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Turbidimetric inhibition immunoassay revisited to enhance its sensitivity via an optofluidic laser
摘要： Turbidimetric inhibition immunoassay (TIIA) is a classic immunodiagnostic method that has been extensively used for biomarker detection. However, the low sensitivity of this technique hinders its applications in the early diagnosis of diseases. Here, a new concept, optofluidic laser TIIA (OFL-TIIA), is proposed and demonstrated for sensitive protein detection. In contrast to the immunoreaction in traditional TIIA, in which the single-pass laser loss is detected, the immunoreaction in the OFL-TIIA method takes place in a laser cavity, which considerably increases the loss induced by antigen-antibody complexes (AACs) via the amplification effect of the laser. A commercial IgG TIIA kit was selected as a demonstrative model to characterize the performance of OFL-TIIA. A wide dynamic range of five orders of magnitude with an exceptional limit of detection (LOD) (1.8×10-10 g/L) was achieved. OFL-TIIA is a fast, sensitive, and low-cost immunoassay with a simple homogeneous and wash-free process and low-volume sample consumption, thus providing a new detection platform for disease diagnostics.
关键词： Biomarker detection,Optofluidic laser,Turbidimetric inhibition immunoassay,Antigen-antibody complexes,Laser dye
更新于2025-11-25 10:30:42
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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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Preclinical stress originates in the rat optic nerve head during development of autoimmune optic neuritis
摘要： Optic neuritis is a common manifestation of multiple sclerosis, an inflammatory demyelinating disease of the CNS. Although it is the presenting symptom in many cases, the initial events are currently unknown. However, in the earliest stages of autoimmune optic neuritis in rats, pathological changes are already apparent such as microglial activation and disturbances in myelin ultrastructure of the optic nerves. αB-crystallin is a heat-shock protein induced in cells undergoing cellular stress and has been reported to be up-regulated in both multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. Therefore, we wished to investigate the timing and localization of its expression in autoimmune optic neuritis. Although loss of oligodendrocytes was not observed until the later disease stages accompanying immune cell infiltration and demyelination, an increase in oligodendrocyte αB-crystallin was observed during the preclinical stages. This was most pronounced within the optic nerve head and was associated with areas of IgG deposition. Since treatment of isolated oligodendrocytes with sera from myelin oligodendrocyte glycoprotein (MOG)-immunized animals induced an increase in αB-crystallin expression, as did passive transfer of sera from MOG-immunized animals to unimmunized recipients, we propose that the partially permeable blood–brain barrier of the optic nerve head may present an opportunity for blood-borne components such as anti-MOG antibodies to come into contact with oligodendrocytes as one of the earliest events in disease development.
关键词： EAE,optic nerve head,optic neuritis,oligodendrocyte,αB-crystallin,auto-antibody
更新于2025-11-21 11:08:18
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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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Homogeneous immunoassay for alpha-fetoprotein based on the quenching of the fluorescence of quantum dots by antibody labelled with complexed copper ion tags
摘要： A homogeneous fluorescent immunoassay is described for the determination of alpha fetoprotein (AFP) relying on the interaction between copper ion complex and quantum dots (QDs). The copper ion complex-labelled antibody can be employed as a quencher of fluorescence of QDs and capture probe of AFP in homogeneous solution. The labelled antibody is mixed with QDs to form the immune ensemble probe. Upon the addition of AFP, the labelled antibody is stripped away from QDs by antigen-antibody combination leading to the increase in the fluorescence signal. Thus, the determination of AFP can be realized by fluorometry (best measured at excitation/emission wavelengths of 360/520 nm). The fluorescence intensity shows a good linear relationship with the AFP concentration ranging from 40 to 640 ng mL?1, and the LOD is 26 ng mL?1. The proposed method provides a new approach to incorporate metal complexes into QD-based biomolecule sensing.
关键词： Stern-Volmer plot,Metal complex,DTPAA,Copper ion complex-labelled antibody,Fluorescence quenching
更新于2025-11-19 16:46:39
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Photoelectrochemical biosensor for microRNA detection based on a MoS2/g-C3N4/black TiO2 heterojunction with Histostar@AuNPs for signal amplification
摘要： Herein, a novel photoelectrochemical (PEC) biosensor was developed for the ultrasensitive detection of microRNA-396a based on a MoS2/g-C3N4/black TiO2 heterojunction as the photoactive material and gold nanoparticles carrying Histostar antibodies (Histostar@AuNPs) for signal amplification. Briefly, MoS2/g-C3N4/black TiO2 was deposited on an indium tin oxide (ITO) electrode surface, after which gold nanoparticles (AuNPs) and probe DNA were assembled on the modified electrode. Hybridization with miRNA-396a resulted in a rigid DNA:RNA hybrid being formed, which was recognized by the S9.6 antibody. The captured antibody can further conjugate with the secondary IgG antibodies of Histostar@AuNPs, thereby leading to the immobilization of horse radish peroxidase (HRP). In the presence of HRP, the oxidation of 4-chloro-1-naphthol (4-CN) by H2O2 was accelerated, producing the insoluble product benzo-4-chlorohexadienone on the electrode surface and causing a significant decrease in the photocurrent. The developed biosensor could detect miRNA-396a at concentrations from 0.5 fM to 5000 fM, with a detection limit of 0.13 fM. Further, the proposed method can also be used to investigate the effect of heavy metal ions on the expression level of miRNAs. Results suggest that the biosensor developed herein offers a promising platform for the ultrasensitive detection of miRNA.
关键词： S9.6 antibody,Histostar@AuNPs,MicroRNA detection,MoS2/g-C3N4/black TiO2 heterojunction,Photoelectrochemical biosensor
更新于2025-11-14 17:04:02
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Rapid preimplantation genetic screening (PGS) using a handheld, nanopore-based, DNA sequencer
摘要： Background/aims: A birth dose of hepatitis B immunoglobulin (HBIG), in combination with hepatitis B vaccine (HepB), is recommended for infants born to hepatitis B surface antigen (HBsAg)-positive mothers. However, the optimal dosage of HBIG remains to be resolved. This prospective cohort study aimed to compare the efficacy of two dosages of HBIG combined with HepB to prevent mother-to-child transmission (MTCT) of HBV. Methods: From 2009 to 2011, we prospectively enrolled mother-infant pairs with positive maternal HBsAg in China. Infants were assigned to receive one dose of 100 IU or 200 IU HBIG within 12 h of birth according to maternal numbering, followed by completion of the 3-dose 10 μg HepB series. At 7 months, post-vaccination serologic testing (PVST) was performed in 545 and 632 infants in 100 IU and 200 IU HBIG groups, respectively, among whom, 451 and 529 were followed up to 12 months. Results: Maternal and birth characteristics were comparable between infants in 100 IU and 200 IU HBIG groups. At 7 months, the rates of perinatal infection were 1.5% (8/545) and 1.9% (12/632) in 100 IU and 200 IU HBIG groups, respectively (p = .568). One non-responder infant in 200 IU HBIG group became newly infected at 12 months. The antibody to hepatitis B surface antigen (anti-HBs) positive rates were 98.5% (529/537) and 98.2% (609/620) in 100 IU and 200 IU HBIG groups at 7 months, respectively (p = .704), and the corresponding figures were 98.2% (431/439) and 97.1% (496/511) at 12 months (p = .266). The anti-HBs geometric mean concentrations were comparable between two groups at 7 months (707.95 mIU/mL vs. 602.56 mIU/mL, p = .062) and 12 months (245.47 mIU/mL vs. 229.09 mIU/mL, p = .407). Conclusions: One birth dose of 100 IU HBIG, combined with the HepB series, might be enough for preventing MTCT of HBV in infants born to HBsAg-positive mothers.
关键词： Antibody to hepatitis B surface antigen,Mother-to-child transmission,Hepatitis B virus,Hepatitis B immunoglobulin,Hepatitis B vaccine
更新于2025-09-23 15:23:52
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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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Biosensor for point-of-care analysis of immunoglobulins in urine by metal enhanced fluorescence from gold nanoparticles
摘要： Biosensors are easy-to-use and cost-effective devices that are emerging as an attracting tool not only in settling diagnosis or in disease monitoring, but also in mass screening tests, a timely topic that impacts on daily life of the whole society. Nanotechnologies lend themselves to the development of highly sensitive device whose realization has become a very interdisciplinary topic. Relying on the enhancement of the fluorescence signal detected at the surface of patterned gold nanoparticles, we report the behavior of an analytical device in detecting immunoglobulins in real urine samples that shows a limit of detection of approximately 8 μg/L and a linear range of 10-100 μg/L well below the detection limit of nephelometric method, which is the reference method for this analysis. These performances have been reached thanks to an effective surface functionalization technique and can be improved even more if superhydrophobic features of the substrate we produce will be exploited. Since the analyte recognition is realized by antibodies the specificity is very high and, in fact, no interference has been detected by other compounds also present in the real urine samples. The device has been assessed on serum samples by comparing IgG concentrations values obtained by the biosensor with those provided by a nephelometer. In this step we found that our approach allows the analysis of the whole blood without any pretreatment; moreover, it is inherently extendable to the analysis of most biochemical markers in biological fluids.
关键词： antibody,point-of-care device,nanostructured gold surface,photochemical immobilization technique,gold nanoparticles,metal enhanced fluorescence,biosensors
更新于2025-09-23 15:22:29
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Paired-Agent Fluorescence Molecular Imaging of Sentinel Lymph Nodes Using Indocyanine Green as a Control Agent for Antibody-Based Targeted Agents
摘要： Purpose. Paired-agent molecular imaging methods, which employ coadministration of an untargeted, “control” imaging agent with a targeted agent to correct for nonspecific uptake, have been demonstrated to detect 200 cancer cells in a mouse model of metastatic breast cancer. This study demonstrates that indocyanine green (ICG), which is approved for human use, is an ideal control agent for future paired-agent studies to facilitate eventual clinical translation. Methods. The kinetics of ICG were compared with a known ideal control imaging agent, IRDye-700DX-labeled antibody in both healthy and metastatic rat popliteal lymph nodes after coadministration, intradermally in the footpad. Results. The kinetics of ICG and antibody-based imaging agent in tumor-free rat lymph nodes demonstrated a strong correlation with each other (r = 0.98, p < 0.001) with a measured binding potential of -0.102 ± 0.03 at 20 min postagent injection, while the kinetics of ICG and targeted imaging agent shows significant separation in the metastatic lymph nodes. Conclusion. This study indicated a potential for microscopic sensitivity to cancer spread in sentinel lymph nodes using ICG as a control agent for antibody-based molecular imaging assays.
关键词： Paired-agent imaging,Antibody-based targeted agents,Control agent,Indocyanine green,Fluorescence molecular imaging,Sentinel lymph node
更新于2025-09-23 15:22:29