Commercial mAb Research Articles

Characterization of a Panel of Monoclonal Antibodies Targeting the F-Protein of the Respiratory Syncytial Virus (RSV) for the Typing of Contemporary Circulating Strains.

Respiratory syncytial virus (RSV) is the most common cause of upper and lower respiratory tract infections in infants and young children. Virus-specific monoclonal antibodies (mAbs) can be used for diagnosis, prophylaxis, and research of RSV pathogenesis. A panel of 16 anti-RSV mAbs was obtained from mice immunized by RSV strain Long. Half of them had virus-neutralizing activity. According to Western blot all of these mAbs effectively bound native oligomeric (homodimeric and homotrimeric) forms of the RSV fusion (F) protein. Only five of the mAbs interacted with the monomeric form, and only one of these possessed neutralizing activity. None of these mAbs, nor the commercial humanized neutralizing mAb palivizumab, reacted with the denaturated F protein. Thus, interaction of all these mAbs with F protein had clear conformational dependence. Competitive ELISA and neutralization assays allowed the identification of nine antigenic target sites for the interaction of mAb with the F protein. Five partially overlapping sites may represent a complex spatial structure of one antigenic determinant, including one neutralizing and four non-neutralizing epitopes. Four sites (three neutralizing and one non-neutralizing) were found to be distinct. As a result of virus cultivation RSV-A, strain Long, in the presence of a large amount of one of the neutralizing mAbs, an escape mutant with a substitution, N240S, in the F protein, was obtained. Thus, it was shown for the first time that position 240 is critical for the protective effect of an anti-RSV antibody. To assess the ability of these mAbs to interact with modern RSV strains circulating in St. Petersburg (Russia) between 2014 and 2022, 73 RSV-A and 22 RSV-B isolates were analyzed. Six mAbs were directed to conserved epitopes of the F protein as they interacted most efficiently with both RSV subtypes in a fixed cell-ELISA and could be used for diagnostic assays detecting RSV.

THU267 A Novel In Vitro Assay Correlates Insulin Receptor Autoantibodies To Fasting Insulin In Type B Insulin Resistance

Abstract Disclosure: B. Abel: None. W. Minich: Stock Owner; Self; ImmunometriX GmbH. C. Schwiebert: Employee; Self; Bruker Corporation. T. Welsink: Employee; Self; Bruker Corporation. P. Seemann: Stock Owner; Self; ImmunometriX GmbH, selenOmed GmbH. L. Schomburg: Stock Owner; Self; selenOmed GmbH. R.J. Brown: None. Severe insulin resistance (IR) in the presence of insulin receptor autoantibodies (InsR-aAb) is known as type B IR (TBIR). TBIR has a spectrum of phenotypes, including severe IR with uncontrolled diabetes (DM), milder IR without uncontrolled DM, hypoglycemia, and remission states. Considerable progress in therapy has been achieved, but diagnosis and monitoring of InsR-aAb remains a challenge. We aimed to develop a robust in vitro method for InsR-Ab quantification and correlate it with clinical outcomes. 28 longitudinal serum samples from 9 patients with TBIR were collected before, during, and after immunosuppression. Samples included 11 in states of remission, 4 in hypoglycemia, 6 in mild IR, and 7 in severe IR. Samples from 8 patients with IR not due to TBIR (type 1 DM, N=1; type 2 DM, N=4; lipodystrophy, N=2; and INSR mutation, N=1) were used as controls. A bridge-assay for InsR-aAb detection was established using recombinant human insulin receptor as bait and detector and validated using monoclonal antibodies as positive controls. The assay consists of an immobilized recombinant InsR at a plastic surface and an InsR-Luc fusion protein for detection of InsR-aAb. There was dose-dependent increase in intensity over >2 orders of magnitude with InsR-specific commercial mAb, but not with control mAb. In the presence of insulin, strong InsR phosphorylation was observed in cells incubated with Ig from control sera, but not those incubated with Ig from TBIR patient sera. Fasting insulin was highest in severe TBIR (median 1000 [IQR 273-1000] mcU/mL), followed by mild (71[32-230]), and lower in hypoglycemia (6 [3,103]) and remission (14[6,35]), P=0.0014, and was moderately elevated in insulin resistant controls (55[11,120]). A1c was elevated in severe TBIR & controls (9.9±3.0 & 9.1±3.8%) and near-normal in mild, hypoglycemic, and remission states of TBIR (5.7±1.3, 6.0±0.7, 5.7±1.1). InsR-aAb titers changed in parallel to changes in TBIR state and severity: highest in severe TBIR (median 700 [IQR 365-977]), followed by mild (176[98-254]), and lower in hypoglycemia (14[1.3-189]) and remission (17[3-28]). All TBIR samples showed clearly elevated signal intensities versus controls (0.9[0.7-1.1], Bonferroni corrected P<0.05 for all TBIR states vs controls). During treatment, InsR-aAb for each subject declined with amelioration of disease symptoms. Fasting insulin strongly correlated with InsR-aAb titers (r=0.825, p<0.001). The assay correctly identified all samples from patients with TBIR vs controls with non-autoimmune IR, and thus is an easy to interpret diagnostic tool. InsR-aAb titers correlated with fasting insulin, supporting direct relevance of InsR-aAb as causal for disease severity and as therapeutic targets for Ab depletion. By easing diagnosis and monitoring of TBIR, the new assay for InsR-aAb quantification should enhance access to timely and effective treatment for TBIR. Presentation: Thursday, June 15, 2023

Blockade of mIL-6R alleviated lipopolysaccharide-induced systemic inflammatory response syndrome by suppressing NF-κB-mediated Ccl2 expression and inflammasome activation.

Systemic inflammatory response syndrome (SIRS) is characterized by dysregulated cytokine release, immune responses and is associated with organ dysfunction. IL‐6R blockade indicates promising therapeutic effects in cytokine release storm but still remains unknown in SIRS. To address the issue, we generated the human il‐6r knock‐in mice and a defined epitope murine anti‐human membrane‐bound IL‐6R (mIL‐6R) mAb named h‐mIL‐6R mAb. We found that the h‐mIL‐6R and the commercial IL‐6R mAb Tocilizumab significantly improved the survival rate, reduced the levels of TNF‐α, IL‐6, IL‐1β, IFN‐γ, transaminases and blood urea nitrogen of LPS‐induced SIRS mice. Besides, the h‐mIL‐6R mAb could also dramatically reduce the levels of inflammatory cytokines in LPS‐treated THP‐1 cells in vitro. RNA‐seq analysis indicated that the h‐mIL‐6R mAb could regulate LPS‐induced activation of NF‐κB/Ccl2 and NOD‐like receptor signaling pathways. Furthermore, we found that the h‐mIL‐6R mAb could forwardly inhibit Ccl2 expression and NLRP3‐mediated pyroptosis by suppressing NF‐κB in combination with the NF‐κB inhibitor. Collectively, mIL‐6R mAbs suppressed NF‐κB/Ccl2 signaling and inflammasome activation. IL‐6R mAbs are potential alternative therapeutics for suppressing excessive cytokine release, over‐activated inflammatory responses and alleviating organ injuries in SIRS.

Development of Astatine-211 (211At)-Based Anti-CD123 Radioimmunotherapy for Acute Leukemias and Other CD123+ Hematologic Malignancies

Background: Radioimmunotherapy (RIT) has long been pursued to improve outcomes in acute leukemia. Of current interest are alpha-particle emitting radionuclides as they deliver a very large amount of radiation over just a few cell diameters, enabling efficient and selective target cell kill. So far, alpha-emitters including astatine-211 (211At) have been primarily explored with monoclonal antibodies (mAbs) targeting CD45 or CD33 but their broad display on non-malignant target-expressing cells can lead to marked “on-target, off tumor cell” toxicities. To overcome this limitation, we developed a novel form of 211At-based RIT targeting CD123. CD123 is displayed widely on acute leukemia cells, including underlying leukemic stem cells, but is expressed only on a discrete subset of normal hematopoietic cells and is virtually absent on non-blood cells.Methods: We immunized BALB/c mice with peptides consisting of the extracellular domain of human CD123 to generate anti-CD123 mAbs. Flow cytometry-based assays with human acute leukemia cell lines were used to characterize binding of hybridoma supernatants and mAbs to CD123. mAbs were conjugated with isothiocyantophenethyl-ureido-closo-decaborate(2-) (B10), a boron cage molecule for subsequent astatination, and were then labeled with 211At. In vivo leukemia cell targeting (“biodistribution”) and efficacy studies were conducted in immunodeficient NOD-Rag1 null IL2rɣ null/J (NRG) mice xenografted with MOLM-13 cells, a CD123+ human acute myeloid leukemia cell line.Results: Based on initial hybridoma screening studies, we selected 4 mAbs (10C4, 5G4, 11F11, and 1H8) for further characterization. Phenotyping studies with CD123+ and CD123- human acute leukemia cell lines (including CD123+ cell lines in which CD123 was deleted via CRISPR/Cas9) confirmed specific binding of all mAbs to human CD123 (binding intensity: 10C4>5G4=11F11=1H8), with 10C4 yielding a higher median fluorescence intensity than the widely used commercial anti-CD123 mAb clones, 7G3 and 6H6 (Figure 1). In vitro internalization with a panel of human acute leukemia cell lines studies demonstrated uptake of all mAbs by CD123+ target cells with a kinetic slower than that for anti-CD33 antibodies (typically, 30-50% of the anti-CD123 mAb internalized over 2-4 hours). All 4 anti-CD123 mAbs could be conjugated to B10 and subsequently labeled with 211At. Unlike a non-binding 211At-labeled control mAb, 211At-labeled anti-CD123 mAbs showed uptake at MOLM-13 flank tumors in NRG mice carrying MOLM-13 xenografts. After additional leukemia cell targeting studies to optimize the dosing of 10C4, we conducted proof-of-concept efficacy studies in NRG mice injected intravenously with luciferase-transduced MOLM-13 cells (disseminated leukemia model). Animals were either untreated or treated with 10 µCi, 20 µCi, or 40 µCi of 211At-labeled 10C4-B10 mAb (9-11 animals/group). This was followed by the infusion of bone marrow cells from donor mice as stem cell support 3 days later. As shown in Figure 2 and Figure 3, 211At-10C4-B10 led to a dose dependent decrease in tumor burden. Further, the treatment significantly prolonged survival compared to untreated animals (median survival: 49 days [40 µCi of 211At] vs. 31 days [10 µCi of 211At] vs. 21 days [Ctrl]; P<0.0001 for Ctrl vs. 10 µCi, P<0.004 for 10 µCi vs. 40 µCi), demonstrating potent in vivo anti-leukemia efficacy of a single dose of 211At-CD123 RIT.Conclusion: Our data support the further development of 211At-CD123 RIT for the treatment of patients with acute leukemia and other CD123+ hematologic malignancies. [Display omitted] DisclosuresGreen: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Cellectar Biosciences: Research Funding; GSK: Membership on an entity's Board of Directors or advisory committees; JANSSEN Biotech: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno Therapeutics: Patents & Royalties, Research Funding; Legend Biotech: Consultancy; Neoleukin Therapeutics: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; SpringWorks Therapeutics: Research Funding. Walter: Kite: Consultancy; Janssen: Consultancy; Genentech: Consultancy; BMS: Consultancy; Astellas: Consultancy; Agios: Consultancy; Amphivena: Consultancy, Other: ownership interests; Selvita: Research Funding; Pfizer: Consultancy, Research Funding; Jazz: Research Funding; Macrogenics: Consultancy, Research Funding; Immunogen: Research Funding; Celgene: Consultancy, Research Funding; Aptevo: Consultancy, Research Funding; Amgen: Research Funding.

Subvisible Particle Analysis of 17 Monoclonal Antibodies Approved in China Using Flow Imaging and Light Obscuration

In the study, subvisible particles in 205 samples from 17 commercial mAb drug products approved in China were analyzed using light obscuration (LO) and flow imaging microscopy (FIM) methods. For each method, a total 633 tests (runs) were performed. In the tests, samples in state of lyophilized powder or syringe package had significantly higher particle concentrations. It was confirmed by analyzing the 205 drug product samples that FIM particle counts are generally higher than LO counts. The cause of the higher counts of FIM method than LO counts was examined by looking into the contribution of proteinaceous, translucent particles in the samples. The data of the study showed that the number of proteinaceous, translucent particles was a factor in the elevated counts of FIM method compared to LO method.

Towards a simple on-line coupling of ion exchange chromatography and native mass spectrometry for the detailed characterization of monoclonal antibodies

This work describes the direct hyphenation of cation exchange chromatography (CEX) with a compact, easy-to-use benchtop Time of Flight mass spectrometer (ToF/MS) for the analytical characterization of monoclonal antibodies (mAbs). For this purpose, a wide range of commercial mAb products (including expired samples and mAb biosimilars) were selected to draw reliable conclusions. From a chromatographic point of view, various buffers and column dimensions were tested. When considering pH response, buffer stability over time and MS compatibility, the best compromise is represented by the following recipe: 50 mM ammonium acetate, titrated to pH 5.0 (mobile phase A) and 160 mM ammonium acetate, titrated to pH 8.5 (mobile phase B). Despite the broader peaks observed with the 2.1 mm i.d. CEX column, this was preferentially selected for CEX-MS operation, since the efficiency loss (caused by extra-column dispersion) was still acceptable while MS compatibility was strongly enhanced (thanks to low flow rate). In terms of MS, it was important to avoid the use of glass-bottled mobile phases, laboratory glassware and glass vials to minimize loss of MS resolution, sensitivity, and mass accuracy due to metal contaminants. With this new CEX-MS setup, straightforward and rapid analysis (in less than 10 min) of charge variants was possible, allowing the separation and identification of several charge variants.

Interlaboratory method validation of capillary electrophoresis sodium dodecyl sulfate (CE-SDS) methodology for analysis of mAbs.

Capillary electrophoresis sodium dodecyl sulfate (CE-SDS) is an analytical method to assess the purity of proteins, commonly applied to monoclonal antibodies (mAbs) in the biopharmaceutical industry. To address the need to standardize the CE-SDS method in the pharmaceutical industry and to enhance the confidence in method transfer between laboratories operating different commercial capillary electrophoresis (CE) instrument platforms, an interlaboratory CE-SDS method validation was organized involving 13 laboratories in 13 companies on four different types of commercial capillary electrophoresis instruments. In the validation, a commercial mAb therapeutic was used as the sample. The validation process followed the analytical guidelines set by the ICH guidelines (International Conference for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use). The method's precision, accuracy, linearity and range, and limit of quantitation (LOQ) were validated in the study. Variations of all the parameters validated in the study passed the pre-set criteria defined at the beginning of the study. The definition was based on previously published works and the intended application purpose of the CE-SDS method for mAbs. The study proved that the CE-SDS method fits its intended application purpose as a size impurity assay and size heterogeneity characterization assay for mAb therapeutic products. This study is the first time a CE-SDS method is validated by multiple laboratories using different commercial CE instrument platforms and on a commercial mAb therapeutic. Its results will enhance the confidence of the biopharmaceutical industry to develop CE-SDS methods and transfer CE-SDS methods between different laboratories.

Characterization of Opalescence in low Volume Monoclonal Antibody Solutions Enabled by Microscale Nephelometry

Monoclonal antibody (mAb)-based drugs are often prone to unfavorable solution behaviors including high viscosity, opalescence, phase separation, and aggregation at the high concentrations needed to enable patient-centric subcutaneous dosage forms. Given that these can have a detrimental impact on manufacturability, stability, and delivery, approaches to identifying, monitoring, and controlling these behaviors during drug development are critical. Opalescence presents a significant challenge due to its relationship to liquid-liquid phase separation. Quantitative characterization of opalescence via turbidimetry is often restrictive due to large volume requirements (>2mL) and alternative microscale approaches based on light transmittance (Eckhardt etal., J Pharm Sci Technol. 1994, 48: 64-70) may pose challenging with respect to accuracy. To address the need for accurate and quantitative microscale opalescence measurements, we have evaluated the use of a 'de-tuned' static light scattering detector which requires <10 μL sample per measurement. We show that tuning of the laser power to a range far below that of traditional light scattering measurements results in a stable detector response that can be accurately calibrated to the nephelometric turbidity unit (NTU) scale using appropriate standards. The calibrated detector signal yields NTU values for mAbs and other protein solutions that are comparable to a commercial turbidimeter. We used this microscale approach to characterize the opalescence of 48 commercial mAb drug products and found that the majority have opalescence below 15 NTU. However, in products with mAb concentrations greater than 75mg/mL, a broad range of opalescence was observed, in a few cases greater than 20 NTU. These measurements as well as nephelometric characterization of several IgG1 and IgG4 mAbs across a broad pH range highlight subclass-specific tendencies toward opalescence in high concentration solutions.

Quantifying the synaptic vesicle‐associated protein, VAMP2, to verify changes in cerebrospinal fluid in preclinical stages of Alzheimer’s disease

AbstractBackgroundSynapse loss, a pathological hallmark of Alzheimer’s disease (AD), is quantified in the brain using synaptophysin in western blot and immunohistochemistry, while the in vivo imaging compound 11C‐UCB‐J targets synaptic vesicle glycoprotein 2A (SV2A). Both proteins are characteristic of synaptic vesicles (SVs) and are found in the majority of synapses. Since synaptophysin is not stable in CSF or serum, it is not a suitable marker to monitor synaptic integrity in fluids (Schlaf et al., 1998). Vesicle‐associated membrane protein 2 (VAMP2), another SV component critical for vesicular trafficking, was identified as potential fluid surrogate for synapse loss using relative quantification (Lleo et al., 2019). Here, we have developed a new digital immunoassay for VAMP2 and evaluated its CSF profile in 226 samples from the AD continuum. For comparison the more established post‐synaptic marker Neurogranin (Ng) was included, which is expressed in dendrites of excitatory glutaminergic neurons.MethodThe immunoassay (Simoa) for VAMP2 was established using a commercial rabbit mAb and a novel high‐affinity mouse mAb 15E4. CSF was measured in duplicate on the HD‐1 analyzer across cognitively normal controls (n=68), preclinical AD stage 1 (Amyloidosis +, n=31), preclinical AD stage 2 (Amyloidosis +, neurodegeneration +, n=8), prodromal AD (n=80) and AD dementia (n=39). Ng was quantified with ELISA (EUROIMMUN).ResultThe assay was able to quantify VAMP2 in all CSF samples with an average CV below 10%. Three quality control samples had an inter‐/intra‐assay variability below 10%. VAMP2 levels correlated strongly with Ng in all AD stages (r=0.78‐0.96, p&lt;0.0001). VAMP2 (p&lt;0.0001) and Ng (p&lt;0.0001) were decreased by 40% in preclinical AD stage 1. They were both elevated in the prodromal (VAMP2 25%, p=0.0009; Ng 70%, p&lt;0.0001) and dementia group (VAMP2 25%, p=0.014; Ng 50%, p&lt;0.0001).ConclusionA novel immunoassay for VAMP2 is capable of detecting changes in different AD stages. CSF changes in VAMP2 and Ng precede clinical symptoms and CSF neurodegeneration markers in the AD continuum. In line with observations that CSF‐tau and CSF‐Aβ abnormality precede plaque and tangle load via PET imaging, future longitudinal studies relating synaptic fluid markers with synaptic PET ligands (i.e. 11C‐UCB‐J) could extend these findings.

Development of Monoclonal Antibody Specific to Foot-and-Mouth Disease Virus Type A for Serodiagnosis.

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease affecting cloven-hoofed livestock worldwide. FMD virus (FMDV) type A is one of the most common causes of FMD outbreaks among the seven FMDV serotypes, and its serological diagnosis is therefore important to confirm FMDV type A infection and to determine FMD vaccine efficacy. Here, we generated monoclonal antibodies (mAbs) specific to FMDV type A via hybridoma systems using an inactivated FMDV type A (A22/Iraq/1964) and found 4 monoclones (#29, #106, #108, and #109) with high binding reactivity to FMDV type A among 594 primary clones. In particular, the #106 mAb had a higher binding reactivity to the inactivated FMDV type A than the other mAbs and a commercial mAb. Moreover, the #106 mAb showed no cross-reactivity to inactivated FMDV type South African territories 1, 2, and 3, and low reactivity to inactivated FMDV type O (O1 Manisa). Importantly, the solid-phase competitive ELISA (SPCE) using horseradish peroxidase (HRP)-conjugated #106 mAb detected FMDV type A-specific Abs in sera from FMD type A-vaccinated cattle more effectively than a commercial SPCE. These results suggest that the newly developed FMDV type A-specific mAb might be useful for diagnostic approaches for detecting Abs against FMDV type A.

Shedding Light into the Subcutis: A Mass Spectrometry Based Immunocapture Assay Enabling Full Characterization of Therapeutic Antibodies after Injection in Vivo.

An understanding of what happens to therapeutic antibodies in vivo after subcutaneous injection is of high interest. Therefore, we applied the open flow microperfusion technique to extract interstitial fluid from the subcutaneous tissue. In order to analyze those biological samples, a specific and sensitive workflow was required. In this study, we present a complete workflow that enables full characterization of therapeutic antibodies after subcutaneous injection. Compared to classical pharmacokinetic approaches where only a limited number of peptides are detected, our workflow provides full sequence coverage and even enables the identification of potential quality attributes. The efficiency to purify therapeutic antibodies from biological matrixes of two different antibody capture molecules and two types of magnetic beads was compared. Furthermore, several desalting protocols were tested in the development of a miniaturized peptide map procedure. The best results were achieved using a commercial anti-human capture mAb fragment in combination with streptavidin coated magnetic beads, providing capture efficiencies of 90-100%. The optimized peptide map protocol that requires <1 μg of mAb includes two desalting steps and showed sequence coverages of 95-100%. The final method was successfully used for analysis of interstitial fluid and serum samples after a subcutaneous injection of a therapeutic antibody into a domestic pig.

Precise definition of PTEN C-terminal epitopes and its implications in clinical oncology

Anti-PTEN monoclonal antibodies (mAb) are arising as important tools for immunohistochemistry (IHC) and protein quantification routine analysis in clinical oncology. Although an effort has been made to document the reliability of tumor tissue section immunostaining by anti-PTEN mAb, and to standardize their IHC use in research and in the clinical practice, the precise topological and biochemical definition of the epitope recognized by each mAb has been conventionally overlooked. In this study, six commercial anti-PTEN mAb have been validated and characterized for sensitivity and specificity by IHC and FISH, using a set of prostate and urothelial bladder tumor specimens, and by immunoblot, using PTEN positive and PTEN negative human cell lines. Immunoblot precise epitope mapping, performed using recombinant PTEN variants and mutations, revealed that all mAb recognized linear epitopes of 6–11 amino acid length at the PTEN C-terminus. Tumor-associated or disease-associated mutations at the PTEN C-terminus did not affect subcellular localization or PIP3 phosphatase activity of PTEN in cells, although resulted in specific loss of reactivity for some mAb. Furthermore, specific mimicking-phosphorylation mutations at the PTEN C-terminal region also abolished binding of specific mAb. Our study adds new evidence on the relevance of a precise epitope mapping in the validation of anti-PTEN mAb for their use in the clinics. This will be substantial to provide a more accurate diagnosis in clinical oncology based on PTEN protein expression in tumors and biological fluids.

Development and evaluation of a gold nanoparticle-based immunochromatographic strip test for the detection of canine parvovirus

Canine parvovirus (CPV) is the leading viral cause of enteritis in dogs and occurs mainly in 6- to 8-week-old pups. Rapid diagnosis of CPV under field conditions is now possible due to commercially available immunochromatographic (IC) assays. However, these commercial kits are somewhat expensive because they utilize a minimum of two monoclonal antibodies (mAbs) targeting different epitopes as capture and detector antibodies. Using only a singlemAb for both capture and detection purposemay reduce the sensitivity of the assay. In the present study, efforts were made to develop an economical assay that can be utilized for diagnosis of CPV under Indian conditions with a high level of confidence. Rabbit polyclonal antibodies (pAbs) generated against recombinant truncated VP2 proteins of CPV were used as capture antibodies because they can be produced economically, while a commercial anti-CPV mAb was used as the detector antibody. The detection limit of the test strip was 6.6×105 TCID50/ml, and it specifically detected CPV-2, CPV-2a and CPV-2b while displaying no cross-reactivity with other common canine enteric pathogens. The relative sensitivity/specificity of pAb based strip test was 71%/92% and 71%/100% in relation to the hemagglutination test and a commercial IC kit, respectively, with substantial agreement. In addition, two commercially available mAbs targeting different epitopes were also used for development of another IC assay, which showed sensitivity, and specificity of 82%/87% and 90%/98% in relation to the hemagglutination test and commercial kit. Hence, the present strip test based on a combination of mAb and pAb provides an acceptable alternative for onsite and cost-effective diagnosis of CPV infection.

Chimeric fab fragments of antibodies to recombinant Ebola virus glycoprotein

Previously, we have determined the nucleotide and amino acid sequences of the variable domains of three mouse monoclonal antibodies specific to the individual epitopes of the Ebola virus glycoprotein: GPE118 (IgG), GPE325 (IgM) and GPE534 (IgG) [1]. In the present paper, chimeric Fab fragments of Fab118, Fab325, and Fab534 antibodies were obtained based on the variable domains of murine antibodies by attaching CH1 and CL constant regions of human kappa-IgG1 to them. The recombinant chimeric Fab fragments were synthesized in the heterologous expression system Escherichia coli, isolated and purified using metal chelate affinity chromatography. The immunochemical properties of the obtained Fab fragments were studied by immunoblotting techniques as well as indirect and competitive ELISA using recombinant Ebola virus proteins: EBOV rGPdTM (recombinant glycoprotein of Ebola hemorrhagic fever virus without the transmembrane domain), NP (nucleoprotein) and VP40 (structural protein). The identity of recombinant chimeric Fab fragments, as well as their specificity to the recombinant glycoprotein of Ebola hemorrhagic fever virus (EBOV GP) was proved. The results of indirect ELISA evidence the absence of immunological cross-reactivity to NP and VP40 proteins of Ebola virus. The dissociation constants of the antigen-antibody complex K d equal to 5.0, 1.0 and 1.0 nM for Fab118, Fab325 and Fab534, respectively, were determined; they indicate high affinity of the obtained experimental samples to EBOV GP. The epitope specificity of Fab fragments was studied using a panel of commercial neutralizing antibodies. It was found that all studied antibodies to EBOV GP are targeted to different epitopes, while the epitopes of the recombinant chimeric Fab fragments and original murine monoclonal antibodies (mAbs) coincide. All the obtained and studied mAbs to EBOV GP are specific to epitopes that coincide or overlap the epitopes of three commercial neutralizing mAbs to Ebola virus: epitopes Fab118 and Fab325 overlap the epitope of the known commercial mAb h13F6; Fab325 epitope also overlaps mAb c6D8 epitope; Fab534 epitope is located near mAb KZ52 conformational epitope, in the formation of which amino acid residues of GP1 and GP2 domains of EBOV GP are involved.

Expression of NKp46 Splice Variants in Nasal Lavage Following Respiratory Viral Infection: Domain 1-Negative Isoforms Predominate and Manifest Higher Activity.

The natural killer (NK) cell activating receptor NKp46/NCR1 plays a critical role in elimination of virus-infected and tumor cells. The NCR1 gene can be transcribed into five different splice variants, but the functional importance and physiological distribution of NKp46 isoforms are not yet fully understood. Here, we shed light on differential expression of NKp46 splice variants in viral respiratory tract infections and their functional difference at the cellular level. NKp46 was the most predominantly expressed natural cytotoxicity receptor in the nasal lavage of patients infected with four respiratory viruses: respiratory syncytia virus, adenovirus, human metapneumovirus, or influenza A. Expression of NKp30 was far lower and NKp44 was absent in all patients. Domain 1-negative NKp46 splice variants (i.e., NKp46 isoform d) were the predominantly expressed isoform in nasal lavage following viral infections. Using our unique anti-NKp46 mAb, D2-9A5, which recognizes the D2 extracellular domain, and a commercial anti-NKp46 mAb, 9E2, which recognizes D1 domain, allowed us to identify a small subset of NKp46 D1-negative splice variant-expressing cells within cultured human primary NK cells. This NKp46 D1-negative subset also showed higher degranulation efficiency in term of CD107a surface expression. NK-92 cell lines expressing NKp46 D1-negative and NKp46 D1-positive splice variants also showed functional differences when interacting with targets. A NKp46 D1-negative isoform-expressing NK-92 cell line showed enhanced degranulation activity. To our knowledge, we provide the first evidence showing the physiological distribution and functional importance of human NKp46 splice variants under pathological conditions.

MONOCLONAL ANTIBODIES ICO-401 AGAINST CD133 ANTIGEN

Monoclonal antibody (mAb) against CD133 antigen is a stem marker of human tumor cells. Strain ICO-401 was prepared by cell fusion of murine myeloma NS-1 cells with splenocytes of BALB/ C mice, pre-immunized three times with an interval of two weeks with the cells of human melanoma mel IbrEE34RMCR. Merging was conducted with solution PEG/DMSO. Screening of mAb ICO-401 was performed on human melanoma cell lines from FSBI «N.N. Blokhin RCRC» collection which differ in the expression of CD133 antigen. Antigen expression was evaluated in immunofluorescence reaction by flow cytometer BD FACSCanto TM II. ICO-401 was compared with commercial mAb against CD133 antigen. The results indicated that both mAb recognize the same antigen, but bind to different epitops.

P38 MAP kinase–dependent phosphorylation of the Gp78 E3 ubiquitin ligase controls ER–mitochondria association and mitochondria motility

Gp78 is an ERAD-associated E3 ubiquitin ligase that induces degradation of the mitofusin mitochondrial fusion proteins and mitochondrial fission. Gp78 is localized throughout the ER; however, the anti-Gp78 3F3A monoclonal antibody (mAb) recognizes Gp78 selectively in mitochondria-associated ER domains. Epitope mapping localized the epitope of 3F3A and a commercial anti-Gp78 mAb to an 8-amino acid motif (533-541) in mouse Gp78 isoform 2 that forms part of a highly conserved 41-amino acid region containing 14-3-3- and WW-binding domains and a p38 MAP kinase (p38 MAPK) consensus site on Ser-538 (S538). 3F3A binds selectively to nonphosphorylated S538 Gp78. Using 3F3A as a reporter, we induced Gp78 S538 phosphorylation by serum starvation and showed it to be mediated by p38 MAPK. Mass spectroscopy analysis of Gp78 phosphopeptides confirmed S538 as a major p38 MAPK phosphorylation site on Gp78. Gp78 S538 phosphorylation limited its ability to induce mitochondrial fission and degrade MFN1 and MFN2 but did not affect in vitro Gp78 ubiquitin E3 ligase activity. Phosphomimetic Gp78 S538D mutation prevented Gp78 promotion of ER-mitochondria interaction, and SB203580 inhibition of p38 MAPK increased ER-mitochondria association. p38 MAPK phosphorylation of Gp78 S538 therefore regulates Gp78-dependent ER-mitochondria association and mitochondria motility.

Preparation and diagnostic use of a novel recombinant single-chain antibody against rabies virus glycoprotein

Rabies virus (RABV) causes a fatal infectious disease, but effective protection may be achieved with the use of rabies immunoglobulin and a rabies vaccine. Virus-neutralizing antibodies (VNA), which play an important role in the prevention of rabies, are commonly evaluated by the RABV neutralizing test. For determining serum VNA levels or virus titers during the RABV vaccine manufacturing process, reliability of the assay method is highly important and mainly dependent on the diagnostic antibody. Most diagnostic antibodies are monoclonal antibodies (mAbs) made from hybridoma cell lines and are costly and time consuming to prepare. Thus, production of a cost-effective mAb for determining rabies VNA levels or RABV titers is needed. In this report, we describe the prokaryotic production of a RABV-specific single-chain variable fragment (scFv) protein with a His-tag (scFv98H) from a previously constructed plasmid in a bioreactor, including the purification and refolding process as well as the functional testing of the protein. The antigen-specific binding characteristics, affinity, and relative affinity of the purified protein were tested. The scFv98H antibody was compared with a commercial RABV nucleoprotein mAb for assaying the VNA level of anti-rabies serum samples from different sources or testing the growth kinetics of RABV strains for vaccine manufactured in China. The results indicated that scFv98H may be used as a novel diagnostic tool to assay VNA levels or virus titers and may be used as an alternative for the diagnostic antibody presently employed for these purposes.

High resolution reversed phase analysis of recombinant monoclonal antibodies by ultra-high pressure liquid chromatography column coupling

Monoclonal antibodies (mAbs) represent one of the fastest growing areas of new drug development. However, their analytical characterization is complex and generally requires an array of orthogonal analytical techniques. Reversed phase liquid chromatography is a valuable strategy due to its high resolving power and straightforward compatibility to mass spectrometry. The present study demonstrates that high peak capacity can be attained with intact mAb of ∼150kDa, reduced mAb fragments of ∼25–50kDa and also digested mAb generating numerous peptides of ∼0.5–3kDa. Several long columns packed with fully porous wide-pore sub-2μm particles were coupled in series to evaluate the effect of column length on peak capacity. By using three columns of 150mm length, a mobile phase temperature of 80°C and a gradient time of around 20min, peak capacities of 117 and 128 were obtained for a commercial intact mAb and its reduced mAb fragments, respectively. On the other hand, when peptide mapping was performed at 50°C, with a gradient time of 270min and a column length of 450mm, a peak capacity of more than 700 was achieved.

Comparison of an antibody and its recombinant derivative for the detection of the small molecule explosive 2,4,6-trinitrotoluene

Antibodies are commonly used as recognition elements in immunoassays because of their high specificity and affinity, and have seen extensive use in competitive assays for the detection of small molecules. However, these complex molecules require production either in animals or by mammalian cell cultures, and are not easily tailored through genetic manipulation. Single chain antibodies (scFv), recombinantly expressed molecules consisting of only the antibody's binding region joined via a linking peptide, can provide an alternative to intact antibodies. We describe the characterization of a new monoclonal antibody (mAb), 2G5B5, able to detect the small molecule explosive 2,4,6-trinitrotoluene (TNT) and the scFv derived from its variable regions. The mAb and scFv were tested by surface plasmon resonance to determine their affinity for an immobilized TNT surrogate; dissociation constants were determined to be 1.5×10−13M and 4.8×10−10M respectively. Circular dichroism was used to determine their melting temperatures. The mAb is more stable melting at ∼75°C while the scFv melts at ∼65°C. The recognition elements were incorporated into a competitive assay format using a bead-based multiplexing platform to examine their sensitivity and specificity. The scFv was able to detect TNT ∼10-fold more sensitively than the mAb in this assay format, allowing detection of TNT concentrations down to at least 1μgL−1. The 2G5B gave similar detection limits to a commercial anti-TNT mAb, but was less specific, recognizing 1,3,5-trinitrobenzene (TNB) equally well as TNT.