Antibody Complexes Research Articles

PRODUCTION OF POLYCLONAL ANTIBODIES AGAINST INDIAN PAPER WASP ROPALIDIA MARGINATA VENOM TOXINS AND THEIR EFFICACY IN THE REVERSAL OF TOXIC EFFECTS

Objective: In this study, albino mice were injected with a sub-lethal dosage of purified wasp Ropalidia Marginata venom toxins to assess the effectiveness of polyclonal anti-venom antibodies. Methods: To neutralize the toxic effects, polyclonal antibodies were generated by immunizing albino mice. The antibody underwent partial purification using ammonium sulphate treatment and octanoic acid precipitation. To detect the presence of antibodies in the antiserum, an immunodouble diffusion test was conducted using Ouchterlony's method (1962). This involved allowing both antigens and antibodies to diffuse radially towards each other from their respective wells. When they reached an equivalence zone, a precipitation complex of antigen and antibody became visible as a concentric band, indicating the development of the combination. To quantitatively determine the amount of antibodies in the antiserum, the equivalency zone approach was used. Results: Experimental mice were injected with a combination containing 400, 800, and 1200 µg of pure antibody, which had been treated serum biomolecules, including metabolic enzymes, completely reversed in the experimental with 40% of the LD50 of wasp venom the elevated serum parameters were glucose, pyruvic acid, lipid, protein and free amino acid, reached to normal (100%) in the treated with 40% of LD50 of the venom and polyclonal treated after 6 h of administration. Anti-serum treatment also successfully normalized the alteration in serum enzyme just after 4h. Similarly, anti-serum treatment also successfully normalized the alteration in serum enzyme just after 4h treated with 40% of LD50 of the venom. Serum ACP content was obtained as 125.35% after 40% of LD50 venom injection, which was get normalized up to 102.81% after 4 h of the anti-venom treatment. Serum ALP content of 114.8% elevation was reversed back to 102.40% after anti-venom treatment. The GPT level significantly reversed up to 102.5%, while it was 130% in the venom-treated mice. A complete reversal was obtained in GPT level, which was obtained as 104.54% in the venom-treated animal. Similarly, LDH which was elevated up to 112.45 % in venom-injected mice was successfully reversed up to 100.25% after anti-venom treatment. Similarly, Ache concentration was fully recovered after anti-venom treatment 6 h, all animals (group B-E) that had received 40% of the LD50 of venom treated with pure antiserum. Conclusion: The venom-injected group showed a complete restoration of serum protein, free amino acid, uric acid, cholesterol, pyruvic acid, total lipid, and glucose level in experimental mice.

The Mathematical Modeling of the Host-Virus Interaction in Dengue Virus Infection: A Quantitative Study.

Infectious diseases, such as Dengue fever, pose a significant public health threat. Developing a reliable mathematical model plays a crucial role in quantitatively elucidating the kinetic characteristics of antibody-virus interactions. By integrating previous models and incorporating the antibody dynamic theory, we have constructed a novel and robust model that can accurately simulate the dynamics of antibodies and viruses based on a comprehensive understanding of immunology principles. It explicitly formulates the viral clearance effect of antibodies, along with the positive feedback stimulation of virus-antibody complexes on antibody regeneration. In addition to providing quantitative insights into the dynamics of antibodies and viruses, the model exhibits a high degree of accuracy in capturing the kinetics of viruses and antibodies in Dengue fever patients. This model offers a valuable solution to modeling the differences between primary and secondary Dengue infections concerning IgM/IgG antibodies. Furthermore, it demonstrates that a faster removal rate of antibody-virus complexes might lead to a higher peak viral loading and worse clinical symptom. Moreover, it provides a reasonable explanation for the antibody-dependent enhancement of heterogeneous Dengue infections. Ultimately, this model serves as a foundation for constructing an optimal mathematical model to combat various infectious diseases in the future.

A Novel Label-Free Electrochemical Immunosensor for the Detection of Thyroid Transcription Factor 1 Using Ribbon-like Tungsten Disulfide-Reduced Graphene Oxide Nanohybrids and Gold Nanoparticles.

Thyroid transcription factor 1 (TTF1) is an important cancer-related biomarker for clinical diagnosis, especially for carcinomas of lung and thyroid origin. Herein, a novel label-free electrochemical immunosensor was prepared for TTF1 detection based on nanohybrids of ribbon-like tungsten disulfide-reduced graphene oxide (WS2-rGO) and gold nanoparticles (AuNPs). The proposed immunosensor employed H2O2 as the electrochemical probe because of the excellent peroxidase-like activity of ribbon-like WS2-rGO. The introduction of AuNPs not only enhanced the electrocatalytic activity of the immunosensor, but also provided immobilization sites for binding TTF1 antibodies. The electrochemical signals can be greatly amplified due to their excellent electrochemical performance, which realized the sensitive determination of TTF1 with a wide linear range of 0.025-50 ng mL-1 and a lower detection limit of 0.016 ng mL-1 (S/N = 3). Moreover, the immunosensor exhibited high selectivity, good reproducibility, and robust stability, as well as the ability to detect TTF1 in human serum with satisfactory results. These observed properties of the immunosensor enhance its potential practicability in clinical applications. This method can also be used for the detection of other tumor biomarkers by using the corresponding antigen-antibody complex.

Insulin-induced Panniculitis in a Child: A Rare Entity

Abstract Insulin-induced panniculitis is a rare entity. It represents the early stage of development of lipoatrophy. The complex of insulin and insulin antibodies leads to the activation of macrophages, which is responsible for the initiation of autoimmune process and leading to panniculitis followed by subsequent development of lipoatrophy. Here, we report the case of insulin-induced panniculitis with lipoatrophy associated with Type 1 diabetes mellitus in an 11-year-old female child.

The Adsorption of P2X2 Receptors Interacting with IgG Antibodies Revealed by Combined AFM Imaging and Mechanical Simulation.

The adsorption of proteins onto surfaces significantly impacts biomaterials, medical devices, and biological processes. This study aims to provide insights into the irreversible adsorption process of multiprotein complexes, particularly focusing on the interaction between anti-His6 IgG antibodies and the His6-tagged P2X2 receptor. Traditional approaches to understanding protein adsorption have centered around kinetic and thermodynamic models, often examining individual proteins and surface coverage, typically through Molecular Dynamics (MD) simulations. In this research, we introduce a computational approach employing Autodesk Maya 3D software for the investigation of multiprotein complexes' adsorption behavior. Utilizing Atomic Force Microscopy (AFM) imaging and Maya 3D-based mechanical simulations, our study yields real-time structural and kinetic observations. Our combined experimental and computational findings reveal that the P2X2 receptor-IgG antibody complex likely undergoes absorption in an 'extended' configuration. Whereas the P2X2 receptor is less adsorbed once is complexed to the IgG antibody compared to its individual state, the opposite is observed for the antibody. This insight enhances our understanding of the role of protein-protein interactions in the process of protein adsorption.

IL-4/IL-4 Ab complex enhances the accumulation of both antigen-specific and bystander CD8 T cells in mouse lungs infected with influenza A virus

BackgroundUnlike conventional T cells, innate and virtual-memory CD8 T cells in naïve mice acquire their memory phenotypes and functions in the absence of antigenic encounters in a cytokine-dependent manner. The relevant cytokines include interleukin-4 (IL-4), type I interferon, and interleukin-15 (IL-15). Moreover, exogenous IL-4 can also induce de novo generation and/or expansion of the virtual-memory CD8 T cell population. In this study, we investigated whether exogenous IL-4 could enhance the immune response to a viral infection.ResultsIn vivo administration of IL-4 and an anti-IL-4 antibody complex (IL-4C) increased CXCR3 expression in both memory and naïve phenotype CD8 T cells in the absence of antigenic stimulation, and protected mice from lethal influenza infection. Flow cytometric analysis of lung-infiltrating immune cells on day 5 after virus infection revealed higher numbers of antigen-specific and bystander CD8 T cells in IL-4C-treated mice than in control mice. In particular, the bystander CD8 T cells were a naïve or evident memory phenotypes. Crucially, an anti-CXCR3 blocking antibody abrogated this IL-4C effect, reflecting that the increased accumulation of CD8 T cells in the lungs after IL-4C treatment is dependent on CXCR3.ConclusionsThese data demonstrate that exogenous IL-4C plays a protective role by enhancing CXCR3-dependent migration of CD8 T cells into influenza-infected lungs.

The cytosolic antibody receptor TRIM21 is required for effective tau immunotherapy

AbstractBackgroundUsing a variety of cell and animal model systems we demonstrate that the cytosolic antibody receptor and E3 ligase TRIM21 plays a critical role in antibody‐mediated protection against tau pathologyMethodExperiments with organotypic hippocampal slice cultures and in vivo studies in mouse models were conducted to study the role of TRIM21 in tau immunotherapy.ResultWe demonstrate that tau:antibody complexes were internalised to the cytosol of neurons, enabling TRIM21 engagement and thereby protecting against seeded aggregation. Crucially, antibody‐mediated protection against tau pathology was lost in mice lacking TRIM21ConclusionThe cytosolic compartment provides a site of immunotherapeutic protection, which can help in the design of antibody‐based therapies in neurodegenerative disease.

Effect of Heparin on Antigen-Antibody Interaction in a Model System of the Bevacizumab Therapeutic Antibody And its Target VEGF-А165

In this work, we studied the effect of heparin on the binding process of VEGF-A165, the most common and important growth factor, and the monoclonal antibody bevacizumab by bio-layer interferometry. A complex of the full-length bevacizumab antibody with VEGF-A165 was modeled. The data obtained can be used in therapy with this antibody, as well as in the development of other therapeutic antibodies with maximum target specificity under various conditions.

Predicting the immune escape of SARS-CoV-2 neutralizing antibodies upon mutation

COVID-19 has resulted in millions of deaths and severe impact on economies worldwide. Moreover, the emergence of SARS-CoV-2 variants presented significant challenges in controlling the pandemic, particularly their potential to avoid the immune system and evade vaccine immunity. This has led to a growing need for research to predict how mutations in SARS-CoV-2 reduces the ability of antibodies to neutralize the virus. In this study, we assembled a set of 1813 mutations from the interface of SARS-CoV-2 spike protein's receptor binding domain (RBD) and neutralizing antibody complexes and developed a machine learning model to classify high or low escape mutations using interaction energy, inter-residue contacts and predicted binding free energy change. Our approach achieved an Area under the Receiver Operating Characteristics (ROC) Curve (AUC) of 0.91 using the Random Forest classifier on the test dataset with 217 mutations. The model was further utilized to predict the escape mutations on a dataset of 29,165 mutations located at the interface of 83 RBD-neutralizing antibody complexes. A small subset of this dataset was also validated based on available experimental data. We found that top 10 % high escape mutations were dominated by charged to nonpolar mutations whereas low escape mutations were dominated by polar to nonpolar mutations. We believe that the present method will allow prioritization of high/low escape mutations in the context of neutralizing antibodies targeting SARS-CoV-2 RBD region and assist antibody design for current and emerging variants.

Predicting virus Fitness: Towards a structure-based computational model

Predicting the impact of new emerging virus mutations is of major interest in surveillance and for understanding the evolutionary forces of the pathogens. The SARS-CoV-2 surface spike-protein (S-protein) binds to human ACE2 receptors as a critical step in host cell infection. At the same time, S-protein binding to human antibodies neutralizes the virus and prevents interaction with ACE2. Here we combine these two binding properties in a simple virus fitness model, using structure-based computation of all possible mutation effects averaged over 10 ACE2 complexes and 10 antibody complexes of the S-protein (∼380,000 computed mutations), and validated the approach against diverse experimental binding/escape data of ACE2 and antibodies. The ACE2-antibody selectivity change caused by mutation (i.e., the differential change in binding to ACE2 vs. immunity-inducing antibodies) is proposed to be a key metric of fitness model, enabling systematic error cancelation when evaluated. In this model, new mutations become fixated if they increase the selective binding to ACE2 relative to circulating antibodies, assuming that both are present in the host in a competitive binding situation. We use this model to categorize viral mutations that may best reach ACE2 before being captured by antibodies. Our model may aid the understanding of variant-specific vaccines and molecular mechanisms of viral evolution in the context of a human host.

Learning from prepandemic data to forecast viral escape

Effective pandemic preparedness relies on anticipating viral mutations that are able to evade host immune responses to facilitate vaccine and therapeutic design. However, current strategies for viral evolution prediction are not available early in a pandemic—experimental approaches require host polyclonal antibodies to test against1–16, and existing computational methods draw heavily from current strain prevalence to make reliable predictions of variants of concern17–19. To address this, we developed EVEscape, a generalizable modular framework that combines fitness predictions from a deep learning model of historical sequences with biophysical and structural information. EVEscape quantifies the viral escape potential of mutations at scale and has the advantage of being applicable before surveillance sequencing, experimental scans or three-dimensional structures of antibody complexes are available. We demonstrate that EVEscape, trained on sequences available before 2020, is as accurate as high-throughput experimental scans at anticipating pandemic variation for SARS-CoV-2 and is generalizable to other viruses including influenza, HIV and understudied viruses with pandemic potential such as Lassa and Nipah. We provide continually revised escape scores for all current strains of SARS-CoV-2 and predict probable further mutations to forecast emerging strains as a tool for continuing vaccine development (evescape.org).

Long cruising aptamer-albumin nanobots intelligently capture and restrain circulating tumor cells

Circulating tumor cells (CTC) are the root cause of life-threatening cancer metastases. The rare CTC cannot be specifically killed by anti-cancer agents without producing any side effects, nor be targeted-and-eradicated by antibody or aptamer complexes because of the complexes’ limited circulating half-life in the blood owing to metabolism and immune clearance. Here, we build the biosafe and biostable aptamer-albumin nanobots that can cruise in the blood much long together with the CTC, resulting in the increased capture efficiency and specificity of the nanobots targeting CTC. The intelligent-recognizing unit of the novel nanobots is composed of three EpCAM-targeting aptamers (AP) that can specifically target breast CTC, and their sticky ends are completely closed to form the biostable circular trivalent aptamers (CTA). The binding energy, specificity and stability of the circular three arms CTA are significantly superior to their linear AP. After conjugating CTA to human serum albumin (CTA-HSA) by amination reaction, the blood circulation time of CTA-HSA in mice was significantly prolonged by 3 folds, resulting in the enhanced collision probability and increased capture efficiency of CTA-HSA to CTC. CTA-HSA can escape the immune clearance and inhibit adhesion/invasion and cell cycle of CTC, resulting in CTC apoptosis. This proof-of-concept study provides a promising nanobiomaterial for CTC-induced cancer metastases.

59 DNA-templated antibody complexes provide insights into the geometric requirements of human complement activation

59 DNA-templated antibody complexes provide insights into the geometric requirements of human complement activation

Site‐selective heterodimerization of antibody fragments for bispecific antibody complexes enabled by divinylpyrimidine reagent

AbstractBackgroundBispecific Abs (BsAbs) are useful class of Ab‐based drugs but with difficulty to fabricate them.AimsCross‐linking two Abs by click chemistry.Materials & MethodsBis‐alkynylated divinylpyrimidine (DVP) was synthesized and incorporated into an Ab, which was then cross‐linked with azido‐modified Ab to produce BsAb.ResultsThe heterodimeric anti‐PD‐L1‐HER2 BsAbC was prepared by CuAAC and its formation was confirmed by SDS‐PAGE. BsAbC was purified by biotinylated HER2 antigen coated magnetic bead and was used to fabricate a microarray. In the microarray binding assay, the heterodimeric anti‐PD‐L1‐HER2 BsAbC showed binding activity with PD‐L1 and HER2 antigens.DiscussionThis study represents the first proof‐of‐concept that bis‐alkynylated DVP crosslinker reagent can be used for conjugating half‐antibody fragments site‐selectively by CuAAC. A preliminary characterization revealed that this reagent predominantly incorporates a bioorthogonal functional handle into the desired half‐antibody fragments with minor amounts of a re‐bridged full‐length antibody.ConclusionThis study highlights the utility of DVP crosslinker reagents and validates the use of these molecules for the rapid assembly of switchable complex antibody bioconjugates that enables the control of the intended function of fabricated BsAbC.

Single-Cell Heterogeneity Restorative Chimeric Engineering Nanoparticles for Alleviating Antibody-Mediated Allograft Injury.

Disturbance of single-cell transcriptional heterogeneity is an inevitable consequence of persistent donor-specific antibody (DSA) production and allosensitization. However, identifying and efficiently clearing allospecific antibody repertoires to restore single-cell transcriptional profiles remain challenging. Here, inspired by the high affinity of natural bacterial proteins for antibodies, a genetic engineered membrane-coated nanoparticle termed as DSA trapper by the engineering chimeric gene of protein A/G with phosphatidylserine ligands for macrophage phagocytosis was reported. It has been shown that DSA trappers adsorbed alloreactive antibodies with high saturation and activated the heterophagic clearance of antibody complexes, alleviating IgG deposition and complement activation. Remarkably, DSA trappers increased the endothelial protective lineages by 8.39-fold, reversed the highly biased cytotoxicity, and promoted the proliferative profiles of Treg cells, directly providing an obligate immune tolerant niche for single-cell heterogeneity restoration. In the mice of allogeneic transplantation, the DSA trapper spared endothelial from inflammatory degenerative rosette, improved the glomerular filtration rate, and prolonged the survival of allogeneic mice from 23.6 to 78.3 days. In general, by identifying the lineage characteristics of rejection-related antibodies, the chimeric engineered DSA trapper realized immunoadsorption and further phagocytosis of alloantibody complexes to restore the single-cell genetic architecture of the allograft, offering a promising prospect for the treatment of alloantibody-mediated immune injury.

Suppression of Nasopharyngeal and Gastric Tumor Growth in a Mouse Model by Antibodies to Epstein-Barr Virus LMP1 Protein.

The study aimed to investigate the antitumor efficacy of anti-LMP1 antibodies in EBV-positive nasopharyngeal and stomach cell lines and xenograft models. The study also examined the NF-κB expression and cell cycle activation of NPC-serum-exosome-associated LMP1. Anti-LMP1 antibody treatment before or during cell implantation prevented tumor growth in nude mice. A small dose of antibodies resulted in complete tumor regression for at least three months after the tumors had grown in size. The consumption of antigen-antibody complexes by tumor cells limited tumor growth. In vitro experiments showed that anti-LMP1 antibodies killed EBV-positive NPC- or GC-derived epithelial cell lines and EBV-positive human B-cell lines but not EBV-negative cell lines. Treatment with anti-LMP1 reduced NF-κB expression in cells. The animal model experiments showed that anti-LMP1 inhibited and prevented NPC- or GC-derived tumor growth. The results suggest that LMP1 antibody immunotherapy could cure nasopharyngeal cancer, EBV-positive gastric carcinoma, and EBV-associated lymphomas. However, further validation of these findings is required through human clinical trials.

P-107 MICROFLUIDIC SPERM SEXING: An XLD (X-linked Disorder) model for preventing genetic disorders in newborns

Abstract Study question Can sperm sexing based on unique surface membrane markers – enable genotypic sperm sorting by allosomal content to prevent certain X-linked disorders in newborns? Summary answer In an X-Linked-Disorder prevention model, we utilized a microfluidic device functionalized with H-Y monoclonal antibodies attached to magnetic beads to enrich Y-chromosome bearing sperm sorts. What is known already About 4% of newborns are at risk of being born with an X-linked disease such as Fragile X Syndrome. The current clinically approved mitigation plan for preventing such genetic disorders is to consider Preimplantation Genetic Testing for Monogenetic diseases (PGT-M) which involves an invasive microsurgical procedure on fragile embryos. To minimize this invasiveness, we proposed a less invasive microsurgical exclusion approach to prevent such genetic disorders by genotypically sorting sperm cells that can be used for IUI/IVF/ICSI – with microfluidics – using their allosomal characteristics. Study design, size, duration Semen samples purchased from FairFax Cryobank from anonymous donors were used in immuno-magnetic microfluidic sorting protocols with the use of phycoerythrin (PE) binding Tetrameric Antibody Complexes (TAC) coupled with magnetic beads to tag H-Y monoclonal antibody (mab) binding activities on the sperm surface membrane. Participants/materials, setting, methods Immuno-magnetic sorting was conducted with specially fabricated microfluidic-sperm-sorting (MSS) chips. Sort yield and purities of positively selected sperm populations were assessed by flow-cytometry, immunofluorescence, fluorescent-in-situ-hybridization (FISH) and by quantitative-PCR (qPCR – Cq & Tm). Quantitative experiments were run in triplicates and the results were expressed as absolute means for comparison between paired variables. Statistical analyses were performed using Student’s t-test and a p-value of less than 0.05 was considered to be statistically significant. Main results and the role of chance For the assessment of H-Y sperm sorts, our preliminary data showed that flow cytometry was able to confirm the positive sperm selection (which were mostly Y-chromosome bearing sperm cells) in a sperm genotyping protocol – initially utilizing immuno-magnetic sort tubes prior to flowing through microfluidic chips. Both immuno-magnetic sort tubes and the microfluidic chips were able to optimally confirm from qPCR-Cq means – the negative selection – being the X-chromosome bearing sperm that may have been largely unselected by the H-Y-mab. Values for Cq means compared under H-Y-qPCR data sets were statistically significant with a p value of 0.000023 (p values is significant at < 0.05). Our study was able to elucidate a potential less invasive clinical application of genotypically sorting sperm cells with mab-functionalized microfluidic chips for an XLD-model. Molecular down stream applications like flow cytometry may therefore be utilized to experimentally confirm sort purities of positively selected sperm populations (with H-Y markers) – based on the utility of the SMCX and SMCY gene in the X and Y sperm chromosome bearing sperm cells respectively. Limitations, reasons for caution Physiological effect of magnetic fields of varying tesla or gauss strengths on sperm selected with immuno-magnetic beads may warrant further safety studies prior to actual clinical applications. Optimal selection results may be achieved with the use of freshly collected normospermic rather than frozen-thawed samples. Wider implications of the findings Although we had de-emphasized the clinical application of the microfluidic sperm sexing protocol for legitimate use in sex selection and gender balancing, this study design was solely focused on adapting a less invasive method of preventing X-linked genetic disorders with the potential exclusion of microsurgical procedures leading to PGT-M. Trial registration number NA

Analysis of Adeno-Associated Virus Serotype 8 (AAV8)-antibody complexes using epitope mapping by molecular imprinting leads to the identification of Fab peptides that potentially evade AAV8 neutralisation

Gene therapy is a promising approach for treating genetic disorders by delivering therapeutic genes to replace or correct malfunctioning genes. However, the introduced gene therapy vector can trigger an immune response, leading to reduced efficacy and potential harm to the patient. To improve the efficiency and safety of gene therapy, preventing the immune response to the vector is crucial. This can be achieved through the use of immunosuppressive drugs, vector engineering to evade the immune system, or delivery methods that bypass the immune system altogether. By reducing the immune response, gene therapy can deliver therapeutic genes more effectively and potentially cure genetic diseases. In this study, a novel molecular imprinting technique, combined with mass-spectrometry and bioinformatics, was used to identify four antigen-binding fragments (Fab) sequences of Adeno-Associated Virus (AAV) - neutralising antibodies capable of binding to AAV. The identified Fab peptides were shown to prevent AAV8's binding to antibodies, demonstrating their potential to improve gene therapy efficiency by preventing the immune response.

Coreactant-free electrochemiluminescence of polyfluorene nanoparticle coupling double quencher for β-amyloid1-42 detection.

β-amyloid1-42 (Aβ1-42) is a humoral biomarker for early diagnosis of Alzheimer's disease (AD), and exists at a low level in human body. Its sensitive detection is very valuable. The electrochemiluminescence (ECL) assay of Aβ1-42 has attracted special attention owing to high sensitivity and simple operation. However, currently reported ECL assays for Aβ1-42 usually required the introduction of exogenous coreactants to improve the detection sensitivity. Introducing exogenous coreactants will lead to non-negligible repeatability and stability problems. This work exploited poly [(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1',3}-thiadazole)] nanoparticles (PFBT NPs) as coreactant-free ECL emitters for detecting Aβ1-42. The PFBT NPs, first antibody (Ab1) and antigen Aβ1-42 were successively assembled on glassy carbon electrode (GCE). Silica nanoparticles served as a carrier to grow polydopamine (PDA) in situ, and further assembled Au nanoparticles (Au NPs) and second antibody (Ab2), producing the secondary antibody complex (SiO2@PDA-Au NPs-Ab2). With its assembly on the biosensor, the ECL signal decreased since both PDA and Au NPs could quench ECL emission from PFBT NPs. The limit of detection (LOD) of 0.55fg/mL and limit of quantification (LOQ) of 37.45fg/mL for Aβ1-42 were obtained. PFBT NPs coupling dual-quencher PDA-Au NPs created an excellent ECL system for bioassays, and constructed a sensitive analytical method for Aβ1-42.

Tetrameric Antibody Complexes and Affinity Tag Peptides for the Selective Immobilization and Imaging of Single Quantum Dots.

Colloidal semiconductor quantum dots (QDs) are of widespread interest as fluorescent labels for bioanalysis and imaging applications. Single-particle measurements have proven to be a very powerful tool for better understanding the fundamental properties and behaviors of QDs and their bioconjugates; however, a recurring challenge is the immobilization of QDs in a solution-like environment that minimizes interactions with a bulk surface. Immobilization strategies for QD-peptide conjugates are particularly underdeveloped within this context. Here, we present a novel strategy for the selective immobilization of single QD-peptide conjugates using a combination of tetrameric antibody complexes (TACs) and affinity tag peptides. A glass substrate is modified with an adsorbed layer of concanavalin A (ConA) that binds a subsequent layer of dextran that minimizes nonspecific binding. A TAC with anti-dextran and anti-affinity tag antibodies binds to the dextran-coated glass surface and to the affinity tag sequence of QD-peptide conjugates. The result is spontaneous and sequence-selective immobilization of single QDs without any chemical activation or cross-linking. Controlled immobilization of multiple colors of QDs is possible using multiple affinity tag sequences. Experiments confirmed that this approach positions the QD away from the bulk surface. The method supports real-time imaging of binding and dissociation, measurements of Förster resonance energy transfer (FRET), tracking of dye photobleaching, and detection of proteolytic activity. We anticipate that this immobilization strategy will be useful for studies of QD-associated photophysics, biomolecular interactions and processes, and digital assays.