RESUMEN
Immunoglobulin E (IgE) plays pivotal roles in allergic diseases through interaction with a high-affinity receptor (FcεRI). We established that Fab fragments of anti-IgE antibodies (HMK-12 Fab) rapidly dissociate preformed IgE-FcεRI complexes in a temperature-dependent manner and inhibit IgE-mediated anaphylactic reactions, even after allergen challenge. X-ray crystallographic studies revealed that HMK-12 Fab interacts with each of two equivalent epitopes on the Cε2 homodimer domain involved in IgE F(ab')2. Consequently, HMK-12 Fab-mediated targeting of Cε2 reduced the binding affinity of Fc domains and resulted in rapid removal of IgE from the receptor complex. This unexpected finding of allosteric inhibition of IgE-FcεRI interactions by simultaneous targeting of two epitope sites on the Cε2 homodimer domain of IgE F(ab')2 may have implications for the development of novel therapies for allergic disease.
Asunto(s)
Epítopos , Inmunoglobulina E , Fragmentos Fab de Inmunoglobulinas , Receptores de IgE , Inmunoglobulina E/inmunología , Inmunoglobulina E/metabolismo , Receptores de IgE/inmunología , Receptores de IgE/metabolismo , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/metabolismo , Fragmentos Fab de Inmunoglobulinas/química , Epítopos/inmunología , Animales , Regulación Alostérica , Cristalografía por Rayos X , Ratones , Unión Proteica , Ratones Endogámicos BALB C , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Antiidiotipos/química , Humanos , Anafilaxia/inmunologíaRESUMEN
The quartz tuning fork (QTF) is a promising instrument for biosensor applications due to its advanced properties such as high sensitivity to physical quantities, cost-effectiveness, frequency stability, and high-quality factor. Nevertheless, the fork's small size and difficulty in modifying the prongs' surfaces limit its wide use in experimental research. Our study presents the development of a QTF immunosensor composed of three active layers: biocompatible natural melanin nanoparticles (MNPs), glutaraldehyde (GLU), and anti-IgG layers, for the detection of immunoglobulin G (IgG). Frequency shifts of QTFs after MNP functionalization, GLU activation, and anti-IgG immobilization were measured with an Asensis QTF F-master device. Using QTF immunosensors that had been modified under optimum conditions, the performance of QTF immunosensors for IgG detection was evaluated. Accordingly, a finite element method (FEM)-based model was produced using the COMSOL Multiphysics software program (COMSOL License No. 2102058) to simulate the effect of deposited layers on the QTF resonance frequency. The experimental results, which demonstrated shifts in frequency with each layer during QTF surface functionalization, corroborated the simulation model predictions. A modelling error of 0.05% was observed for the MNP-functionalized QTF biosensor compared to experimental findings. This study validated a simulation model that demonstrates the advantages of a simulation-based approach to optimize QTF biosensors, thereby reducing the need for extensive laboratory work.
Asunto(s)
Técnicas Biosensibles , Inmunoglobulina G , Melaninas , Nanopartículas , Cuarzo , Inmunoglobulina G/química , Inmunoglobulina G/inmunología , Técnicas Biosensibles/métodos , Técnicas Biosensibles/instrumentación , Nanopartículas/química , Melaninas/química , Cuarzo/química , Inmunoensayo/métodos , Inmunoensayo/instrumentación , Simulación por Computador , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Antiidiotipos/química , HumanosRESUMEN
We developed an aptamer-based fluorescence resonance energy transfer (FRET) assay capable of recognizing therapeutic monoclonal antibody bevacizumab and rapidly quantifying its concentration with just one mixing step. In this assay, two fluorescent dyes (fluorescein and tetramethylrhodamine) labeled aptamers bind to two Fab regions on bevacizumab, and FRET fluorescence is observed when both dyes come into close proximity. We optimized this assay in three different formats, catering to a wide range of analytical needs. When applied to hybridoma culture samples in practical settings, this assay exhibited a signal response that was concentration-dependent, falling within the range of 50-2000 µg/mL. The coefficients of determination (r2) ranged from 0.998 to 0.999, and bias and precision results were within ±24.0 % and 20.3 %, respectively. Additionally, during thermal and UV stress testing, this assay demonstrated the ability to detect denatured samples in a manner comparable to conventional Size Exclusion Chromatography. Notably, it offers the added advantage of detecting decreases in binding activity without changes in molecular weight. In contrast to many existing process analytical technology tools, this assay not only identifies bevacizumab but also directly measures the quality attributes related to mAb efficacy, such as the binding activity. As a result, this assay holds great potential as a valuable platform for providing highly reliable quality attribute information in real-time. We consider this will make a significant contribution to the worldwide distribution of high-quality therapeutic mAbs in various aspects of antibody manufacturing, including production monitoring, quality control, commercial lot release, and stability testing.
Asunto(s)
Aptámeros de Nucleótidos , Bevacizumab , Transferencia Resonante de Energía de Fluorescencia , Bevacizumab/análisis , Bevacizumab/química , Transferencia Resonante de Energía de Fluorescencia/métodos , Aptámeros de Nucleótidos/química , Anticuerpos Antiidiotipos/química , Anticuerpos Antiidiotipos/análisis , Humanos , Colorantes Fluorescentes/químicaRESUMEN
BACKGROUND: The screening of umbilical cord blood samples by the Direct Antiglobulin Test (DAT) is the reference tool for the identification of maternal erythrocyte alloantibodies present in erythrocytes; however, its diagnostic usefulness is controversial. OBJECTIVE: To evaluate the diagnostic validity, safety, and efficiency of the eluate testing (detection of antibody in erythrocyte eluates by the Indirect Antiglobulin Test/IAT) in cord blood samples for detection of maternal erythrocyte alloantibodies in comparison with the DAT. MATERIALS AND METHODS: Evaluation study of diagnostic tests. DAT and eluate testing were performed in 306 cord blood samples from neonates born to mothers admitted at Clínica Somer in Rionegro, Colombia; then, antibodies present in the eluates were identified with erythrocyte panels. Percentage of positive results by DAT and IAT were compared with the Pearson's chi-square test and the agreement between both assays with the Cohen's kappa coefficient. The diagnostic sensitivity, specificity, safety, and efficiency of the eluate testing were calculated, taking into account the use of DAT as an imperfect reference test. RESULTS: The DAT detected alloantibodies in 6.21% of samples and the eluate testing in 14.1 %; the strength of agreement between both tests was moderate (k = 0.56) due to 25 discrepancies. The eluate testing showed sensitivity and specificity of 98.83 % and 92.31 % respectively, and a negative predictive value of 99.9 %. The diagnostic efficiency was sufficient for detection of maternal erythrocyte alloantibodies. The antibodies identified in the erythrocyte eluates were anti-A or anti-B (79.5 %), anti-D (136%), anti-C (2,3%), and anti-Fya (2,3%). CONCLUSION: The eluate testing in cord blood samples is a valid, safe, and efficient test for the diagnosis of maternal erythrocyte alloantibodies.
Asunto(s)
Anticuerpos Antiidiotipos/química , Eritrocitos/inmunología , Isoanticuerpos/inmunología , Anemia Hemolítica Autoinmune/prevención & control , Transfusión Sanguínea , Prueba de Coombs/métodos , Pruebas Diagnósticas de Rutina , Sangre Fetal/citología , Humanos , Recién Nacido , Isoanticuerpos/química , Valor Predictivo de las Pruebas , Valores de Referencia , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
The anti-idiotypic antibody is widely used in the field of immunology to simulate structural features or even induce the biological activity of antigens. In this study, we obtained seven anti-idiotypic single-chain variable fragments (scFv) antibodies of Cry2Aa toxin from a phage-displayed mutant library constructed using error-prone PCR technique. A mutant designated 2-12B showed the best binding ability amongst all anti-idiotypic scFv isolates to Plutella xylostella brush border membrane vesicles (BBMVs). 2-12B and Cry2Aa toxin shared a potential receptor of polycalin in P. xylostella BBMVs. Homology modeling and molecular docking demonstrated that 2-12B and Cry2Aa toxin have seven common binding amino acid residues in polycalin. Insect bioassay results suggested that 2-12 had insecticidal efficacy against P. xylostella larvae. These results indicated that the Cry2Aa anti-idiotypic scFv antibody 2-12B partially mimicked the structure and function of Cry2Aa toxin. The anti-idiotypic scFv antibody provides the basic material for the future study of surrogate molecules or new insecticidal materials.
Asunto(s)
Anticuerpos Antiidiotipos/química , Anticuerpos Monoclonales/química , Toxinas de Bacillus thuringiensis/química , Endotoxinas/química , Proteínas Hemolisinas/química , Región Variable de Inmunoglobulina/química , Anticuerpos de Cadena Única/química , Animales , Anticuerpos Antiidiotipos/genética , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Antiidiotipos/metabolismo , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Toxinas de Bacillus thuringiensis/inmunología , Toxinas de Bacillus thuringiensis/metabolismo , Membrana Celular/metabolismo , Endotoxinas/inmunología , Endotoxinas/metabolismo , Proteínas Hemolisinas/inmunología , Proteínas Hemolisinas/metabolismo , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/metabolismo , Simulación del Acoplamiento Molecular , Mariposas Nocturnas , Mutación , Biblioteca de Péptidos , Conformación Proteica , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/metabolismoRESUMEN
The critical role of IgE in allergic diseases is well-documented and clinically proven. Omalizumab, a humanized anti-IgE antibody, was the first approved antibody for the treatment of allergic diseases. Nevertheless, omalizumab still has some limitations, such as product instability and dosage restriction in clinical application. In this study, we attempted to develop an omalizumab biobetter antibody with the potential to overcome its limitations. We removed two aspartic acid isomerization hotspots in CDRs of omalizumab to improve antibody candidate's stability. Meanwhile, several murine amino acids in the framework region of omalizumab were replaced with human source to reduce the potential immunogenicity. Yeast display technology was then applied to screen antibody candidates with high binding affinity to IgE. Moreover, YTE mutation in Fc fragment was introduced into the candidates for extending their serum half-life. A lead candidate, AB1904Am15, was screened out, which showed desired biophysical properties and improved stability, high binding affinity and elevated potency in vitro, prolonged half-life in human FcRn transgenic mouse, and enhanced in vivo efficacy in cynomolgus monkey asthma model. Overall, our study developed a biobetter antibody of omalizumab, AB1904Am15, which has the potential to show improved clinical benefit in the treatment of allergic diseases.
Asunto(s)
Antialérgicos/farmacología , Antialérgicos/uso terapéutico , Anticuerpos Antiidiotipos/farmacología , Anticuerpos Antiidiotipos/uso terapéutico , Hipersensibilidad/tratamiento farmacológico , Omalizumab/farmacología , Omalizumab/uso terapéutico , Antialérgicos/química , Anticuerpos Antiidiotipos/química , Afinidad de Anticuerpos/inmunología , Fenómenos Biofísicos , Cromatografía Liquida , Monitoreo de Drogas , Estabilidad de Medicamentos , Citometría de Flujo , Humanos , Hipersensibilidad/diagnóstico , Hipersensibilidad/inmunología , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Omalizumab/química , Unión Proteica , Espectrometría de Masas en Tándem , Resultado del TratamientoRESUMEN
In this work, we developed a new antibody-targeted and redox-responsive drug delivery system "MSNs-CAIX" by binding the anti-carbonic anhydrase IX antibody (A-CAIX Ab) on the surface of mesoporous silica nanoparticles (MSNs) via disulfide linkages. The design of the composite particles "MSNs-CAIX" involved the synthesis and surface functionalization with thiol groups, 2,2'-dipyridyl disulfide and CAIX antibody. In vitro, CAIX capping the doxorubicin hydrochloric (DOX)-loaded nanoparticles (DOX@MSNs-CAIX) exhibited effectively redox-responsive release in the presence of glutathione (GSH) owing to the cleavage of the disulfide bond. Compared with CAIX negative Mef cells (mouse embryo fibroblast), remarkably more DOX@MSNs-CAIX was internalized into CAIX positive 4T1 cells (mouse breast cancer cells) by receptor-mediation. Tumor targeting in vivo studies clearly demonstrated DOX@MSNs-CAIX accumulated in tumors and induced more tumor cells apoptosis in 4T1 tumor-bearing mice. With great potential, this drug delivery system is a promising candidate for targeted and redox-responsive cancer therapy.
Asunto(s)
Anhidrasa Carbónica IX/antagonistas & inhibidores , Sistemas de Liberación de Medicamentos , Nanopartículas del Metal/química , Neoplasias/tratamiento farmacológico , Animales , Anticuerpos Antiidiotipos/química , Anticuerpos Antiidiotipos/farmacología , Apoptosis/efectos de los fármacos , Anhidrasa Carbónica IX/inmunología , Doxorrubicina/química , Doxorrubicina/farmacología , Xenoinjertos , Humanos , Inmunoconjugados/farmacología , Ratones , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/patología , Oxidación-Reducción/efectos de los fármacos , Porosidad , Dióxido de Silicio/químicaRESUMEN
Immunoglobulin E (IgE) plays a central role in the allergic response, in which cross-linking of allergen by FcεRI-bound IgE triggers mast cell and basophil degranulation and the release of inflammatory mediators. The high-affinity interaction between IgE and FcεRI is a long-standing target for therapeutic intervention in allergic disease. Omalizumab is a clinically approved anti-IgE monoclonal antibody that binds to free IgE, also with high affinity, preventing its interaction with FcεRI. All attempts to crystallize the pre-formed complex between the omalizumab Fab and the Fc region of IgE (IgE-Fc), to understand the structural basis for its mechanism of action, surprisingly failed. Instead, the Fab alone selectively crystallized in different crystal forms, but their structures revealed intermolecular Fab/Fab interactions that were clearly strong enough to disrupt the Fab/IgE-Fc complexes. Some of these interactions were common to other Fab crystal structures. Mutations were therefore designed to disrupt two recurring packing interactions observed in the omalizumab Fab crystal structures without interfering with the ability of the omalizumab Fab to recognize IgE-Fc; this led to the successful crystallization and subsequent structure determination of the Fab/IgE-Fc complex. The mutagenesis strategy adopted to achieve this result is applicable to other intractable Fab/antigen complexes or systems in which Fabs are used as crystallization chaperones.
Asunto(s)
Anticuerpos Antiidiotipos/metabolismo , Cristalización/métodos , Inmunoglobulina E/metabolismo , Fragmentos Fab de Inmunoglobulinas/metabolismo , Fragmentos Fc de Inmunoglobulinas/metabolismo , Omalizumab/metabolismo , Anticuerpos Antiidiotipos/química , Cristalografía por Rayos X/métodos , Humanos , Inmunoglobulina E/química , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/química , Omalizumab/farmacología , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Targeting of immunoglobulin E (IgE) represents an interesting approach for the treatment of allergic disorders. A high-affinity monoclonal anti-IgE antibody, ligelizumab, has recently been developed to overcome some of the limitations associated with the clinical use of the therapeutic anti-IgE antibody, omalizumab. Here, we determine the molecular binding profile and functional modes-of-action of ligelizumab. We solve the crystal structure of ligelizumab bound to IgE, and report epitope differences between ligelizumab and omalizumab that contribute to their qualitatively distinct IgE-receptor inhibition profiles. While ligelizumab shows superior inhibition of IgE binding to FcεRI, basophil activation, IgE production by B cells and passive systemic anaphylaxis in an in vivo mouse model, ligelizumab is less potent in inhibiting IgE:CD23 interactions than omalizumab. Our data thus provide a structural and mechanistic foundation for understanding the efficient suppression of FcεRI-dependent allergic reactions by ligelizumab in vitro as well as in vivo.
Asunto(s)
Antialérgicos/administración & dosificación , Anticuerpos Antiidiotipos/administración & dosificación , Anticuerpos Monoclonales Humanizados/administración & dosificación , Hipersensibilidad/tratamiento farmacológico , Omalizumab/administración & dosificación , Animales , Antialérgicos/química , Anticuerpos Antiidiotipos/química , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Basófilos/efectos de los fármacos , Basófilos/inmunología , Humanos , Hipersensibilidad/inmunología , Inmunoglobulina E/química , Inmunoglobulina E/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Omalizumab/química , Receptores de IgE/inmunologíaRESUMEN
The antibody repertoire of cartilaginous fish comprises an additional heavy-chain-only antibody isotype that is referred to as IgNAR (immunoglobulin novel antigen receptor). Its antigen-binding site consists of one single domain (vNAR) that is reportedly able to engage a respective antigen with affinities similar to those achieved by conventional antibodies. While vNAR domains offer a reduced size, which is often favorable for applications in a therapeutic as well as a biotechnological setup, they also exhibit a high physicochemical stability. Together with their ability to target difficult-to-address antigens such as virus particles or toxins, these shark-derived antibody domains seem to be predestined as tools for biotechnological and diagnostic applications. In the following chapter, we will describe the isolation of anti-idiotypic vNAR domains targeting monoclonal antibody paratopes from semi-synthetic, yeast-displayed libraries. Anti-idiotypic vNAR variants could be employed for the characterization of antibody-based therapeutics (such as antibody-drug conjugates) or as positive controls in immunogenicity assays. Peculiarly, when using semi-synthetic vNAR libraries, we found that it is not necessary to deplete the libraries using unrelated antibody targets, which enables a fast and facile screening procedure that exclusively delivers anti-idiotypic binders.
Asunto(s)
Anticuerpos Antiidiotipos , Proteínas de Peces , Biblioteca de Péptidos , Tiburones , Anticuerpos de Cadena Única , Animales , Anticuerpos Antiidiotipos/química , Anticuerpos Antiidiotipos/genética , Anticuerpos Antiidiotipos/inmunología , Proteínas de Peces/química , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Dominios Proteicos , Tiburones/genética , Tiburones/inmunología , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunologíaRESUMEN
Surface plasmon resonance (SPR) has become a leading technique for in situ bioaffinity assay of diverse targets without need of fluorescent or enzymatic labeling. Nanomaterials-enhanced SPR sensors have developed rapidly and widened the application scope of SPR sensing technology. In this report we describe an ultrasensitive SPR biosensor for detecting carcinoembryonic antigen (CEA). Our SPR biosensor utilizes a Ti3C2-MXene-based sensing platform and multi-walled carbon nanotube (MWCNTs)-polydopamine (PDA)-Ag nanoparticle (AgNPs) signal enhancer. Ti3C2-MXene, a new class of two-dimensional (2D) transition metal carbides, offers a large hydrophilic-biocompatible surface ideal for SPR biosensing. Ti3C2-MXene/AuNPs composites after synthesis are then decorated with staphylococcal protein A (SPA) to orient and immobilize monoclonal anti-CEA antibody (Ab1) through its Fc region. By introducing MWCNTs-PDA-AgNPs-polyclonal anti-CEA antibody (MWPAg-Ab2) conjugate combined with a sandwich format, the present method provides a dynamic range for CEA determination of 2×10-16 to 2×10-8â¯M and a detection limit of 0.07â¯fM. This biosensing approach demonstrates good reproducibility and high specificity for CEA in real serum samples providing a promising method to evaluate CEA in human serum for early diagnosis and monitoring of cancer.
Asunto(s)
Técnicas Biosensibles , Antígeno Carcinoembrionario/aislamiento & purificación , Nanopartículas del Metal/química , Resonancia por Plasmón de Superficie , Anticuerpos Antiidiotipos/química , Anticuerpos Antiidiotipos/inmunología , Antígeno Carcinoembrionario/química , Oro/química , Humanos , Nanoestructuras/química , Nanotubos de Carbono/química , Plata/química , Elementos de Transición/químicaRESUMEN
Zika virus (ZIKV) has been declared a public health emergency of international concern. ZIKV has been associated with some neurological disorders, and their long-term effects are not completely understood. The majority of the methods for ZIKV diagnosis are based on the detection of IgM antibodies, which are the first signs of immunological response. However, the detection of IgG antibodies can be an important approach for ZIKV past infection diagnosis, especially for pregnant women, helping the comprehension/treatment of this disease. There has been a growing interest in applying nanoparticles for efficient ZIKV or antibodies detection. Quantum dots (QD) are unique fluorescent semiconductor nanoparticles, highly versatile for biological applications. In the present study, we explored the special QD optical properties to develop an immunofluorescence assay for anti-ZIKV IgG antibodies detection. Anti-IgG antibodies were successfully conjugated with QDs and applied in a fluorescence sensing nanoplatform. After optimization using IgG antibodies, the conjugates were employed to detect anti-ZIKV IgG antibodies in polystyrene microplates sensitized with ZIKV envelope E protein. The nanoplatform was able to detect anti-ZIKV IgG antibodies in a concentration at least 100-fold lower than the amount expected for protein E immune response. Moreover, conjugates were able to detect the antibodies for at least 4â¯months. Thus, our results showed that this QDs-based fluoroimmunoplatform can be considered practical, simple and promising to detect Zika past infections and/or monitoring immune response in vaccine trials.
Asunto(s)
Anticuerpos Antiidiotipos/análisis , Anticuerpos Antiidiotipos/química , Fluoroinmunoensayo/métodos , Puntos Cuánticos/química , Virus Zika/inmunología , Anticuerpos Antiidiotipos/inmunología , Compuestos de Cadmio/química , Telurio/química , Virus Zika/aislamiento & purificaciónRESUMEN
Dysregulated vascular inflammation is the underlying cause of acute lung inflammation/injury (ALI). Bacterial infections and trauma cause ALI that may rapidly lead to acute respiratory distress syndrome (ARDS). There are no pharmacological therapies available to patients with ALI/ARDS, partially as drugs cannot specifically target the lungs. Herein, we developed a stimuli-responsive nanoparticle (NP) to target inflammatory lungs for ALI therapies. The NP is composed of a sharp acid-sensitive segment poly(ß-amino esters) as a core for drug loading and controlled release and a polyethylene glycol-biotin on the particle surface available for bioconjugation, enabling lung targeting and extended circulation. The studies on dissipative particle dynamics simulation and characteristics of NPs suggest that anti-ICAM-1 antibodies can be coated to the particle surface and this coating is required to enhance lung targeting of NPs. A model drug of anti-inflammatory agent TPCA-1 is encapsulated in NPs with a high drug-loading content at 24% (w/w). In the mouse ALI model, our TPCA-1-loaded NPs coated with anti-ICAM-1 can target inflamed lungs after intravenous injection, followed by drug release triggered by the acid environment, thus mitigating lung inflammation and injury. Our studies reveal the rational design of nanotherapeutics for improved therapy of ALI, which may be applied to treating a wide range of vascular inflammation.
Asunto(s)
Lesión Pulmonar Aguda/tratamiento farmacológico , Nanopartículas/administración & dosificación , Neumonía/tratamiento farmacológico , Síndrome de Dificultad Respiratoria/tratamiento farmacológico , Lesión Pulmonar Aguda/complicaciones , Lesión Pulmonar Aguda/patología , Amidas/administración & dosificación , Amidas/química , Animales , Anticuerpos Antiidiotipos/administración & dosificación , Anticuerpos Antiidiotipos/química , Infecciones Bacterianas/complicaciones , Infecciones Bacterianas/tratamiento farmacológico , Infecciones Bacterianas/microbiología , Portadores de Fármacos/administración & dosificación , Portadores de Fármacos/química , Humanos , Concentración de Iones de Hidrógeno , Molécula 1 de Adhesión Intercelular/efectos de los fármacos , Pulmón/efectos de los fármacos , Pulmón/patología , Ratones , Nanopartículas/química , Neumonía/complicaciones , Neumonía/patología , Polietilenglicoles/química , Ratas , Tiofenos/administración & dosificación , Tiofenos/químicaRESUMEN
The association of systemic sclerosis with anti-Topoisomerase 1 antibody (ATASSc) with specific alleles of human leukocyte antigen (HLA)-DR has been observed among various ethnics. The anti-Topoisomerase 1 antibody is a common autoantibody in SSc with diffuse cutaneous scleroderma, which is one of the clinical subtypes of SSc. On the other hand, an immunodominant peptide of topoisomerase 1 (Top1) self-protein (residues 349-368) was reported to have strong association with ATASSc. In this study, molecular dynamics simulation was performed on the complexes of Top1 peptide with various HLA-DR subtypes divided into ATASSc-associated alleles (HLA-DRB1*08:02, HLA-DRB1*11:01 and HLA-DRB1*11:04), suspected allele (HLA-DRB5*01:02), and non-associated allele (HLA-DRB1*01:01). The unique interaction for each system was compared to the others in terms of dynamical behaviors, binding free energies and solvation effects. Our results showed that three HLA-DR/Top1 complexes of ATASSc association mostly exhibited high protein stability and increased binding efficiency without solvent interruption, in contrast to non-association. The suspected case (HLA-DRB5*01:02) binds Top1 as strongly as the ATASSc association case, which implied a highly possible risk for ATASSc development. This finding might support ATASSc development mechanism leading to a guideline for the treatment and avoidance of pathogens like Top1 self-peptide risk for ATASSc.
Asunto(s)
ADN-Topoisomerasas de Tipo I/genética , Cadenas HLA-DRB1/química , Cadenas HLA-DRB5/química , Esclerodermia Sistémica/genética , Alelos , Anticuerpos Antiidiotipos/química , Anticuerpos Antiidiotipos/genética , Anticuerpos Antiidiotipos/inmunología , ADN-Topoisomerasas de Tipo I/química , ADN-Topoisomerasas de Tipo I/inmunología , Epítopos/genética , Epítopos/inmunología , Predisposición Genética a la Enfermedad , Cadenas HLA-DRB1/genética , Cadenas HLA-DRB1/inmunología , Cadenas HLA-DRB5/genética , Cadenas HLA-DRB5/inmunología , Humanos , Simulación de Dinámica Molecular , Péptidos/química , Péptidos/genética , Péptidos/inmunología , Unión Proteica/genética , Estabilidad Proteica , Factores de Riesgo , Esclerodermia Sistémica/inmunología , Esclerodermia Sistémica/patologíaRESUMEN
In this work, the gram-negative bacterium Escherichia coli strain BL21(DE3) (with lipopolysaccharide (LPS) in its outer membrane) and its modified ClearColi™ strain (lacking LPS) were used for the separation of anti-LPS antibodies from human serum by the following steps: (1) binding of the serum proteins to BL21(DE3); (2) dissociation of the bound proteins (including anti-LPS antibodies) from BL21(DE3) with acid; (3) filtering of the dissociated proteins using ClearColi to remove unwanted proteins; and (4) separation of the antibody fraction by protein-A column chromatography. The binding properties of the separated antibodies were analyzed by fluorescence-activated cell sorting to confirm their selective binding to LPS on the outer membrane of BL21(DE3), and by thermophoretic immunoassay to estimate their dissociation constant. The in vitro applicability of the separated anti-LPS antibodies was demonstrated through a fluorescence assay of BL21(DE3), after immobilizing the antibodies onto a modified microplate surface. The electrochemical detection of BL21(DE3) was also achieved after immobilizing the anti-LPS antibodies onto a gold electrode.
Asunto(s)
Anticuerpos Antiidiotipos/sangre , Técnicas Biosensibles , Escherichia coli/aislamiento & purificación , Lipopolisacáridos/aislamiento & purificación , Anticuerpos Antiidiotipos/química , Humanos , Inmunoensayo , Lipopolisacáridos/sangreRESUMEN
Glycans and anti-glycan antibodies (AGAs) are essential for infiltration of inflammatory cells in various allergies. The glycocalyx structure of the cells is modified during disease progression, and this modification is possible to evaluate by assessment of AGAs. A printed glycan array with 55 immobilized glycans and immobilized antibodies to IgG, IgA, and IgM was used to study the changes in AGA profiles in bronchial asthma (BA). Levels of antibodies to certain glycans in BA patients statistically differed from levels in healthy donors (p < 0.0007 by the Mann-Whitney test); the glycan set included 6Su-6`-SiaLec, Sia LeX, Sia6Htype2; Tαα, Manß1-4GlcNAc, and Manα1-4Manß. The obtained results help to better understand the mechanisms of the cell-mediated immune response in bronchial asthma and other types of allergic reactions.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Asma/inmunología , Hipersensibilidad/inmunología , Polisacáridos/inmunología , Adolescente , Anticuerpos Antiidiotipos/química , Anticuerpos Inmovilizados/química , Anticuerpos Inmovilizados/inmunología , Asma/sangre , Asma/patología , Niño , Preescolar , Femenino , Humanos , Hipersensibilidad/sangre , Hipersensibilidad/patología , Inmunidad Celular/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina M/sangre , Inmunoglobulina M/inmunología , Inflamación/sangre , Inflamación/inmunología , Inflamación/patología , Masculino , Polisacáridos/sangre , Polisacáridos/químicaRESUMEN
Immunoglobulin E (IgE) antibodies play a central role in the allergic response: interaction with FcεRI on mast cells and basophils leads to immediate hypersensitivity reactions upon allergen challenge, while interaction with CD23/FcεRII, expressed on a variety of cells, regulates IgE synthesis among other activities. The receptor-binding IgE-Fc region has recently been found to display remarkable flexibility, from acutely bent to extended conformations, with allosteric communication between the distant FcεRI and CD23 binding sites. We report the structure of an anti-IgE antibody Fab (8D6) bound to IgE-Fc through a mixed protein-carbohydrate epitope, revealing further flexibility and a novel extended conformation with potential relevance to that of membrane-bound IgE in the B cell receptor for antigen. Unlike the earlier, clinically approved anti-IgE antibody omalizumab, 8D6 inhibits binding to FcεRI but not CD23; the structure reveals how this discrimination is achieved through both orthosteric and allosteric mechanisms, supporting therapeutic strategies that retain the benefits of CD23 binding.
Asunto(s)
Anticuerpos Antiidiotipos/química , Anticuerpos Antiidiotipos/metabolismo , Inmunoglobulina E/química , Inmunoglobulina E/metabolismo , Receptores de IgE/metabolismo , Linfocitos B/inmunología , Cristalografía por Rayos X , Células HEK293 , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/metabolismo , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/metabolismo , Mastocitos/inmunología , Unión Proteica , Conformación ProteicaRESUMEN
To achieve the dual-channel (analog and digital) encoding, microbeads assembled with quantum dots (QDs) and element coding nanoparticles (ECNPs) have been prepared. Dual-spectra, including fluorescence generated from quantum dots (QDs) and laser induced breakdown spectrum obtained from the plasma of ECNPs, including AgO, MgO and ZnO nanoparticles, has been adopted to provide more encoding amounts and more accurate dual recognition for encoded microbeads in multiplexed utilization. The experimental results demonstrate that the single microbead can be decoded in two optical channels. Multiplexed analysis and contrast adsorption experiment of anti-IgG verified the availability and specificity of dual-channel-coded microbeads in bioanalysis. In gradient detection of anti-IgG, we obtained the linear concentration response to target biomolecules from 3.125â¯×â¯10-10â¯M to 1â¯×â¯10-8â¯M, and the limit of detection was calculated to be 2.91â¯×â¯10-11â¯M.
Asunto(s)
Anticuerpos Antiidiotipos/análisis , Nanopartículas del Metal/química , Microesferas , Puntos Cuánticos/química , Animales , Anticuerpos Antiidiotipos/química , Complejo Antígeno-Anticuerpo/química , Fluorescencia , Límite de Detección , Óxido de Magnesio/química , Ratones , Fenómenos Ópticos , Óxidos/química , Poliestirenos/química , Conejos , Ratas , Compuestos de Plata/química , Óxido de Zinc/químicaRESUMEN
A novel catechol-bearing polypeptide (CtP) was synthesized and used as a component of electrochemical biosensor involving both enzymatic activity and affinity-based sensing systems. Glucose oxidase (GOx) and anti-immunoglobulin G (Anti-IgG) were selected as model biorecognition elements for the selective analysis of glucose and IgG. Step-by-step surface modifications were followed using various techniques such as cyclic voltammetry (CV) and electrochemical impedance spectrometry (EIS) as well as X-ray photoelectron spectroscopy (XPS). Additionally, contact angles were measured in order to observe surface properties. Amperometric measurements using the GOx biosensor were performed at -0.7 V by following the oxygen consumption due to the enzymatic reaction in different glucose concentrations. Affinity-based interactions via IgG sensor were monitored using the differential pulse voltammetry (DPV) technique. As the "surface design with CtP" approach employed herein is generally applicable and easily adaptable to obtain functional matrices for biomolecule immobilization, CtP-coated surfaces can be promising platforms for the fabrication of various biobased sensing systems.
Asunto(s)
Técnicas Biosensibles/métodos , Catecoles/química , Glucosa/análisis , Inmunoglobulina G/análisis , Péptidos/química , Anticuerpos Antiidiotipos/química , Anticuerpos Antiidiotipos/metabolismo , Glucosa Oxidasa/química , Glucosa Oxidasa/metabolismo , Propiedades de SuperficieRESUMEN
The incretin hormone glucagon-like peptide-1 (GLP-1) has been subject to substantial pharmaceutical research regarding the treatment of type 2 diabetes mellitus. However, quantification of GLP-1 levels remains complicated due to the low circulation concentration and concurrent existence of numerous metabolites, homologous peptides, and potentially introduced GLP-1 receptor agonists. Surface plasmon resonance (SPR) facilitates real-time monitoring allowing a more detailed characterisation of the interaction compared with conventional enzyme-linked immunosorbent assays (ELISA). In this paper, we describe the development of the first SPR assays for characterisation of anti-GLP-1 antibodies for ELISA purposes. Binding responses were obtained on covalently immobilised anti-GLP-1 antibodies at 12°C, 25°C, and 40°C and fitted to a biomolecular (1:1) interaction model showing association rates of 1.01 × 103 to 4.54 × 103 M-1 s-1 and dissociation rates of 3.56 × 10-5 to 1.56 × 10-3 s-1 leading to affinities of 35.2 to 344 nM, depending on the temperature. Determination of thermodynamic properties revealed an enthalpy driven interaction (ΔH < ΔS < 0) with higher affinities at lower temperatures due to the formation and stabilisation of hydrogen bonds within the binding site primarily composed of polar amino acids (ΔCp < 0). Pair-wise epitope mapping was performed on captured anti-GLP-1 antibodies followed by subsequent interaction with GLP-1 (7-36) and other anti-GLP-1 antibodies. A global evaluation of every binding response led to an epitope map elucidating the potential of various anti-GLP-1 antibody pairs for sandwich ELISA and hence pinpointing the optimal antibody combinations. The SPR assays proved capable of providing vital information for ELISA development endorsing it as a useful optimisation tool.