RESUMEN
Increasing the binding affinity of an antibody to its target antigen is a crucial task in antibody therapeutics development. This paper presents a pretrainable geometric graph neural network, GearBind, and explores its potential in in silico affinity maturation. Leveraging multi-relational graph construction, multi-level geometric message passing and contrastive pretraining on mass-scale, unlabeled protein structural data, GearBind outperforms previous state-of-the-art approaches on SKEMPI and an independent test set. A powerful ensemble model based on GearBind is then derived and used to successfully enhance the binding of two antibodies with distinct formats and target antigens. ELISA EC50 values of the designed antibody mutants are decreased by up to 17 fold, and KD values by up to 6.1 fold. These promising results underscore the utility of geometric deep learning and effective pretraining in macromolecule interaction modeling tasks.
Asunto(s)
Afinidad de Anticuerpos , Redes Neurales de la Computación , Humanos , Anticuerpos/inmunología , Anticuerpos/química , Simulación por Computador , Aprendizaje Profundo , Antígenos/inmunología , Unión Proteica , Ensayo de Inmunoadsorción Enzimática , Modelos MolecularesRESUMEN
Protein-glutamine gamma-glutamyltransferase 2 (TGM2) is a Ca 2+ dependent enzyme that catalyzes transglutaminase cross-linking modifications. TGM2 is involved in various diseases, either in a protective or contributory manner, making it a crucial protein to study and determine its therapeutic potential. Identifying high-performing TGM2 antibodies would facilitate these investigations. Here we have characterized seventeen TGM2 commercial antibodies for western blot and sixteen for immunoprecipitation, and immunofluorescence. The implemented standardized experimental protocol is based on comparing read-outs in knockout cell lines against their isogenic parental controls. This study is part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While the use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.
Asunto(s)
Anticuerpos , Western Blotting , Técnica del Anticuerpo Fluorescente , Inmunoprecipitación , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transglutaminasas , Humanos , Transglutaminasas/inmunología , Técnica del Anticuerpo Fluorescente/métodos , Inmunoprecipitación/métodos , Anticuerpos/inmunología , Proteínas de Unión al GTP/inmunologíaRESUMEN
Leukemia inhibitory factor (LIF) is involved in the progression of different cancers. In this study, we investigated the effect of anti-LIF antibodies on immune-related gene expression in the Balb/c mouse model of breast cancer. To immunize mice against LIF, recombinant LIF with Freund adjuvant was injected into the test group, whereas the control group received phosphate-buffered saline with adjuvant. Tumor induction (4T1 cell line) was performed by increasing the antibody titer. The expression of immune-related genes was evaluated by real-time PCR. The anti-LIF titer was significantly increased in the immunized group. The expression of genes related to the differentiation of T helper (Th)-1, Th-2, and Th-17 cells was significantly higher in the immunized group than in the control group. In addition, anti-LIF did not have a significant effect on the expression of genes related to the differentiation of regulatory T cells, and immune checkpoint-associated genes. Additionally, the test group had higher survival and lower tumor development rates. The results demonstrated that the anti-LIF antibody may potentially play a role in the differentiation of immune cells or immune responses. However, further studies utilizing advanced techniques are necessary to validate its function.
Asunto(s)
Neoplasias de la Mama , Factor Inhibidor de Leucemia , Ratones Endogámicos BALB C , Animales , Femenino , Factor Inhibidor de Leucemia/genética , Factor Inhibidor de Leucemia/metabolismo , Factor Inhibidor de Leucemia/inmunología , Ratones , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/genética , Modelos Animales de Enfermedad , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Anticuerpos/inmunologíaRESUMEN
Huntingtin encodes a 3144 amino acid protein, with a polyglutamine repeat tract at the N-terminus. Expansion of this repeat tract above a pathogenic threshold of 36 repeats is the causative mutation of Huntington's disease, a neurodegenerative disorder characterized by loss of striatal neurons. Here we have characterized twenty Huntingtin commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.
Asunto(s)
Anticuerpos , Western Blotting , Técnica del Anticuerpo Fluorescente , Proteína Huntingtina , Inmunoprecipitación , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/inmunología , Inmunoprecipitación/métodos , Técnica del Anticuerpo Fluorescente/métodos , Anticuerpos/inmunología , Animales , Enfermedad de Huntington/inmunología , Enfermedad de Huntington/diagnóstico , Enfermedad de Huntington/genética , Células HEK293RESUMEN
Small molecules find extensive application in medicine, food safety, and environmental studies, particularly in biomedicine. Immunoassay technology, leveraging the specific recognition between antigens and antibodies, offers a superior alternative to traditional physical and chemical analysis methods. This approach allows for the rapid and accurate detection of small molecular compounds, owing to its high sensitivity, specificity, and swift analytical capabilities. However, small molecular compounds often struggle to effectively stimulate an immune response due to their low molecular weight, weak antigenicity, and limited antigenic epitopes. To overcome this, coupling small molecule compounds with macromolecular carriers to form complete antigens is typically required to induce specific antibodies in animals. Consequently, the preparation of small-molecule artificial antigens and the production of efficient specific antibodies are crucial for achieving precise immunoassays. This paper reviews recent advancements in small molecule antibody preparation technology, emphasizing the design and synthesis of haptens, the coupling of haptens with carriers, the purification and identification of artificial antigens, and the preparation of specific antibodies. Additionally, it evaluates the current technological shortcomings and limitations while projecting future trends in artificial antigen synthesis and antibody preparation technology.
Asunto(s)
Anticuerpos , Antígenos , Haptenos , Antígenos/inmunología , Animales , Anticuerpos/inmunología , Anticuerpos/química , Haptenos/química , Haptenos/inmunología , Humanos , Inmunoensayo/métodosRESUMEN
Traditional computational methods for antibody design involved random mutagenesis followed by energy function assessment for candidate selection. Recently, diffusion models have garnered considerable attention as cutting-edge generative models, lauded for their remarkable performance. However, these methods often focus solely on the backbone or sequence, resulting in the incomplete depiction of the overall structure and necessitating additional techniques to predict the missing component. This study presents Antibody-SGM, an innovative joint structure-sequence diffusion model that addresses the limitations of existing protein backbone generation models. Unlike previous models, Antibody-SGM successfully integrates sequence-specific attributes and functional properties into the generation process. Our methodology generates full-atom native-like antibody heavy chains by refining the generation to create valid pairs of sequences and structures, starting with random sequences and structural properties. The versatility of our method is demonstrated through various applications, including the design of full-atom antibodies, antigen-specific CDR design, antibody heavy chains optimization, validation with Alphafold3, and the identification of crucial antibody sequences and structural features. Antibody-SGM also optimizes protein function through active inpainting learning, allowing simultaneous sequence and structure optimization. These improvements demonstrate the promise of our strategy for protein engineering and significantly increase the power of protein design models.
Asunto(s)
Cadenas Pesadas de Inmunoglobulina , Modelos Moleculares , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/inmunología , Secuencia de Aminoácidos , Ingeniería de Proteínas , Regiones Determinantes de Complementariedad/química , Conformación Proteica , Anticuerpos/química , Anticuerpos/inmunologíaRESUMEN
The comprehensive understanding of the orientation of antibodies on a solid surface is crucial for affinity-based sensing mechanisms. In this study, we demonstrated that the orientation of primary antibodies modified on carboxy-functionalized polystyrene (PS) particles can be analyzed using zeta potential behavior at different pH based on the combined Gouy-Chapman-Stern model and the acid dissociation of carboxy groups and antibodies. We observed that at low surface concentrations of the primary antibody, a side-on orientation was predominant. However, at higher concentrations (approximately 30000 antibodies per PS particle), the orientation shifted to an end-on type due to steric hindrance. Furthermore, the reaction mechanism of the secondary antibody exhibited pH-dependent behavior. At pH > 7, the zeta potential changes were attributed to the antibody-antibody reaction, whereas at pH < 7, adsorption of secondary antibody onto the PS particle was observed, leading to a change in the orientation of the primary antibody modified on the PS particle to an end-on type. The change in zeta potential due to secondary antibody binding indicated a detection limit of 37000 antibodies per PS particle. As a result, we revealed that the analysis of zeta potential behavior enables the evaluation of antibody orientation and the detection of zeptomole order antibodies. This study represents the first demonstration of this capability. We anticipate that the present concept and results will broaden the quantitative application of zeta potential measurements and have significant implications for research areas, including physical chemistry and analytical chemistry.
Asunto(s)
Anticuerpos , Poliestirenos , Poliestirenos/química , Concentración de Iones de Hidrógeno , Anticuerpos/química , Anticuerpos/inmunología , Propiedades de Superficie , Tamaño de la PartículaRESUMEN
Exosomes are small extracellular vesicles produced by almost all cell types in the human body, and exosomal microRNAs (miRNAs) are small non-coding RNA molecules that are known to serve as important biomarkers for diseases such as cancer. Given that the upregulation of miR-106b is closely associated with several types of malignancies, the sensitive and accurate detection of miR-106b is important but difficult. In this study, a surface acoustic wave (SAW) biosensor was developed to detect miR-106b isolated from cancer cells based on immunoaffinity separation technique using our unique paddle screw device. Our novel SAW biosensor could detect a miR-106b concentration as low as 0.0034 pM in a linear range from 0.1 pM to 1.0 µM with a correlation coefficient of 0.997. Additionally, we were able to successfully detect miR-106b in total RNA extracted from the exosomes isolated from the MCF-7 cancer cell line, a model system for human breast cancer, with performance comparable to commercial RT-qPCR methods. Therefore, the exosome isolation by the paddle screw method and the miRNA detection using the SAW biosensor has the potential to be used in basic biological research and clinical diagnosis as an alternative to RT-qPCR.
Asunto(s)
Técnicas Biosensibles , Exosomas , MicroARNs , Humanos , Exosomas/química , Técnicas Biosensibles/métodos , Técnicas Biosensibles/instrumentación , MicroARNs/aislamiento & purificación , MicroARNs/genética , Células MCF-7 , Anticuerpos/inmunología , Anticuerpos/químicaRESUMEN
Synaptotagmin-1 is a synaptic vesicle transmembrane protein that senses calcium influx via its tandem C2-domains, triggering synchronous neurotransmitter release. Disruption to SYT1 is associated with neurodevelopmental disorders, highlighting the importance of identifying high-quality research reagents to enhance understanding of Synaptotagmin-1 in health and disease. Here we have characterized thirteen Synaptotagmin-1 commercial antibodies for western blot, immunoprecipitation, immunofluorescence and flow cytometry using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.
Asunto(s)
Anticuerpos , Western Blotting , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Inmunoprecipitación , Sinaptotagmina I , Sinaptotagmina I/inmunología , Sinaptotagmina I/metabolismo , Humanos , Citometría de Flujo/métodos , Inmunoprecipitación/métodos , Técnica del Anticuerpo Fluorescente/métodos , Anticuerpos/inmunologíaRESUMEN
Macroporous three-dimensional (3D) framework structured melamine foam-based Enzyme-Linked Immunosorbent Assay (f-ELISA) biosensors were developed for rapid, reliable, sensitive, and on-site detection of trace amount of biomolecules and chemicals. Various ligands can be chemically immobilized onto the melamine foam, which brings in the possibility of working with antibodies, nanobodies, and peptides, respectively, as affinity probes for f-ELISA biosensors with improved stability. Different chemical reagents can be used to modify the foam materials, resulting in varied reactivities with antibodies, nanobodies, and peptides. As a result, the f-ELISA sensors produced from these modified foams exhibit varying levels of sensitivity and performance. This study demonstrated that the chemical reagents used for immobilizing antibodies, nanobodies, and peptides could affect the sensitivities of the f-ELISA sensors, and their storage stabilities under different temperatures varied depending on the sensing probes used, with f-ELISA sensors employing nanobodies as probes exhibiting the highest stability. This study not only showcases the versatility of the f-ELISA system but also opens new avenues for developing cost-effective, portable, and user-friendly diagnostic tools with optimized sensitivity and stability.
Asunto(s)
Técnicas Biosensibles , Ensayo de Inmunoadsorción Enzimática , Anticuerpos de Dominio Único , Triazinas , Triazinas/análisis , Triazinas/química , Ensayo de Inmunoadsorción Enzimática/métodos , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/química , Técnicas Biosensibles/métodos , Péptidos/química , Anticuerpos/inmunología , Anticuerpos/química , Anticuerpos Inmovilizados/inmunología , Anticuerpos Inmovilizados/química , Límite de DetecciónRESUMEN
The ability to accurately predict antibody-antigen complex structures from their sequences could greatly advance our understanding of the immune system and would aid in the development of novel antibody therapeutics. There have been considerable recent advancements in predicting protein-protein interactions (PPIs) fueled by progress in machine learning (ML). To understand the current state of the field, we compare six representative methods for predicting antibody-antigen complexes from sequence, including two deep learning approaches trained to predict PPIs in general (AlphaFold-Multimer and RoseTTAFold), two composite methods that initially predict antibody and antigen structures separately and dock them (using antibody-mode ClusPro), local refinement in Rosetta (SnugDock) of globally docked poses from ClusPro, and a pipeline combining homology modeling with rigid-body docking informed by ML-based epitope and paratope prediction (AbAdapt). We find that AlphaFold-Multimer outperformed other methods, although the absolute performance leaves considerable room for improvement. AlphaFold-Multimer models of lower quality display significant structural biases at the level of tertiary motifs (TERMs) toward having fewer structural matches in non-antibody-containing structures from the Protein Data Bank (PDB). Specifically, better models exhibit more common PDB-like TERMs at the antibody-antigen interface than worse ones. Importantly, the clear relationship between performance and the commonness of interfacial TERMs suggests that the scarcity of interfacial geometry data in the structural database may currently limit the application of ML to the prediction of antibody-antigen interactions.
Asunto(s)
Complejo Antígeno-Anticuerpo , Complejo Antígeno-Anticuerpo/química , Conformación Proteica , Anticuerpos/química , Anticuerpos/inmunología , Simulación del Acoplamiento Molecular , Modelos Moleculares , HumanosRESUMEN
Binding of anti-PEG antibodies to poly(ethylene glycol) (PEG) on the surface of PEGylated liposomal doxorubicin (PLD) in vitro and in rats can activate complement and cause the rapid release of doxorubicin from the liposome interior. Here, we find that irinotecan liposomes (IL) and L-PLD, which have 16-fold lower levels of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-PEG2000 in their liposome membrane as compared to PLD, generate less complement activation but remain sensitive to destabilization and drug release by anti-PEG antibodies. Complement activation and liposome destabilization correlated with the theoretically estimated number of antibody molecules bound per liposome. Drug release from liposomes proceeded through the alternative complement pathway but was accelerated by the classical complement pathway. In contrast to PLD destabilization by anti-PEG immunoglobulin G (IgG), which proceeded by the insertion of membrane attack complexes in the lipid bilayer of otherwise intact PLD, anti-PEG IgG promoted the fusion of L-PLD, and IL to form unilamellar and oligo-vesicular liposomes. Anti-PEG immunoglobulin M (IgM) induced drug release from all liposomes (PLD, L-PLD, and IL) via the formation of unilamellar and oligo-vesicular liposomes. Anti-PEG IgG destabilized both PLD and L-PLD in rats, indicating that the reduction of PEG levels on liposomes is not an effective approach to prevent liposome destabilization by anti-PEG antibodies.
Asunto(s)
Doxorrubicina , Liposomas , Polietilenglicoles , Polietilenglicoles/química , Liposomas/química , Doxorrubicina/química , Doxorrubicina/farmacología , Doxorrubicina/análogos & derivados , Animales , Ratas , Anticuerpos/química , Anticuerpos/inmunología , Activación de Complemento/efectos de los fármacos , Fosfatidiletanolaminas/química , Liberación de FármacosRESUMEN
Peptides are a very critical class of pharmaceutical compounds that can control several signaling pathways and thereby affect many physiological and biochemical processes. Previous research suggests that both peptides and antibodies may serve as potent tools for research, diagnostics, vaccination, and therapeutics across diverse domains. The distinct attributes of peptides, like their profound tissue penetration, efficient cellular internalization, reduced immunogenicity, and adaptability to chemical modification, underscore their significance in biomedical applications. However, they also possess drawbacks such as lower affinity, poor absorption, low stability to proteolytic digestion, and rapid clearance. The advent of peptibodies is a significant advance that improves the limitations of both peptides and antibodies. Peptibodies, or Peptide-Fc fusions, represent a promising therapeutic modality comprising biologically active peptides fused to an Fc domain. The stability and efficacy of the peptide are enhanced by this fusion strategy, which overcomes some of the inherent limitations. Many peptibodies have been developed to treat conditions like cancer, diabetes, and lupus. Romiplostim and Dulaglutide are the only ones approved by the EMA and FDA, respectively. Given the growing significance of peptibodies in the pharmaceutical landscape, this investigation aims to explain key aspects encompassing the intrinsic properties of peptides, the intricacies of peptibody production, and their potential therapeutic applications.
Asunto(s)
Péptidos , Humanos , Péptidos/química , Péptidos/inmunología , Péptidos/uso terapéutico , Animales , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/uso terapéutico , Fragmentos Fc de Inmunoglobulinas/química , Anticuerpos/química , Anticuerpos/inmunologíaRESUMEN
Adipose tissue, or fat tissue, can now be classified as an endocrine organ as it responds to stimuli by secreting a range of hormones, termed adipokines, which regulate the functions of various other tissues and organs. Because novel adipokines continue to be discovered and characterized by researchers, there is an enduring need for the development of new analytical assays that target these hormones. Discovered recently, asprosin is an adipokine hormone secreted by white adipose tissue (WAT) during fasting which has been implicated for its important effects on the liver, skeletal muscle, hypothalamus, pancreas, and possibly other tissues. While standard immunoassays have been developed, the continued surge in research on asprosin's function would greatly benefit from an assay with homogeneous, mix-and-read workflow, and the nanomolar clinical range makes this goal more feasible. In this work, we developed such an assay for asprosin using our thermofluorimetric analysis (TFA) methods with antibody-oligonucleotide conjugate probes. The assay, achievable in less than one hour, was successfully validated by quantifying native levels of asprosin in human serum collected from fasting, nonfasting, type II diabetic, and obese donors.
Asunto(s)
Fibrilina-1 , Humanos , Fibrilina-1/química , Inmunoensayo/métodos , Anticuerpos/inmunología , Anticuerpos/química , Oligonucleótidos/química , AdipoquinasRESUMEN
A nanohybrid-modified glassy carbon electrode based on conducting polypyrrole doped with carbon quantum dots (QDs) was developed and used for the electrochemical detection of anti-tissue transglutaminase (anti-tTG) antibodies. To improve the polypyrrole conductivity, carrier mobility, and carrier concentration, four types of carbon nanoparticles were tested. Furthermore, a polypyrrole-modified electrode doped with QDs was functionalized with a PAMAM dendrimer and transglutaminase 2 protein by cross-linking with N-hydroxysuccinimide (NHS)/N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC). The steps of electrode surface modification were surveyed via electrochemical measurements (differential pulse voltammetry (DPV), impedance spectroscopy, and X-ray photoelectron spectroscopy (XPS)). The surface characteristics were observed by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, and contact angle measurements. The obtained modified electrode exhibited good stability and repeatability. DPV between - 0.1 and 0.6 V (vs. Ag/AgCl 3 M KCl reference electrode) was used to evaluate the electrochemical alterations that occur after the antibody interacts with the antigen (transglutaminase 2 protein), for which the limit of detection was 0.79 U/mL. Without the use of a secondary label, (anti-tTG) antibodies may be detected at low concentrations because of these modified electrode features.
Asunto(s)
Dendrímeros , Proteína Glutamina Gamma Glutamiltransferasa 2 , Pirroles , Puntos Cuánticos , Transglutaminasas , Humanos , Anticuerpos/inmunología , Anticuerpos/química , Técnicas Biosensibles/métodos , Carbono/química , Dendrímeros/química , Técnicas Electroquímicas/métodos , Electrodos , Proteínas de Unión al GTP/inmunología , Polímeros/química , Pirroles/química , Puntos Cuánticos/química , Transglutaminasas/inmunología , Transglutaminasas/químicaRESUMEN
Synergistic factors can enhance the toxicity of Bt toxins and delay the development of Bt resistance. Previous research has demonstrated that a Helicoverpa armigera cadherin fragment (HaCad-TBR) increased the toxicity of Cry1Ac in Plutella xylostella larvae but did not have a synergistic effect on Cry1B, Cry1C, and Cry1F toxins. In this study, a fusion protein (HaCad-TBR-2D3 VL) derived from HaCad-TBR and a Bt Cry1-specific antibody peptide was expressed in Escherichia coli. The HaCad-TBR-2D3 VL enhanced Cry1Ac toxicity more efficiently in insects and Sf9 cells than HaCad-TBR and also significantly increased the toxicity of Cry1B, Cry1C, and Cry1F toxins in insects. Further investigation indicated that the improved stability in insect midguts and higher binding capacity with Bt toxins contributed to the enhanced synergism of HaCad-TBR-2D3 VL over HaCad-TBR. This study suggested that Bt antibody fragments can potentially broaden the synergistic range of Bt receptor fragments, providing a theoretical foundation for developing broad-spectrum synergists for other biopesticides.
Asunto(s)
Toxinas de Bacillus thuringiensis , Proteínas Bacterianas , Cadherinas , Endotoxinas , Proteínas Hemolisinas , Proteínas de Insectos , Larva , Mariposas Nocturnas , Proteínas Recombinantes de Fusión , Animales , Cadherinas/genética , Cadherinas/metabolismo , Cadherinas/inmunología , Cadherinas/química , Proteínas Hemolisinas/química , Proteínas Hemolisinas/farmacología , Proteínas Hemolisinas/inmunología , Proteínas Hemolisinas/genética , Endotoxinas/inmunología , Endotoxinas/química , Endotoxinas/farmacología , Endotoxinas/metabolismo , Endotoxinas/genética , Toxinas de Bacillus thuringiensis/química , Toxinas de Bacillus thuringiensis/farmacología , Mariposas Nocturnas/efectos de los fármacos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/farmacología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Insectos/inmunología , Proteínas de Insectos/química , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Larva/crecimiento & desarrollo , Péptidos/química , Péptidos/inmunología , Péptidos/farmacología , Anticuerpos/inmunología , Anticuerpos/química , Bacillus thuringiensis/química , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Insecticidas/química , Insecticidas/farmacología , Control Biológico de VectoresRESUMEN
The aggregation of α-Synuclein (αSyn) is strongly linked to neuronal death in Parkinson's disease and other synucleinopathies. The spreading of aggregated αSyn between neurons is at least partly dependent on electrostatic interactions between positively charged stretches on αSyn fibrils and the negatively charged heparan sulphate proteoglycans on the cell surface. To date there is still no therapeutic option available that could halt the progression of Parkinson's disease and one of the major limitations is likely the relatively low proportion of αSyn aggregates accessible to drugs in the extracellular space. Here, we investigated whether a negatively charged peptide tail fused to the αSyn aggregate-specific antibodies SynO2 and 9E4 could enhance the antibodies' avidity to αSyn aggregates in order to improve their potential therapeutic effect through inhibiting cell-to-cell spreading and enhancing the clearance of extracellular aggregates. We performed ELISAs to test the avidity to αSyn aggregates of both monovalent and bivalent antibody formats with and without the peptide tail. Our results show that the addition of the negatively charged peptide tail decreased the binding strength of both antibodies to αSyn aggregates at physiological salt conditions, which can likely be explained by intermolecular repulsions between the tail and the negatively charged C-terminus of αSyn. Additionally, the tail might interact with the paratopes of the SynO2 antibody abolishing its binding to αSyn aggregates. Conclusively, our peptide tail did not fulfil the required characteristics to improve the antibodies' binding to αSyn aggregates. Fine-tuning the design of the peptide tail to avoid its interaction with the antibodies' CDR and to better mimic relevant characteristics of heparan sulphates for αSyn aggregate binding may help overcome the limitations observed in this study.
Asunto(s)
alfa-Sinucleína , alfa-Sinucleína/inmunología , alfa-Sinucleína/metabolismo , alfa-Sinucleína/química , Humanos , Afinidad de Anticuerpos , Agregado de Proteínas , Unión Proteica , Péptidos/química , Péptidos/inmunología , Anticuerpos/inmunología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/inmunologíaRESUMEN
Natural antibodies are used to compare immune systems across taxa, to study wildlife disease ecology, and as selection markers in livestock breeding. These immunoglobulins are present prior to immune stimulation. They are described as having low antigen specificity or polyreactive binding and are measured by binding to self-antigens or novel exogenous proteins. Most studies use only one or two antigens to measure natural antibodies and ignore potential effects of antigen specificity in analyses. It remains unclear how different antigen-specific natural antibodies are related or how diversity among natural antibodies may affect analyses of these immunoglobulins. Using genetically distinct lines of chickens as a model system, we tested the hypotheses that (1) antigen-specific natural antibodies are independent of each other and (2) antigen specificity affects the comparison of natural antibodies among animals. We used blood cell agglutination and enzyme-linked immunosorbent assays to measure levels of natural antibodies binding to four antigens: (i) rabbit erythrocytes, (ii) keyhole limpet hemocyanin, (iii) phytohemagglutinin, or (iv) ovalbumin. We observed that levels of antigen specific natural antibodies were not correlated. There were significant differences in levels of natural antibodies among lines of chickens, indicating genetic variation for natural antibody production. However, line distinctions were not consistent among antigen specific natural antibodies. These data show that natural antibodies are a pool of relatively distinct immunoglobulins, and that antigen specificity may affect interpretation of natural antibody function and comparative immunology.
Asunto(s)
Pollos , Animales , Pollos/inmunología , Conejos , Antígenos/inmunología , Eritrocitos/inmunología , Especificidad de Anticuerpos/inmunología , Ovalbúmina/inmunología , Anticuerpos/inmunología , Hemocianinas/inmunología , Fitohemaglutininas/inmunología , Ensayo de Inmunoadsorción EnzimáticaRESUMEN
Antibodies are widely used as therapeutic agents to tackle various diseases. In the present study, to enhance their clinical values, we rationally designed pH-responsivity by exploiting the idiosyncratic protonation/deprotonation profiles of non-natural amino acids. 3-Nitro-L-tyrosine, 3-cyano-L-tyrosine, and 3, 5-halogenated-L-tyrosine, each with near neutral pKa, were thus incorporated into Fab fragments in place of tyrosines and other residues in the variable regions. Cell-based assays showed that these modifications achieved up to 140-fold tighter binding to antigens and several-fold tighter cytotoxicity to antigen-expressing cell at pH 6.0 than pH 7.4. The pH-dependent binding effect was retained in full-length antibodies. In silico structural analyses revealed electrostatic repulsion at neutral pH between antigens and antibodies or inside the antibody as the underlying mechanisms of the acid preference, and this finding increases the designability of pH-dependent antigen binding. The development of antibodies responsive to the microenvironments of diseased tissues will allow more disease-related antigens to be targeted in treatments, because of the reduced cross-reactivity toward healthy tissues.
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Aminoácidos , Fragmentos Fab de Inmunoglobulinas , Concentración de Iones de Hidrógeno , Aminoácidos/química , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Anticuerpos/química , Anticuerpos/inmunología , Animales , Tirosina/química , Diseño de Fármacos , Electricidad EstáticaRESUMEN
BACKGROUND: Cell-surface proteins, which are closely associated with various physiological and pathological processes, have drawn much attention in drug discovery and disease diagnosis. Thus, wash-free imaging of the target cell-surface protein under its native environment is critical and helpful for early detection and prognostic evaluation of diseases. RESULTS: To minimize the interference from autofluorescence and fit the penetration depth towards tissue samples, we developed a fluorogenic antibody-based probe, Ab-Cy5.5, which will liberate > 5-fold turn-on near-infrared (NIR) emission in the presence of its target antigen within 10 min. SIGNIFICANCE: By taking advantage of the fluorescence-quenched dimeric H-aggregation of Cy5.5, Ab-Cy5.5 with Cy5.5 attached at the N-terminus showed negligible background signal, allowing direct imaging of the target cell-surface protein in both living cells and tissue samples without washing.