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Cancer Cell Targeting With Functionalized Quantum Dot-Encoded Polyelectrolyte Microcapsules
摘要： Imaging agents and drug carriers are commonly targeted toward cancer cell through functionalization with specific recognition molecules. Quantum dots (QDs) are fluorescent semiconductor nanocrystals whose extraordinary brightness and photostability make them attractive for direct fluorescent labeling of biomolecules or optical encoding of the membranes and cells. Here, we analyse the cytotoxicity of QD-encoded microcapsules, validate an approach to the activation of further functionalization with monoclonal antibody Trastuzumab, a humanized monoclonal antibody targeting the extracellular domain of the human epidermal growth factor receptor 2 (HER2) and already in clinical use for the treatment of HER2 positive breast cancer. In addition, we characterize the cell-specific targeting activity of the resultant bio-conjugate by immunofluorescence assay (IFA) and real-time analysis of interaction of the conjugates with live HER2 overexpressing human breast cancer cells. We demonstrate, that encapsulation of QDs into the polymer shell using the layer-by-layer deposition method yields highly fluorescent polyelectrolyte microcapsules with a homogeneous size distribution and biocompatibility upon in vitro treatment of cancer cells. Carbodiimide surface activation ensures optimal disperse and optical characteristics of the QD-encoded microcapsules before antibody conjugation. The prepared conjugates of the microcapsules with cancer-specific monoclonal antibody targeting HER2 provide sufficiently sensitive and specific antibody-mediated binding of the microcapsules with live cancer cells, which demonstrated their potential as prospective cancer cell–targeting agents.
关键词： cytotoxicity,monoclonal antibody,polyelectrolyte microcapsules,quantum dots,cancer cell targeting
更新于2025-11-21 11:24:58
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Protein-Protein Interactions of Highly Concentrated Monoclonal Antibody Solutions via Static Light Scattering and Influence on the Viscosity
摘要： The ability to design and formulate mAbs to minimize attractive interactions at high concentrations is important for protein processing, stability and administration, particularly in subcutaneous delivery, where high viscosities are often challenging. The strength of protein-protein interactions (PPI) of an IgG1 and IgG4 monoclonal antibody (mAb) from low to high concentration were determined by static light scattering (SLS) and used to understand viscosity data. The PPI were tuned using NaCl and five organic ionic co-solutes. The PPI strength was quantified by the normalized structure factor S(0)/S(0)HS and Kirkwood-Buff integral G22/G22,HS (HS = hard sphere) determined from the SLS data, and also by fits with (1) a spherical Yukawa potential and (2) an interacting hard sphere (IHS) model, which describes attraction in terms of hypothetical oligomers. The IHS model was better able to capture the scattering behavior of the more strongly-interacting systems (mAb and/or co-solute) than the spherical Yukawa potential. For each descriptor of PPI, linear correlations were obtained between the viscosity at high concentration (200 mg/mL) and the interaction strengths evaluated both at low (20 mg/mL) and high concentration (200 mg/mL) for a given mAb. However, the only parameter that provided a correlation across both mAbs was the oligomer mass ratio (moligomer/mmonomer+dimer) from the IHS model, indicating the importance of self-association (in addition to the direct influence of the attractive PPI) on the viscosity.
关键词： Protein-protein interactions,static light scattering,co-solutes,monoclonal antibody,viscosity,interacting hard sphere model,Yukawa potential
更新于2025-09-23 15:23:52
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Impact of Tryptophan Oxidation in Complementarity-Determining Regions of Two Monoclonal Antibodies on Structure-Function Characterized by Hydrogen-Deuterium Exchange Mass Spectrometry and Surface Plasmon Resonance
摘要： Purpose Tryptophan’s (Trp) unique hydrophobic and structural properties make it an important antigen binding motif when positioned in complementarity-determining regions (CDRs) of monoclonal antibodies (mAbs). Oxidation of Trp residues within the CDR can deleteriously impact antigen binding, particularly if the CDR conformation is altered. The goal of this study was to evaluate the conformational and functional impact of Trp oxidation for two mAb subtypes, which is essential in determining the structure-function relationship and establishing appropriate analytical control strategies during protein therapeutics development. Methods Selective Trp oxidation was induced by 2,2′-Azobis(2-amidinopropane) dihydrochloride (AAPH) treatment in the presence of free methionine (Met). The native and chemically oxidized mAbs were characterized by hydrogen-deuterium exchange mass spectrometry (HDX-MS) for conformational changes and surface plasmon resonance (SPR) for antigen-antibody binding. Results Treatment of mAbs with AAPH selectively oxidized solvent accessible Trp residues. Oxidation of Trp within or in proximity of CDRs increased conformational flexibility in variable domains and disrupted antigen binding. Conclusions Trp oxidation in CDRs can adversely impact mAbs’ conformation and antigen binding. Trp oxidation should be carefully evaluated as part of critical quality attribute assessments. Oxidation susceptible Trp should be closely monitored during process development for mAbs to establish appropriate analytical control for manufacturing of drug substance and drug product.
关键词： Tryptophan oxidation,surface plasmon resonance,complementarity-determining region,hydrogen-deuterium exchange mass spectrometry,monoclonal antibody
更新于2025-09-23 15:19:57
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A Model for the Binding of Fluorescently Labeled Anti-Human CD4 Monoclonal Antibodies to CD4 Receptors on Human Lymphocytes
摘要： The CD4 glycoprotein is a component of the T cell receptor complex which plays an important role in the human immune response. This manuscript describes the measurement and modeling of the binding of fluorescently labeled anti-human CD4 monoclonal antibodies (mAb; SK3 clone) to CD4 receptors on the surface of human peripheral blood mononuclear cells (PBMC). CD4 mAb fluorescein isothiocyanate (FITC) and CD4 mAb allophycoerythrin (APC) conjugates were obtained from commercial sources. Four binding conditions were performed, each with the same PBMC sample and different CD4 mAb conjugate. Each binding condition consisted of the PBMC sample incubated for 30 min in labeling solutions containing progressively larger concentrations of the CD4 mAb-label conjugate. After the incubation period, the cells were re-suspended in PBS-based buffer and analyzed using a flow cytometer to measure the mean fluorescence intensity (MFI) of the labeled cell populations. A model was developed to estimate the equilibrium concentration of bound CD4 mAb-label conjugates to CD4 receptors on PBMC. A set of parameters was obtained from the best fit of the model to the measured MFI data and the known number of CD4 receptors on PBMC surface. Divalent and monovalent binding had to be invoked for the APC and FITC CD4 mAb conjugates, respectively. This suggests that the mAb binding depends on the size of the label, which has significant implications for quantitative flow cytometry. The study supports the National Institute of Standards and Technology program to develop quantitative flow cytometry measurements.
关键词： fluorescence spectroscopy,PBMC,FITC,antibody binding,CD4 monoclonal antibody,flow cytometry,APC,cooperative binding
更新于2025-09-12 10:27:22
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Rapid immunological detection of copper ions using fluorescence immunochromatographic assay
摘要： One-step fluorescence immunochromatographic assay (FICA) has been developed using fluorescent microspheres-labelled monoclonal antibody (McAb) probe for the rapid detection of copper ions in herbal plants and soil samples were investigated in this study. The results can be read by the fluorescence reader according to the fitted standard curve equation. Under optimal conditions, detecting copper ions with FICA can be done within 15 min with a LOD (limit of detection) of 0.2 μg/L, and was very useful for the rapid onsite testing. A total of 31 solid samples were pre-treated by microwave digestion combined with cation exchange solid phase extraction and prepared as test samples for FICA and ICP-AES. The two methods showed good relativity with correlation coefficients of 0.924 and 0.969 in herbal plants and soil samples for copper detection, respectively. We hope that our research can help promote the daily monitoring of heavy metal pollution effectively.
关键词： Copper ions,fluorescence immunochromatographic assay,monoclonal antibody,rapid onsite testing
更新于2025-09-09 09:28:46
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Activatable Near-Infrared Fluorescence Imaging Using PEGylated Bacteriochlorin-Based Chlorin and BODIPY-Dyads as Probes for Detecting Cancer
摘要： Near infrared (NIR) fluorescent probes are attractive tools for biomedical in vivo imaging due to the relatively deeper tissue penetration and lower background autofluorescence. Activatable probes are turned on only after binding to their target, further improving target to background ratios. However, the number of available activatable NIR probes is limited. In this study, we introduce two types of activatable NIR fluorophores derived from bacteriochlorin; chlorin-bacteriochlorin energy-transfer dyads and boron-dipyrromethene (BODIPY)-bacteriochlorin energy-transfer dyads. These fluorophores are characterized by multiple narrow excitation bands with relatively strong emission in the NIR. Targeted bacteriochlorin-based antibody or peptide probes have been previously limited by aggregation after conjugation. Polyethylene glycol (PEG) chains were added to improve the hydrophilicity without altering pharmacokinetics of the targeting moieties. These PEGylated bacteriochlorin-based activatable fluorophores have potential as targeted activatable, multi-color NIR fluorescent probes for in vivo applications.
关键词： Monoclonal antibody,Molecular imaging,Bacteriochlorin,Cancer
更新于2025-09-09 09:28:46