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Fractional laser ablation for the targeted cutaneous delivery of an anti-CD29 monoclonal antibody – OS2966
摘要： Monoclonal antibodies targeting cytokines are administered parenterally for the systemic treatment of severe psoriasis. However, systemic exposure to the biologic increases the risk of side-effects including immunosuppression, whereas only a small fraction of the active molecules actually reaches the target organ, the skin. This preclinical study examines the feasibility of delivering a humanized anti-CD29 monoclonal antibody (OS2966) topically to skin using minimally-invasive fractional laser ablation. This approach would enable the targeted use of a biologic for the treatment of recalcitrant psoriatic plaques in patients with less widespread disease while minimizing the risk of systemic exposure. First, the effect of a wide range of laser poration conditions on skin permeation and deposition of OS2966 was tested in vitro to determine optimal microporation parameters. Subsequently, confocal laser scanning microscopy was employed to visualize the distribution of fluorescently-labelled OS2966 in skin. The results demonstrated that delivery of OS2966 into and across skin was feasible. Above fluences of 35.1 J/cm2, skin deposition and permeation were statistically superior to passive delivery reaching values up to 3.7 ± 1.2 μg/cm2 at the most aggressive condition. Selective targeting of the skin was also possible since ≥70% of the OS2966 was delivered locally to the skin. Although nanogramme quantities were able to permeate across skin, these amounts were orders of magnitude lower than levels seen following subcutaneous or intravenous injection and would result in minimal systemic exposure in vivo. The diffusion of fluorescently-labelled OS2966 into the skin surrounding the pores was clearly higher than in intact skin and demonstrated the feasibility of delivering the antibody at least as deep as the dermo-epithelial junction, a critical border region where inflammatory cells cross to promote disease progression. These preliminary results confirm that fractional laser ablation can be used for the cutaneous delivery of OS2966 and now preclinical/clinical studies are required to demonstrate therapeutic efficacy.
关键词： fractional laser ablation,cutaneous delivery,OS2966,psoriasis,monoclonal antibodies
更新于2025-11-21 11:08:12
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A RAGE-Targeted Antibody-Drug Conjugate: Surface Plasmon Resonance as a Platform for Accelerating Effective ADC Design and Development
摘要： Antibodies, antibody-like molecules, and therapeutics incorporating antibodies as a targeting moiety, such as antibody-drug conjugates, offer significant potential for the development of highly efficacious drugs against a wide range of disorders. Despite some success, truly harnessing the superior targeting properties of these molecules requires a platform from which to effectively identify the best candidates for drug development. To streamline the development of antibody-drug conjugates targeting gynecological cancers within our laboratory, we incorporated surface plasmon resonance analysis (Biacore? T200) into our development toolkit. Antibodies, selected based on positive ELISA screens as suitable for development as antibody-drug conjugates, were evaluated using surface plasmon resonance to determine a wide range of characteristics including specificity, kinetics/affinity, the effect of linker binding, the impact of the drug to antibody ratio, and the effect of endosomal pH on antibody-antigen binding. Analysis revealed important kinetics data and information regarding the effect of conjugation and endosomal pH on our antibody candidates that correlated with cell toxicity and antibody internalization data. As well as explaining observations from cell-based assays regarding antibody-drug conjugate efficacies, these data also provide important information regarding intelligent antibody selection and antibody-drug conjugate design. This study demonstrates the application of surface plasmon resonance technology as a platform, where detailed information can be obtained, supporting the requirements for rapid and high-throughput screening that will enable enhanced antibody-drug conjugate development.
关键词： antibodies,antibody-drug conjugates,gynecological cancers,binding kinetics,surface plasmon resonance
更新于2025-11-21 11:08:12
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Super-resolution microscopy reveals significant impact of M2e-specific monoclonal antibodies on influenza A virus filament formation at the host cell surface
摘要： Influenza A virions are highly pleomorphic, exhibiting either spherical or filamentous morphology. The influenza A virus strain A/Udorn/72 (H3N2) produces copious amounts of long filaments on the surface of infected cells where matrix protein 1 (M1) and 2 (M2) play a key role in virus filament formation. Previously, it was shown that an anti-M2 ectodomain (M2e) antibody could inhibit A/Udorn/72 virus filament formation. However, the study of these structures is limited by their small size and complex structure. Here, we show that M2e-specific IgG1 and IgG2a mouse monoclonal antibodies can reduce influenza A/Udorn/72 virus plaque growth and infectivity in vitro. Using Immuno-staining combined with super-resolution microscopy that allows us to study structures beyond the diffraction limit, we report that M2 is localized at the base of viral filaments that emerge from the membrane of infected cells. Filament formation was inhibited by treatment of A/Udorn/72 infected cells with M2e-specific IgG2a and IgG1 monoclonal antibodies and resulted in fragmentation of pre-existing filaments. We conclude that M2e-specific IgGs can reduce filamentous influenza A virus replication in vitro and suggest that in vitro inhibition of A/Udorn/72 virus replication by M2e-specific antibodies correlates with the inhibition of filament formation on the surface of infected cells.
关键词： influenza A virus,viral replication,super-resolution microscopy,filament formation,M2e-specific monoclonal antibodies
更新于2025-11-21 11:08:12
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Harmonizing by reducing inter-run variability: performance evaluation of a quality assurance program for antinuclear antibody detection by indirect immunofluorescence
摘要： Background: The introduction of automated anti-nuclear antibody (ANA) indirect immunofluorescence (IIF) analysis may allow for more harmonized ANA IIF reporting, provided that a thorough quality assurance program controls this process. The aim of this study was to evaluate various quality indicators used for ANA IIF analysis with the final goal of optimizing the iQC program. Methods: In an experimental setup, we introduced artificial errors, mimicking plausible problems during routine practice on a QUANTA-Lyser-NOVA View? system (Inova Diagnostics, San Diego, CA, USA). Predetermined quality indicators were evaluated against predefined acceptance criteria. In addition, we retrospectively investigated the applicability of the selected quality indicators in the daily routine practice during three pre-defined periods. Results: Both the experimental as the retrospective study revealed that pre-analytical, analytical and post-analytical errors were not highlighted by company internal quality control (iQC) materials. The use of patient derived iQC samples, median fluorescence intensity results per run and the percentage of positive ANA IIF results as additional quality indicators ensured a more adequate ANA IIF quality assurance. Furthermore, negative and moderate positive sample iQC materials merit clinical validation, as titer changes of >1 correspond to clinically important shifts. Traditional Westgard rules, including a clinically defined stop limit, revealed to be useful in monitoring of the supplemental quality indicators. Conclusions: A thorough ANA IIF quality assurance for daily routine practice necessitates the addition of supplemental quality indicators in combination with well-defined acceptance criteria.
关键词： automation,indirect immunofluorescence,antinuclear antibodies,quality control
更新于2025-09-23 15:23:52
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Real Time Monitoring of a UV Light-Assisted Biofunctionalization Protocol Using a Nanophotonic Biosensor
摘要： A protocol for the covalent biofunctionalization of silicon-based biosensors using a UV light-induced thiol–ene coupling (TEC) reaction has been developed. This biofunctionalization approach has been used to immobilize half antibodies (hIgG), which have been obtained by means of a tris(2-carboxyethyl)phosphine (TCEP) reduction at the hinge region, to the surface of a vinyl-activated silicon-on-insulator (SOI) nanophotonic sensing chip. The response of the sensing structures within the nanophotonic chip was monitored in real time during the biofunctionalization process, which has allowed us to confirm that the bioconjugation of the thiol-terminated bioreceptors onto the vinyl-activated sensing surface is only initiated upon UV light photocatalysis.
关键词： UV light photocatalysis,biofunctionalization,silicon on insulator,nanophotonic sensor,half antibodies
更新于2025-09-23 15:23:52
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Real-time profiling Anti-EpCAM Based Immune capture, from Molecules to Cells using MP-SPR
摘要： Antibodies of epithelial cell-adhesion-molecule (anti-EpCAM)-based interfaces have proven to be highly efficient at capturing circulating tumor cells (CTCs). To achieve the bonding of anti-EpCAM to the interface, biotin and streptavidin are used to modify the surface. These processes are critical to subsequent cell-capture efficiencies. However, quantitative research on the interactions between biotin, streptavidin and biotinylated anti-EpCAM on the interface is lacking. In this work, the thermodynamics and kinetics of biomolecular interactions were determined by using surface plasmon resonance. The equilibrium binding affinities for biotinylated anti-EpCAM to streptavidin and streptavidin to biotin (illustrated by biotin-PEG400-thiol) were found to be 2.75×10^6 M^-1 and 8.82×10^6 M^-1, respectively. Each streptavidin can bind up to 2.30 biotinylated anti-EpCAM under thermodynamic equilibrium. The findings provide useful information to optimize the modification of anti-EpCAM and improve the capture efficiency of CTCs.
关键词： surface plasmon resonance,antibodies of epithelial cell-adhesion-molecule,cell capture,biotin–streptavidin interaction,thermodynamics,kinetics
更新于2025-09-23 15:23:52
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[Methods in Molecular Biology] Barley Volume 1900 (Methods and Protocols) || Preparation of Barley Pollen Mother Cells for Confocal and Super Resolution Microscopy
摘要： Recombination (crossover) drives the release of genetic diversity in plant breeding programs. However, in barley, recombination is skewed toward the telomeric ends of its seven chromosomes, restricting the re-assortment of about 30% of the genes located in the centromeric regions of its large 5.1 Gb genome. A better understanding of meiosis and recombination could provide ways of modulating crossover distribution and frequency in barley as well as in other grasses, including wheat. While most research on recombination has been carried out in the model plant Arabidopsis thaliana, recent studies in barley (Hordeum Vulgare) have provided new insights into the control of crossing over in large genome species. A major achievement in these studies has been the use of cytological procedures to follow meiotic events. This protocol provides detailed practical steps required to perform immunostaining of barley meiocytes (pollen mother cells) for confocal or structured illumination microscopy.
关键词： Antibodies,Barley,3D-SIM,Immuno-cytochemistry,Confocal Microscopy,Meiosis
更新于2025-09-23 15:21:21
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Development, optimization and structural characterization of an efficient peptide-based photoaffinity crosslinking reaction for generation of homogeneous conjugates from wild-type antibodies
摘要： Site-specific conjugation of small molecules to antibodies represents an attractive goal for the development of more homogeneous targeted therapies and diagnostics. Most site-specific conjugation strategies require modification or removal of antibody glycans or interchain disulfide bonds or engineering of an antibody mutant that bears a reactive handle. While such methods are effective, they complicate the process of preparing antibody conjugates and can negatively impact biological activity. Herein, we report the development and detailed characterization of a robust photoaffinity crosslinking method for site-specific conjugation to fully-glycosylated wild-type antibodies. The method employs a benzoylphenylalanine (Bpa) mutant of a previously-described 13-residue peptide derived from phage display to bind tightly to the Fc domain; upon UV irradiation, the Bpa residue forms a diradical that reacts with the bound antibody. First, we describe the initial discovery of an effective Bpa mutant peptide and optimization of reaction conditions to enable efficient conjugation without concomitant UV-induced photodamage of the antibody. Second, we assessed the scope of the photoconjugation reaction across different human and non-human antibodies and antibody mutants. Third, the specific site of conjugation on a human antibody was characterized in detail by mass spectrometry experiments and at atomic resolution by X-ray crystallography. Finally, we adapted the photoconjugation method to attach a cytotoxic payload site-specifically to a wild-type antibody and show that the resulting conjugate is both stable in plasma and as potent as a conventional antibody drug conjugate in cells, portending well for future biological applications.
关键词： wild-type antibodies,Fc domain,photoaffinity crosslinking,site-specific conjugation,antibody-drug conjugates
更新于2025-09-23 15:19:57
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Rapid and Straightforward Quantification of Recombinant Proteins Using Fluorescence Polarization
摘要： Biotechnology enables the production and commercialization of recombinant proteins for diagnostic, therapeutic and research applications. For instance, monoclonal antibodies nowadays represent a fast growing market that accounts for almost half of the biopharmaceutical compounds business with $75 billion turnover in 2013. Quantification of proteins is an important task in order to optimize cell culture conditions and ensure that every step of the production process is in line with the specifications. For therapeutics, precise and reliable dosage is mandatory to avoid adverse effects for the patients. Current analytical techniques such as ELISA or HPLC are time-consuming and often require purification prior to analysis. In this context, there is a strong demand for a technology that would be to protein quantification what a balance is to weight measurement: rapid, precise, accurate, robust, and straightforward. To this effect, we have developed an assay for the rapid quantification of recombinant proteins. It is based on fluorescence polarization and requires little or no pretreatment of samples. Fluorescence anisotropy of a small specific fluorescent ligand increased upon binding to its recombinant target protein, e.g. a monoclonal antibody, enabling quantification. The assay did not require prior separation of biotechnological cultures making it as straightforward as weighing using a balance. Monitoring of the production of a representative His-tagged protein during E.coli fermentation was obtained by regular sampling and assaying using the fluorescence polarization assay.
关键词： Fluorescence,Recombinant proteins,Assays,Biotechnology,Antibodies
更新于2025-09-19 17:15:36
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Nanobody Detection of Standard Fluorescent Proteins Enables Multi-Target DNA-PAINT with High Resolution and Minimal Displacement Errors
摘要： DNA point accumulation for imaging in nanoscale topography (PAINT) is a rapidly developing fluorescence super-resolution technique, which allows for reaching spatial resolutions below 10 nm. It also enables the imaging of multiple targets in the same sample. However, using DNA-PAINT to observe cellular structures at such resolution remains challenging. Antibodies, which are commonly used for this purpose, lead to a displacement between the target protein and the reporting fluorophore of 20–25 nm, thus limiting the resolving power. Here, we used nanobodies to minimize this linkage error to ~4 nm. We demonstrate multiplexed imaging by using three nanobodies, each able to bind to a different family of fluorescent proteins. We couple the nanobodies with single DNA strands via a straight forward and stoichiometric chemical conjugation. Additionally, we built a versatile computer-controlled microfluidic setup to enable multiplexed DNA-PAINT in an efficient manner. As a proof of principle, we labeled and imaged proteins on mitochondria, the Golgi apparatus, and chromatin. We obtained super-resolved images of the three targets with 20 nm resolution, and within only 35 minutes acquisition time.
关键词： DNA-PAINT,microfluidics,super-resolution microscopy,fluorescent proteins,molecular localization,multi-color imaging,multiplexing,single domain antibodies (sdAb),linkage error,nanobodies
更新于2025-09-19 17:15:36