically difficult targets for mAb generation, being limited by unsatisfactory affinity and low specificity. Using the heavily glycosylated CD45 protein as an example, we demonstrate the entire process of expressing the protein in eukaryotic cells and using it as an antigen to generate CD45-targeting mAbs in mice. The mAbs generated showed robust affinity and specificity, which are crucial factors for differentiate circulating tumor cells from white blood cells in human breast cancer patient samples. Only 1 cell fusion and 2 cyclic sub-cloning steps were necessary before mAbs with satisfactory performance were obtained.

Evaluation of the fusion partner cell line SPYMEG for obtaining human monoclonal antibodies against influenza B virus.

Influenza B virus has been known to infect humans and other animals, including seals. Vaccination efficacy varies across seasons. Human monoclonal antibodies (mAbs) can be useful for developing novel vaccines, guided by epitope analysis, and can be used therapeutically. Hybridoma technology has been used to make mAbs. Here we evaluated SPYMEG as a fusion partner cell line for human mAb generation specific to influenza B hemagglutinin (HA). SPYMEG is a human/murine myeloma partner cell line that has previously been used to generate human mAbs that recognize the HA of influenza A and B viruses. Peripheral blood mononuclear cells were obtained from 16 volunteers, previously vaccinated with the 2014–2015 trivalent seasonal influenza vaccine, and were fused with SPYMEG to yield hybridomas. The resulting hybridomas were screened for antigen-specific antibody secretion and cloned by limiting dilution. We obtained 32 stable clones secreting anti-influenza B HA human IgG, although most of these clones were obtained from one volunteer (SeaV-29) who had a robust immune response. We conclude that SPYMEG is a good fusion partner cell line, although cloning by limiting dilution may lead to significant loss of hybridomas.

Next-generation anti-CD20 monoclonal antibodies in autoimmune disease treatment

The clinical success of anti-CD20 monoclonal antibody (mAb)-mediated B cell depletion therapy has contributed to the understanding of B cells as major players in several autoimmune diseases. The first therapeutic anti-CD20 mAb, rituximab, is a murine–human chimera to which many patients develop antibodies and/or experience infusion-related reactions. A second generation of anti-CD20 mAbs has been designed to be more effective, better tolerated, and of lower immunogenicity. These include the humanized versions: ocrelizumab, obinutuzumab, and veltuzumab, and the fully human, ofatumumab. We conducted a literature search of relevant randomized clinical trials in the PubMed database and ongoing trials in Clinicaltrials.gov. Most of these trials have evaluated intravenous ocrelizumab or subcutaneous ofatumumab in rheumatoid arthritis, multiple sclerosis, or systemic lupus erythematosus. Understanding how newer anti-CD20 mAbs compare with rituximab in terms of efficacy, safety, convenience, and cost is important for guiding future management of anti-CD20 mAb therapy in autoimmune diseases.

Crystallizable Fragment Glycoengineering for Therapeutic Antibodies Development.

Monoclonal antibody (mAb)-based therapeutics are the fastest growing class of human pharmaceuticals. They are typically IgG1 molecules with N-glycans attached to the N297 residue on crystallizable fragment (Fc). Different Fc glycoforms impact their effector function, pharmacokinetics, stability, aggregation, safety, and immunogenicity. Fc glycoforms affect mAbs effector functions including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) by modulating the Fc–FcγRs and Fc–C1q interactions. While the terminal galactose enhances CDC activity, the fucose significantly decreases ADCC. Defucosylated immunoglobulin Gs (IgGs) are thus highly pursued as next-generation therapeutic mAbs with potent ADCC at reduced doses. A plethora of cell glycoengineering and chemoenzymatic glycoengineering strategies is emerging to produce IgGs with homogenous glycoforms especially without core fucose. The chemoenzymatic glycosylation remodeling also offers useful avenues for site-specific conjugations of small molecule drugs onto mAbs. Herein, we review the current progress of IgG-Fc glycoengineering. We begin with the discussion of the structures of IgG N-glycans and biosynthesis followed by reviewing the impact of IgG glycoforms on antibody effector functions and the current Fc glycoengineering strategies with emphasis on Fc defucosylation. Furthermore, we briefly discuss two novel therapeutic mAbs formats: aglycosylated mAbs and Fc glycan specific antibody–drug conjugates (ADCs). The advances in the understanding of Fc glycobiology and development of novel glycoengineering technologies have facilitated the generation of therapeutic mAbs with homogenous glycoforms and improved therapeutic efficacy.

Conformational Fingerprinting Using Monoclonal Antibodies (on the Example of Angiotensin I-Converting Enzyme-ACE).

During the past 30 years my laboratory has generated 40+ monoclonal antibodies (mAbs) directed to structural and conformational epitopes on human ACE as well as ACE from rats, mice and other species. These mAbs were successfully used for detection and quantification of ACE by ELISA, Western blotting, flow cytometry and immunohistochemistry. In all these applications mainly single mAbs were used. We hypothesized that we can obtain a completely new kind of information about ACE structure and function if we use the whole set of mAbs directed to different epitopes on the ACE molecule. When we finished epitope mapping of all mAbs to ACE (and especially, those recognizing conformational epitopes), we realized that we had obtained a new tool to study ACE. First, we demonstrated that binding of some mAbs is very sensitive to local conformational changes on the ACE surface—due to local denaturation, inactivation, ACE inhibitor or mAbs binding or due to diseases. Second, we were able to detect, localize and characterize several human ACE mutations. And, finally, we established a new concept—conformational fingerprinting of ACE using mAbs that in turn allowed us to obtain evidence for tissue specificity of ACE, which has promising scientific and diagnostic perspectives. The initial goal for the generation of mAbs to ACE 30 years ago was obtaining mAbs to organ-specific endothelial cells, which could be used for organ-specific drug delivery. Our systematic work on characterization of mAbs to numerous epitopes on ACE during these years has lead not only to the generation of the most effective mAbs for specific drug/gene delivery into the lung capillaries, but also to the establishment of the concept of conformational fingerprinting of ACE, which in turn gives a theoretical base for the generation of mAbs, specific for ACE from different organs. We believe that this concept could be applicable for any glycoprotein against which there is a set of mAbs to different epitopes.

Generation and selection of naïve Fab library for parasitic antigen: Anti-BmSXP antibodies for lymphatic filariasis.

Phage display has been applied successfully as a tool for the generation of monoclonal antibodies (mAbs). Naive antibody libraries are unique as they are able to overcome several limitations associated with conventional mAb generation methods like the hybridoma technology. Here, we performed an in vitro selection and generation of Fab antibodies against Brugia malayi SXP protein (BmSXP), a recombinant antigen for the detection of lymphatic filariasis. We developed a naïve multi ethnic Fab antibody library with an estimated diversity of 2.99 × 109 . The antibody library was used to screen for mAbs against BmSXP recombinant antigen. Soluble monoclonal Fab antibodies against BmSXP were successfully isolated from the naïve library. The Fab antibodies obtained were expressed and analyzed to show its binding capability. The diversity obtained from a pool of donors from various ethnic groups allowed for a diverse antibody library to be generated. The mAbs obtained were also functional in soluble form, which makes it useful for further downstream applications. We believe that the Fab mAbs are valuable for further studies and could also contribute to improvements in the diagnosis of filariasis.

Development of novel monoclonal antibodies against starch and ulvan - implications for antibody production against polysaccharides with limited immunogenicity

Monoclonal antibodies (mAbs) are widely used and powerful research tools, but the generation of mAbs against glycan epitopes is generally more problematic than against proteins. This is especially significant for research on polysaccharide-rich land plants and algae (Viridiplantae). Most antibody production is based on using single antigens, however, there are significant gaps in the current repertoire of mAbs against some glycan targets with low immunogenicity. We approached mAb production in a different way and immunised with a complex mixture of polysaccharides. The multiplexed screening capability of carbohydrate microarrays was then exploited to deconvolute the specificities of individual mAbs. Using this strategy, we generated a set of novel mAbs, including one against starch (INCh1) and one against ulvan (INCh2). These polysaccharides are important storage and structural polymers respectively, but both are generally considered as having limited immunogenicity. INCh1 and INCh2 therefore represent important new molecular probes for Viridiplantae research. Moreover, since the α-(1-4)-glucan epitope recognised by INCh1 is also a component of glycogen, this mAb can also be used in mammalian systems. We describe the detailed characterisation of INCh1 and INCh2, and discuss the potential of a non-directed mass-screening approach for mAb production against some glycan targets.

Abstract 54: The many faces of antibody in G protein-coupled receptor biology

Abstract G protein-coupled receptors (GPCRs), the largest family of cell-surface receptor proteins mediating signal transmission, play a pivotal role in many physiological functions and are involved in multiple diseases. Although receptor activities of GPCRs are successfully modulated by many small molecules which represent 30% of all marketed drugs today, in fact only a few GPCR members are targeted and many more GPCRs of interest are intractable targets of small molecules, like orphan GPCRs and GPCRs with large binding sites. Besides, small molecule drugs rarely trigger GPCR-mediated apoptosis or induce direct cell killing, which is crucial for cancer therapies. Therefore, for these GPCRs, antibody-based drug would be a good alternative. Despite being eagerly sought for, the production of monoclonal antibody (mAb) targeting GPCR is hindered by the low expression of recombinant GPCR on cell surface, and relatively small exposed regions with glycosylation and conformational heterogeneity. Therefore those conventional methods that were well established for generation of mAbs targeting soluble proteins usually showed frustrating results in the case of GPCRs. To bypass the production and purification of recombinant GPCR proteins, we developed a novel approach of transplanting immunogenic conformational epitopes of GPCR into an antibody scaffold to make a water soluble surrogate antigen of GPCR, named as GPCR-antigenized antibody which can be easily and abundantly produced in bacteria for animal immunization. In coupled with phage display technology and cell panning strategy, which allow the retrieval of specific binders from a huge number of candidates, we have successfully isolated scFv antibodies for two human GPCRs, the MAS1 receptor and the chemokine receptor CXCR4. These antibodies showed good specificity in immunofluorescent staining, flow cytometry analysis, western protein immunoblot and immunoprecipitation assay. The results demonstrated this novel approach may offer a generic and effective method to generate specific monoclonal antibody targeting GPCR for diagnostic and therapeutic applications. [The project is partly funded by a CUHK direct grant (4054300)] Citation Format: Lei Chen, Wing-Tai Cheung. The many faces of antibody in G protein-coupled receptor biology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 54. doi:10.1158/1538-7445.AM2017-54

CD20-based Immunotherapy of B-cell Derived Hematologic Malignancies.

CD20 is a surface antigen, which is expressed at certain stages of B-cell differentiation. Targeting the CD20-positive B-cells with therapeutic monoclonal antibodies (MAbs) has been an effectual strategy in the treatment of hematologic malignancies such as non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL). Initial success with Rituximab (RTX) has encouraged the creation and development of more effective CD20 based therapeutics. However, treatment with conventional MAbs has not been adequate to overcome the problems such as refractory/ relapsed disease. In this regard, new generations of MAbs with enhanced affinity or improved anti-tumor properties have been developed. CD20 directed therapeutics have heterogeneous features and mechanisms of action. Hence, having sufficient knowledge on the immunological and molecular aspects of CD20 based cancer therapy is necessary for predicting the clinical outcomes and taking the necessary measures. An extensive search was performed in PubMed and similar databases for peer-reviewed articles concerning the biology, function and characteristics of CD20 molecule as well as the mechanisms of action and evolutionary process of CD20 targeting agents. This review provides information about the current situation of CD20 targeting immunotherapeutics including MAbs, bispecific antibodies (which exert multiple functions or involve Tcells in tumor elimination) and CAR T-cells (engineered T-cells armed with chimeric antigen receptors). Moreover, limitations, challenges and available solutions regarding the application of CD20 targeting treatments are addressed. Utilization of CD20-targeted therapeutics, due to their diverse properties, requires special considerations.

Hapten-Specific Single-Cell Selection of Hybridoma Clones by Fluorescence-Activated Cell Sorting for the Generation of Monoclonal Antibodies.

The conventional hybridoma screening and subcloning process is generally considered to be one of the most critical steps in hapten-specific antibody production. It is time-consuming, monoclonality is not guaranteed, and the number of clones that can be screened is limited. Our approach employs a novel hapten-specific labeling technique of hybridoma cells. This allows for fluorescence-activated cell sorting (FACS) and single-cell deposition and thereby eliminates the above-mentioned problems. A two-step staining approach is used to detect antigen specificity and antibody expression: in order to detect antigen specificity, hybridoma cells are incubated with a hapten-horseradish peroxidase conjugate (hapten-HRP), which is subsequently incubated with a fluorophore-labeled polyclonal anti-peroxidase antibody (anti-HRP-Alexa Fluor 488). To characterize the expression of membrane-bound immunoglobulin G (IgG), a fluorophore-labeled anti-mouse IgG antibody (anti-IgG-Alexa Fluor 647) is used. Hundreds of labeled hybridoma cells producing monoclonal antibodies (mAbs) specific for a hapten were rapidly isolated and deposited from a fusion mixture as single-cell clones via FACS. Enzyme-linked immunosorbent assay (ELISA) measurements of the supernatants of the sorted hybridoma clones revealed that all hapten-specific hybridoma clones secrete antibodies against the target. There are significant improvements using this high-throughput technique for the generation of mAbs including increased yield of antibody-producing hybridoma clones, ensured monoclonality of sorted cells, and reduced development times.

A semisynthetic Streptococcus pneumoniae serotype 8 glycoconjugate vaccine.

Glycoconjugate vaccines based on capsular polysaccharides (CPSs) of pathogenic bacteria such as Streptococcus pneumoniae successfully protect from disease but suffer from incomplete coverage, are troublesome to manufacture from isolated CPSs, and lack efficacy against certain serotypes. Defined, synthetic oligosaccharides are an attractive alternative to isolated CPSs but require the identification of immunogenic and protective oligosaccharide antigens. We describe a medicinal chemistry strategy based on a combination of automated glycan assembly (AGA), glycan microarray-based monoclonal antibody (mAb) reverse engineering, and immunological evaluation in vivo to uncover a protective glycan epitope (glycotope) for S. pneumoniae serotype 8 (ST8). All four tetrasaccharide frameshifts of ST8 CPS were prepared by AGA and used in glycan microarray experiments to identify the glycotopes recognized by antibodies against ST8. One tetrasaccharide frameshift that was preferentially recognized by a protective, CPS-directed mAb was conjugated to the carrier protein CRM197. Immunization of mice with this semisynthetic glycoconjugate followed by generation and characterization of a protective mAb identified protective and nonprotective glycotopes. Immunization of rabbits with semisynthetic ST8 glycoconjugates containing protective glycotopes induced an antibacterial immune response. Coformulation of ST8 glycoconjugates with the marketed 13-valent glycoconjugate vaccine Prevnar 13 yielded a potent 14-valent S. pneumoniae vaccine. Our strategy presents a facile approach to develop efficient semisynthetic glycoconjugate vaccines.

Conformational Fingerprinting Using Monoclonal Antibodies (on the Example of Angiotensin I-Converting Enzyme-ACE)

During the past 30 years my laboratory has generated 40+ monoclonal antibodies (mAbs) directed to structural and conformational epitopes on human ACE as well as ACE from rats, mice and other species. These mAbs were successfully used for detection and quantification of ACE by ELISA, Western blotting, flow cytometry and immunohistochemistry. In all these applications mainly single mAbs were used. We hypothesized that we can obtain a completely new kind of information about ACE structure and function if we use the whole set of mAbs directed to different epitopes on the ACE molecule. When we finished epitope mapping of all mAbs to ACE (and especially, those recognizing conformational epitopes), we realized that we had obtained a new tool to study ACE. First, we demonstrated that binding of some mAbs is very sensitive to local conformational changes on the ACE surface-due to local denaturation, inactivation, ACE inhibitor or mAbs binding or due to diseases. Second, we were able to detect, localize and characterize several human ACE mutations. And, finally, we established a new concept - conformational fingerprinting of ACE using mAbs that in turn allowed us to obtain evidence for tissue specificity of ACE, which has promising scientific and diagnostic perspectives. The initial goal for the generation of mAbs to ACE 30 years ago was obtaining mAbs to organ-specific endothelial cells, which could be used for organ-specific drug delivery. Our systematic work on characterization of mAbs to numerous epitopes on ACE during these years has lead not only to the generation of the most effective mAbs for specific drug/gene delivery into the lung capillaries, but also to the establishment of the concept of conformational fingerprinting of ACE, which in turn gives a theoretical base for the generation of mAbs, specific for ACE from different organs. We believe that this concept could be applicable for any glycoprotein against which there is a set of mAbs to different epitopes.

Generation of mAbs to foot-and-mouth disease virus serotype A and application in a competitive ELISA for serodiagnosis.

BackgroundFoot–and–mouth disease (FMD) is an economically devastating disease that severely limits international trade of animals. Of the seven FMD virus (FMDV) serotypes, serotype A is one of the most widespread cross the world. Currently antibodies to FMDV are detected in animals using the virus neutralization test (VNT) and the enzyme-linked immunosorbent assay (ELISA). The VNT is laborious, time–consuming and reliant on live virus and cell cultures, while ELISA has the advantage of using inactivated antigens and often provides more reproducible results. The aim of this study was to develop a reliable and rapid competitive ELISA (cELISA) for the detection of antibodies to FMDV serotype A (FMDV/A).ResultsA panel of FMDV/A specific monoclonal antibodies (mAbs) was generated and their ability to compete with a polyclonal serum from FMDV/A–infected cattle was examined. Two mAbs inhibited the binding of a polyclonal serum to FMDV/A viruses. The binding epitopes of each were determined as conformational and located on the VP2 viral capsid protein. The FMDV/A cELISA was developed using these two mAbs and FMDV/A inactivated virus as antigen. The diagnostic specificity and sensitivity were 99.7 and 99.3% (98.5–100%) respectively, based on a predetermined cut–off of 50% inhibition. When analysing sera from animals experimentally infected with FMDV/A, the cELISA detected antibodies from 5-days post infection (dpi) and remained positive for at least 21–28 days post infection. Comparison based on the Kappa coefficient showed strong agreement (90–94%) between cELISA and VNT.ConclusionThe cELISA results are comparable to the VNT for antibody detection making it a simple and reliable test to detect antibodies against FMDV/A.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0650-z) contains supplementary material, which is available to authorized users.

Lessons from the Crystal Structure of the S. aureus Surface Protein Clumping Factor A in Complex With Tefibazumab, an Inhibiting Monoclonal Antibody

The Staphylococcus aureus fibrinogen binding MSCRAMM (Microbial Surface Components Recognizing Adhesive Matrix Molecules), ClfA (clumping factor A) is an important virulence factor in staphylococcal infections and a component of several vaccines currently under clinical evaluation. The mouse monoclonal antibody aurexis (also called 12-9), and the humanized version tefibazumab are therapeutic monoclonal antibodies targeting ClfA that in combination with conventional antibiotics were effective in animal models but showed less impressive efficacy in a limited Phase II clinical trial. We here report the crystal structure and a biochemical characterization of the ClfA/tefibazumab (Fab) complex. The epitope for tefibazumab is located to the “top” of the N3 subdomain of ClfA and partially overlaps with a previously unidentified second binding site for fibrinogen. A high-affinity binding of ClfA to fibrinogen involves both an interaction at the N3 site and the previously identified docking of the C-terminal segment of the fibrinogen γ-chain in the N2N3 trench. Although tefibazumab binds ClfA with high affinity we observe a modest IC50 value for the inhibition of fibrinogen binding to the MSCRAMM. This observation, paired with a common natural occurring variant of ClfA that is not effectively recognized by the mAb, may partly explain the modest effect tefibazumab showed in the initial clinic trail. This information will provide guidance for the design of the next generation of therapeutic anti-staphylococcal mAbs targeting ClfA.

Identification of a novel Aleutian mink disease virus B-cell epitope using a monoclonal antibody against VP2 protein

Aleutian mink disease virus (AMDV) is a parvovirus that causes an immune complex-mediated disease in minks. Capsid protein VP2 is a major structural viral protein and can be used to diagnose AMDV. In this study, a specific monoclonal antibody, 1M13, was produced against the AMDV VP2 protein (amino acids 291–502). A linear VP2-protein epitope was identified by subjecting a series of partially overlapping synthesized peptides to be enzyme-linked immunosorbent assay (ELISA) analysis. The results indicated that 386HLQQNFSTRYIYD398 was the minimal linear epitope that could be recognized by mAb 1M13. ELISA assays revealed that mink anti-AMDV sera could also recognize the minimal linear epitope. Sequence alignments demonstrated that the linear epitope is highly conserved among AMDV strains except 386H and is less conserved among Raccoon dog amdovirus, Gray fox amdovirus, Red fox amdovirus, Bat parvovirus and Mink enteritis parvovirus. Taken together, the generation of this VP2-specific mAb with a defined linear peptide epitope may have potential applications in the development of suitable diagnostic techniques for AMDV.

Production and Characterization of Monoclonal Antibody Broadly Recognizing Cry1 Toxins by Use of Designed Polypeptide as Hapten.

In this study, by use of synthesized polypeptides as haptens, a monoclonal antibody with broad recognition against seven major Cry1 toxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1E, and Cry1F) has been produced and characterized. First, by comparing the three-dimensional structures of seven Cry1 toxins, analyzing the conserved sequences, and considering the antigenicity and hydrophilicity, three polypeptides (T1, T2, and T3) have been chosen and coupled to keyhole limpet hemocyanin as immunogens for the generic monoclonal antibody (Mab) generation. Thereafter, a double antibody sandwich enzyme-linked immunosorbent assay method (DAS-ELISA) was developed for simultaneous determination of seven Cry1 toxins. The results revealed that the haptens T1, T2, and T3 had different effects in the production of antibodies. Among them, the obtained Mab (strain 2D3) generated by T2 can recognize seven Cry1 toxins simultaneously. Equilibrium dissociation constant (KD) values for seven Cry1 toxins with Mab 2D3 were 1.198 × 10(-8) M for Cry1Aa, 2.197 × 10(-8) M for Cry1Ab, 1.367 × 10(-8) M for Cry1Ac, 2.092 × 10(-8) M for Cry1B, 5.177 × 10(-8) M for Cry1C, 4.016 × 10(-8) M for Cry1E, and 3.497 × 10(-8) M for Cry1F. For 2D3-based DAS-ELISA, the limits of detection (LOD) and limits of quantification (LOQ) can reach 15 and 30 ng·mL(-1) for each Cry1 toxin, respectively. Our study is the first report of a broadly specific immunoassay for multidetermination of seven major Cry1 toxins, and it will provide a new idea and technical routes for development of multidetermination immunoassays.