Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 12.223
Filtrar
1.
Environ Monit Assess ; 195(12): 1462, 2023 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-37955762

RESUMO

Crassostrea virginica is a well-established bivalve species for biomonitoring persistent organic pollutants such as polycyclic aromatic hydrocarbons (PAH) in aquatic environments. Differing biomonitoring methods employing either wild oysters inhabiting sites of interest or naïve cultured oysters deployed to sites for extended periods can be used for site evaluations. However, important differences in total contaminant concentrations accumulated have been observed between the wild and transplanted groups. Furthermore, although rearing cultured triploid oysters is widely popular in commercial farming, the difference in contaminant bioaccumulation potential between triploid and diploid cultured oysters is vastly understudied, particularly for organic contaminants such as PAH. This study explores differences in PAH kinetics between transplanted triploid and diploid cultured oysters and wild oysters at a PAH-impacted site during a 6-week field exposure study using novel immunological techniques: antibody-based biosensor technology and immunofluorescence visualization. Conventional chemical analysis of oyster tissue was also conducted for comparison. While differences were observed in the oyster interstitial fluid between the wild and transplanted oysters throughout the study, whole tissue analysis revealed differing trends at each time point. Our findings suggest that insufficient equilibration time may contribute to the differences observed between groups. Furthermore, when combined with visual evidence via immunofluorescence, internal partitioning of contaminants may be an important determinant for total concentrations measured. A better understanding of the differences observed between wild and transplanted oyster groups is necessary for improved biomonitoring. Our study highlights the value in employing novel immunological techniques to explore possible mechanisms driving these differences.


Assuntos
Ostreidae , Triploidia , Animais , Diploide , Monitoramento Ambiental , Técnicas Imunológicas
2.
Curr Protoc ; 3(8): e867, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37610261

RESUMO

Immunoaffinity chromatography (IAC) is a type of liquid chromatography that uses immobilized antibodies or related binding agents as selective stationary phases for sample separation or analysis. The strong binding and high selectivity of antibodies have made IAC a popular tool for the purification and analysis of many chemicals and biochemicals, including proteins. The basic principles of IAC are described as related to the use of this method for protein purification and analysis. The main factors to consider in this technique are also presented under a discussion of the general strategy to follow during the development of a new IAC method. Protocols, as illustrated using human serum albumin (HSA) as a model protein, are provided for the use of IAC in several formats. This includes both the use of IAC with traditional low-performance supports such as agarose for off-line immunoextraction and supports used in high-performance IAC for on-line immunoextraction. The use of IAC for protein analysis as a flow-based or chromatographic immunoassay is also discussed and described using HSA and a competitive binding assay format as an example. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Off-line immunoextraction by traditional immunoaffinity chromatography Basic Protocol 2: On-line immunoextraction by high-performance immunoaffinity chromatography Basic Protocol 3: Competitive binding chromatographic immunoassay.


Assuntos
Anticorpos Imobilizados , Anticorpos , Humanos , Cromatografia de Afinidade , Técnicas Imunológicas , Cromatografia Líquida , Albumina Sérica Humana
4.
Inmunología (1987) ; 42(1): 30-32, Marzo 2023. graf
Artigo em Espanhol | IBECS | ID: ibc-223910

RESUMO

D69 es una molécula inmunorreguladora que es expresada rápidamente por los leucocitos tras su activación, cuyo papel como inmunoregulador negativo, y no pro-inflamatorio, es cada vez más patente por las investigaciones in vivo que se están realizando. En el artículo publicado en Cell Mol Life Sci por María Jiménez como primera autora, se pone de manifiesto la capacidad de CD69 para unir y responder a lipoproteínas de baja densidad(LDL) aisladas y oxidadas in vitro (oxLDL). Esta unión incrementa la expresión de los receptores nucleares anti-inflamatorios NR4A, especialmente de NR4A3, y de PD1, molécula que actúa como freno para la activación de linfocitos T. Para ello, los autores han usado una línea estable de Jurkat que sobre-expresaCD69, linfocitos CD4 primarios, secuenciación masiva de ARNm, así como otras técnicas de biología molecular como el silenciamiento mediante ARN pequeño de interferencia para NR4A3,receptor nuclear que está implicado en la expresión de PD1. La unión de las oxLD La CD69 produjo el incremento de PD1 tanto a nivel de transcripción (ARNm) como de proteína (citometría deflujo). En este contexto, CD69 señaliza a través NFAT, un factor de transcripción dependiente de calcio. Por otra parte, los autores estudiaron biopsias de pacientes intervenidos por aneurisma de la aorta abdominal; en aquellos con inflamación crónica la expresión de PD1, NR4A3y CD69 está incrementada. Además, se observó una correlación positiva entre los niveles de ARNm de CD69 y de PD1 en estos pacientes, delos que más de la mitad presentaban hiperlipidemia. Estos datos apoyan el papel inmunoregulador negativo de CD69 en patologías como la aterosclerosis, y lo sitúan en el centro de rutas de señalización que actúan como freno de respuestas inflamatorias de origen inmunitario. (AU)


Assuntos
Humanos , Técnicas Imunológicas , Linfócitos/imunologia , Alergia e Imunologia
5.
STAR Protoc ; 4(1): 102063, 2023 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-36853678

RESUMO

Here, we present a protocol combining co-immunoprecipitation (Co-IP) and immunofluorescence approaches with cell cycle stage synchronization to detect cell-cycle-specific complexes. We describe steps to synchronize cells at specific cell cycle stages using drugs. We then detail the preparation of cell extracts from synchronized cells and fractionation of the protein complexes with density centrifugation, followed by Co-IP with specific antibodies. Protein-protein interactions are confirmed by localization using immunofluorescence imaging. This protocol is helpful for visualizing the dynamics of protein complex assembly. For complete details on the use and execution of this protocol, please refer to Habu and Kim (2021).1.


Assuntos
Anticorpos , Ciclo Celular , Divisão Celular , Técnicas Imunológicas , Imunoprecipitação
6.
Vaccimonitor (La Habana, Print) ; 31(3)sept.-dic. 2022.
Artigo em Espanhol | LILACS, CUMED | ID: biblio-1410312

RESUMO

VacciMonitor, producto líder del sello editorial Finlay Ediciones, es una revista arbitrada dedicada a la vacunología y otras disciplinas relacionadas que se reafirma como la única con estas características en Latinoamérica. Existen otras con iguales objetivos editadas por países desarrollados, como Vaccine de la editorial Elsevier (Holanda) y Vacunas, de Doyma (España).1,2 Se fundó en 1992 con el objetivo de divulgar los logros alcanzados por Cuba en el campo de las vacunas; desde esa fecha ha contribuido a visibilizar el impacto de la vacunación en nuestro país y el desarrollo de la industria farmacéutica y biotecnológica que produce gran parte de las vacunas utilizadas en el programa nacional de inmunización.2 En sus inicios tuvo un carácter institucional. En el año 2000 trascendió nuestras fronteras y a partir de ese momento aumentaron los artículos procedentes de otros países1,2. Su frecuencia ha variado, atendiendo principalmente a la disponibilidad de artículos científicos. En los últimos años se ha estabilizado como publicación cuatrimestral.1,2,3 Del año 2005 al 2021 se han publicado 263 artículos científicos, de 31 países y 156 instituciones. El 27,8 por ciento de esta producción es externa a nuestro país y el 72,2 por ciento, interna; de esta última, el 49,4 por ciento corresponde a producción no institucional. En VacciMonitor han publicado autores cubanos y de otros países como México, Venezuela, Malasia, Reino Unido, entre otros. Las Instituciones cubanas que más contribuyen a la revista son: Instituto Finlay de Vacunas, Centro de Ingeniería Genética y Biotecnología, Instituto de Medicina Tropical ¨Pedro Kourí¨, Instituto Superior Politécnico José Antonio Echeverría y Universidad de Ciencias Médicas de La Habana.4) VacciMonitor es una revista a favor de la Ciencia Abierta.5 Acepta manuscritos publicados en archivos de preprints; proporciona un acceso abierto inmediato a su contenido, basado en el principio de que ofrecer al público un acceso libre a las investigaciones ayuda a un mayor intercambio global de conocimiento; el acceso a todos los contenidos de la revista, registro y envío de artículos es totalmente gratuito. Todo el procesamiento de los artículos, su revisión, edición y publicación es libre...(AU)


Assuntos
Humanos , Masculino , Feminino , Vacinas/uso terapêutico , Técnicas Imunológicas , Cuba
7.
J Vis Exp ; (183)2022 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-35575516

RESUMO

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the KIT receptor. Across tumor types, numerous mouse models have been developed in order to investigate the next generation of cancer therapies. However, in GIST, most in vivo studies use xenograft mouse models which have inherent limitations. Here, we describe an immunocompetent, genetically engineered mouse model of gastrointestinal stromal tumor harboring a KitV558Δ/+ mutation. In this model, mutant KIT, the oncogene responsible for most GISTs, is driven by its endogenous promoter leading to a GIST which mimics the histological appearance and immune infiltrate seen in human GISTs. Furthermore, this model has been used successfully to investigate both targeted molecular and immune therapies. Here, we describe the breeding and maintenance of a KitV558Δ/+ mouse colony. Additionally, this paper details the treatment and procurement of GIST, draining mesenteric lymph node, and adjacent cecum in KitV558Δ/+ mice, as well as sample preparation for molecular and immunologic analyses.


Assuntos
Tumores do Estroma Gastrointestinal , Animais , Modelos Animais de Doenças , Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/patologia , Humanos , Técnicas Imunológicas , Camundongos , Mutação , Proteínas Proto-Oncogênicas c-kit/genética
8.
Biosens Bioelectron ; 209: 114237, 2022 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-35447596

RESUMO

Kinetics measurements of antigen-antibody binding interactions are critical to understanding the functional efficiency of SARS-CoV-2 antibodies. Previously reported chaotrope-based avidity assays that rely on artificial disruption of binding do not reflect the natural binding kinetics. This study developed a chaotrope- and label-free biolayer interferometry (BLI) assay for the real-time monitoring of receptor binding domain (RBD) binding kinetics with SARS-CoV-2 spike protein in convalescent COVID-19 patients. An improved conjugation biosensor probe coated with streptavidin-polysaccharide (SA-PS) led to a six-fold increase of signal intensities and two-fold reduction of non-specific binding (NSB) compared to streptavidin only probe. Furthermore, by utilizing a separate reference probe and biotin-human serum albumin (B-HSA) blocking process to subtracted NSB signal in serum, this BLI biosensor can measure a wide range of the dissociation rate constant (koff), which can be measured without knowledge of the specific antibody concentrations. The clinical utility of this improved BLI kinetics assay was demonstrated by analyzing the koff values in sera of 24 pediatric (≤18 years old) and 63 adult (>18 years old) COVID-19 convalescent patients. Lower koff values for SARS-CoV-2 serum antibodies binding to RBD were measured in samples from children. This rapid, easy to operate and chaotrope-free BLI assay is suitable for clinical use and can be readily adapted to characterize SARS-CoV-2 antibodies developed by COVID-19 patients and vaccines.


Assuntos
Técnicas Biossensoriais , COVID-19 , Adolescente , Adulto , Anticorpos Neutralizantes , Anticorpos Antivirais , Criança , Humanos , Técnicas Imunológicas , Interferometria , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Estreptavidina
9.
J Endocrinol Invest ; 45(2): 337-346, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34302683

RESUMO

PURPOSE: Calcium sensing receptor (CaSR), on the surface of normal parathyroid cells, is essential for maintaining serum calcium levels. The normal pattern of CaSR immunostaining remains undefined and is presumptively circumferential. Given the physiological variation in serum calcium, we postulated that CaSR expression could not be uniformly circumferential. Also, cytoplasmic expression has not been evaluated either in normal or pathological tissues. We studied normal parathyroid tissues derived from forensic autopsies and those rimming parathyroid adenomas for membranous and cytoplasmic CaSR immunoexpression. Results were compared with primary hyperparathyroidism (PHPT) to look for any pathogenetic implications. MATERIALS AND METHODS: We evaluated 34 normal parathyroid tissues from 11 autopsies, 30 normal rims, 45 parathyroid adenoma, 10 hyperplasia, and 7 carcinoma cases. Membranous expression was categorized complete/incomplete and weak/moderate/strong; scored using Her2/Neu and Histo-scores; predominant pattern noted. Cytoplasmic expression was categorized negative/weak/moderate/strong; predominant intensity noted. RESULTS: Normal autopsy-derived parathyroid tissues were Her2/Neu 3 + , but incomplete membranous staining predominated in 85%. Their immune-scores were significantly more than the cases (p < < 0.05). The mean histo-score of normal rims was intermediate between the two (p < < 0.05). Cytoplasmic expression was strong in all autopsy-derived tissues, weak/negative in hyperplasia (100%), moderate in 16% adenomas, and 43% carcinomas. CONCLUSIONS: Normal autopsy-derived parathyroid tissues showed strong but predominantly incomplete membranous expression. Surface CaSR expression decreased in PHPT and is probably an early event in parathyroid adenoma, seen even in normal rims. Whether there is a defect in CaSR trafficking from the cytoplasm to the cell surface in adenoma and carcinoma needs further evaluation.


Assuntos
Hiperparatireoidismo Primário , Glândulas Paratireoides , Neoplasias das Paratireoides , Receptores de Detecção de Cálcio/análise , Adulto , Autopsia , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Hiperparatireoidismo Primário/metabolismo , Hiperparatireoidismo Primário/patologia , Imuno-Histoquímica , Técnicas Imunológicas/métodos , Proteínas Sensoras de Cálcio Intracelular/metabolismo , Masculino , Glândulas Paratireoides/metabolismo , Glândulas Paratireoides/patologia , Neoplasias das Paratireoides/metabolismo , Neoplasias das Paratireoides/patologia
11.
Front Immunol ; 12: 735584, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34917073

RESUMO

Common approaches for monitoring T cell responses are limited in their multiplexity and sensitivity. In contrast, deep sequencing of the T Cell Receptor (TCR) repertoire provides a global view that is limited only in terms of theoretical sensitivity due to the depth of available sampling; however, the assignment of antigen specificities within TCR repertoires has become a bottleneck. This study combines antigen-driven expansion, deep TCR sequencing, and a novel analysis framework to show that homologous 'Clusters of Expanded TCRs (CETs)' can be confidently identified without cell isolation, and assigned to antigen against a background of non-specific clones. We show that clonotypes within each CET respond to the same epitope, and that protein antigens stimulate multiple CETs reactive to constituent peptides. Finally, we demonstrate the personalized assignment of antigen-specificity to rare clones within fully-diverse uncultured repertoires. The method presented here may be used to monitor T cell responses to vaccination and immunotherapy with high fidelity.


Assuntos
Separação Celular/métodos , Técnicas Imunológicas/métodos , Receptores de Antígenos de Linfócitos T/análise , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Humanos
12.
J Clin Lab Anal ; 35(12): e24087, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34724262

RESUMO

BACKGROUND: The measurement method for experimental resolution and related data to evaluate analytical performance is poorly explored in clinical research. We established a method to measure the experimental resolution of clinical tests, including biochemical tests, automatic hematology analyzer methods, immunoassays, chemical experiments, and qPCR, to evaluate their analytical performance. METHODS: Serially diluted samples in equal proportions were measured, and correlation analysis was performed between the relative concentration and the measured value. Results were accepted for p ≤ 0.01 of the correlation coefficient. The minimum concentration gradient (eg, 10%) was defined as the experimental resolution. For this method, the smaller the value, the higher the experimental resolution and the better the analytical performance. RESULTS: The experimental resolution of the most common biochemical indices reached 10%, with some even reaching 1%. The results of most counting experiments showed experimental resolution up to 10%, whereas the experimental resolution of the classical chemical assays reached 1%. Unexpectedly, the experimental resolution of more sensitive assays, such as immunoassays was only 25% when using the manual method and 10% for qPCR. CONCLUSION: This study established a method for measuring the experimental resolution of laboratory assays and provides a new index for evaluating the reliability of methods in clinical laboratories.


Assuntos
Análise Química do Sangue/métodos , Técnicas Imunológicas/métodos , Laboratórios Clínicos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Contagem de Células Sanguíneas , Análise Química do Sangue/normas , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Técnicas Imunológicas/normas , Laboratórios Clínicos/normas , Reação em Cadeia da Polimerase em Tempo Real/normas , Reprodutibilidade dos Testes , Espectrofotometria Atômica
13.
STAR Protoc ; 2(4): 100952, 2021 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-34841276

RESUMO

Cell-Specific Mitochondria Affinity Purification (CS-MAP) enables isolation and purification of intact mitochondria from individual cell types of Caenorhabditis elegans. The approach is based on the cell-specific expression of a recombinant hemagglutinin (HA)-tag fused to the TOMM-20 protein that decorates the surface of mitochondria, thereby allowing their immunomagnetic purification. This protocol describes the CS-MAP procedure performed on large populations of animals. The purified mitochondria are suitable for subsequent nucleic acid, protein, and functional analyses. For complete details on the use and execution of this protocol, please refer to Ahier et al. (2018, 2021).


Assuntos
Caenorhabditis elegans/citologia , Técnicas Citológicas/métodos , Técnicas Imunológicas/métodos , Mitocôndrias , Animais , Mitocôndrias/química , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia
14.
Sci Rep ; 11(1): 19603, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34599256

RESUMO

Colorectal cancer (CRC) is a challenging public health problem which successful treatment depends on the stage at diagnosis. Recently, CRC-specific microbiome signatures have been proposed as a marker for CRC detection. Since many countries have initiated CRC screening programs, it would be useful to analyze the microbiome in the samples collected in fecal immunochemical test (FIT) tubes for fecal occult blood testing. Therefore, we investigated the impact of FIT tubes and stabilization buffer on the microbial community structure evaluated in stool samples from 30 volunteers and compared the detected communities to those of fresh-frozen samples, highlighting previously published cancer-specific communities. Altogether, 214 samples were analyzed by 16S rRNA gene sequencing, including positive and negative controls. Our results indicated that the variation between individuals was greater than the differences introduced by the collection strategy. The vast majority of the genera were stable for up to 7 days. None of the changes observed between fresh-frozen samples and FIT tube specimens were related to previously identified CRC-specific bacteria. Overall, we show that FIT tubes can be used for profiling the microbiota in CRC screening programs. This circumvents the need to collect additional samples and can possibly improve the sensitivity of CRC detection.


Assuntos
Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/microbiologia , Detecção Precoce de Câncer/métodos , Microbioma Gastrointestinal , Adulto , Idoso , Bactérias/genética , Estônia , Fezes/microbiologia , Feminino , Congelamento , Humanos , Técnicas Imunológicas/instrumentação , Masculino , Pessoa de Meia-Idade , Sangue Oculto , RNA Ribossômico 16S/genética , Manejo de Espécimes/métodos
15.
Arq. Ciênc. Vet. Zool. UNIPAR (Online) ; 24(2, cont.): e2411, jul-dez. 2021. tab, graf, ilus
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1352319

RESUMO

Salmonellosis is the world's most common foodborne illness. In Brazil, foods contaminated by salmonella lead the statistics. Therefore, the aim of this study is, through biotechnological knowledge, to compile alternative and innovative techniques for the detection of salmonella in foods, such as fish-farming derivatives, immunological and biosensorial techniques. This is a descriptive exploratory data survey of a qualitative nature, aiming at data analysis. Research and data collection were carried out in bibliographic databases: Academic Google, Scielo, CAPES journals and institutional repositories using specific descriptors - in Portuguese and English, with words and terms separated by the Boolean operators 'AND' and 'OR'. Some innovative and alternative methods are available to identify the presence of salmonella in food. Immunological and biosensory techniques, despite being less frequent in the scientific literature than molecular methods, are techniques that present high specificity and sensitivity. These techniques have been the most developed alternative methods in fish in recent years. And, they can employ both molecular and immunological techniques in biorecognition, which is characterized as an advantage of not having a requirement for pre-enrichment of the sample. According to the literature found, the techniques covered in this study are quick to respond, which speeds up decision-making by researchers and technicians, which makes the techniques very promising for industrial application.(AU)


A salmonelose é uma enfermidade de maior ocorrência no mundo veiculada por alimentos. No Brasil, alimentos contaminados por salmonelas lideram as estatísticas. Por isso, o objetivo desse estudo é através dos conhecimentos biotecnológicos compilar técnicas alternativas e inovadoras para a detecção de salmonelas em alimentos, como os derivados da piscicultura, as técnicas imunológicas e biossensoriais. Trata-se de um estudo de levantamento de dados descritivo exploratório de de caráter qualitativo, visando à análise dos dados. As pesquisas e coletas de dados foram realizadas nas bases bibliográficas: Google Acadêmico, Scielo, periódicos da CAPES e repositórios institucionais utilizando os descritores específicos - nos idiomas português e inglês, com palavras e termos separados pelos operadores booleanos 'AND' e 'OR'. São disponibilizados alguns métodos inovadores e alternativos para identificação da presença de salmonelas em alimentos. As técnicas imunológicas e biossensoriais, apesar de serem menos frequentes na literatura científica do que os métodos moleculares são técnicas que apresentaram elevada especificidade e sensibilidade. Essas técnicas têm sido os métodos alternativos mais desenvolvidos em peixes nos últimos anos. E, podem empregar tanto técnicas moleculares como imunológicas no biorreconhecimento, o que se caracteriza como vantagem de não haver requerimento de pré-enriquecimento da amostra. Conforme a literatura encontrada, as técnicas abordadas por esse estudo apresentam rapidez de resposta o que agiliza as tomadas de decisões dos pesquisadores e técnicos, o que torna as técnicas bastante promissora para aplicação industrial.(AU)


La salmonelosis es la enfermedad transmitida por alimentos más común del mundo. En Brasil, los alimentos contaminados por salmonelas lideran las estadísticas. Por tanto, el objetivo de ese estudio fue a través de conocimientos biotecnológicos recopilar técnicas alternativas e innovadoras para la detección de salmonelas en los alimentos, como los derivados de la piscicultura, las técnicas inmunológicas y biosensoriales. Se trata de una encuesta de datos exploratorio descriptivo de carácter cualitativo, cuyo objetivo es el análisis de datos. Las investigaciones y recopilaciones de datos se realizaron en bases de datos bibliográficas: Google Académico, Scielo, revistas CAPES y repositorios institucionales utilizando descriptores específicos, en portugués e inglés, con palabras y términos separados por los operadores booleanos 'AND' y 'OR'. Se encuentran disponibles algunos métodos innovadores y alternativos para identificar la presencia de salmonela en los alimentos. Las técnicas inmunológicas y biosensoriales, a pesar de ser menos frecuentes en la literatura científica que los métodos moleculares, son técnicas de alta especificidad y sensibilidad. Esas técnicas han sido los métodos alternativos más desarrollados en peces en los últimos años. Y pueden emplear técnicas tanto moleculares como inmunológicas en el biorreconocimiento, que se caracteriza por la ventaja de no tener un requisito de preenriquecimiento de la muestra. Según la literatura encontrada, las técnicas abordadas en este estudio son de rápida respuesta, lo que agiliza la toma de decisiones por parte de investigadores y técnicos, lo que hace que las técnicas sean muy prometedoras para la aplicación industrial.(AU)


Assuntos
Animais , Salmonella , Infecções por Salmonella , Técnicas Imunológicas , Sensibilidade e Especificidade , Pesqueiros , Microbiologia de Alimentos , Análise de Dados
16.
MAbs ; 13(1): 1967714, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34491877

RESUMO

Bispecific antibodies have recently attracted intense interest. CrossMab technology was described in 2011 as novel approach enabling correct antibody light-chain association with their respective heavy chain in bispecific antibodies, together with methods enabling correct heavy-chain association using existing pairs of antibodies. Since the original description, CrossMab technology has evolved in the past decade into one of the most mature, versatile, and broadly applied technologies in the field, and nearly 20 bispecific antibodies based on CrossMab technology developed by Roche and others have entered clinical trials. The most advanced of these are the Ang-2/VEGF bispecific antibody faricimab, currently undergoing regulatory review, and the CD20/CD3 T cell bispecific antibody glofitamab, currently in pivotal Phase 3 trials. In this review, we introduce the principles of CrossMab technology, including its application for the generation of bi-/multispecific antibodies with different geometries and mechanisms of action, and provide an overview of CrossMab-based therapeutics in clinical trials.


Assuntos
Anticorpos Biespecíficos , Técnicas Imunológicas , Animais , Anticorpos Monoclonais , Humanos , Engenharia de Proteínas/métodos
17.
Front Immunol ; 12: 728936, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34484239

RESUMO

The use of minimal peptide sets offers an appealing alternative for design of vaccines and T cell diagnostics compared to conventional whole protein approaches. T cell immunogenicity towards peptides is contingent on binding to human leukocyte antigen (HLA) molecules of the given individual. HLA is highly polymorphic, and each variant typically presents a different repertoire of peptides. This polymorphism combined with pathogen diversity challenges the rational selection of peptide sets with broad immunogenic potential and population coverage. Here we propose PopCover-2.0, a simple yet highly effective method, for resolving this challenge. The method takes as input a set of (predicted) CD8 and/or CD4 T cell epitopes with associated HLA restriction and pathogen strain annotation together with information on HLA allele frequencies, and identifies peptide sets with optimal pathogen and HLA (class I and II) coverage. PopCover-2.0 was benchmarked on historic data in the context of HIV and SARS-CoV-2. Further, the immunogenicity of the selected SARS-CoV-2 peptides was confirmed by experimentally validating the peptide pools for T cell responses in a panel of SARS-CoV-2 infected individuals. In summary, PopCover-2.0 is an effective method for rational selection of peptide subsets with broad HLA and pathogen coverage. The tool is available at https://services.healthtech.dtu.dk/service.php?PopCover-2.0.


Assuntos
Epitopos de Linfócito T/imunologia , Antígenos HLA/genética , Antígenos HLA/imunologia , Peptídeos/imunologia , Alelos , Alergia e Imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , COVID-19/imunologia , COVID-19/prevenção & controle , Genótipo , Antígenos HLA/classificação , Humanos , Imunogenicidade da Vacina , Técnicas Imunológicas , Peptídeos/classificação , SARS-CoV-2/imunologia
19.
STAR Protoc ; 2(3): 100758, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34458873

RESUMO

Determining the antigen specificities of the endogenous T-cell repertoire is important for screening naturally occurring or therapy-induced T-cell immunity and may help identify novel targets for T-cell-based therapies. Here, we describe a rapid, sensitive, and high-throughput protocol for expanding antigen-specific T cells from human peripheral blood mononuclear cells in vitro following peptide stimulation and detecting antigen-specific effector cytokine formation by flow cytometry. Our approach can be applied to examining specific T-cell subsets from various tissues. For complete details on the use and execution of this protocol, please refer to Roudko et al. (2020) and Cimen Bozkus et al. (2019).


Assuntos
Citocinas/metabolismo , Técnicas Imunológicas/métodos , Subpopulações de Linfócitos T/imunologia , Técnicas de Cultura de Células/métodos , Criopreservação , Citocinas/farmacologia , Citometria de Fluxo , Humanos , Técnicas Imunológicas/instrumentação , Leucócitos Mononucleares/imunologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/metabolismo
20.
Sci Rep ; 11(1): 17234, 2021 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-34446788

RESUMO

Over the past two decades, there has been a great interest in the study of HLA-E-restricted αß T cells during bacterial and viral infections, including recently SARS-CoV-2 infection. Phenotyping of these specific HLA-E-restricted T cells requires new tools such as tetramers for rapid cell staining or sorting, as well as for the identification of new peptides capable to bind to the HLA-E pocket. To this aim, we have developed an optimal photosensitive peptide to generate stable HLA-E/pUV complexes allowing high-throughput production of new HLA-E/peptide complexes by peptide exchange. We characterized the UV exchange by ELISA and improved the peptide exchange readout using size exclusion chromatography. This novel approach for complex quantification is indeed very important to perform tetramerization of MHC/peptide complexes with the high quality required for detection of specific T cells. Our approach allows the rapid screening of peptides capable of binding to the non-classical human HLA-E allele, paving the way for the development of new therapeutic approaches based on the detection of HLA-E-restricted T cells.


Assuntos
Epitopos de Linfócito T/química , Antígenos de Histocompatibilidade Classe I/química , Complexo Principal de Histocompatibilidade/imunologia , Peptídeos/química , Sequência de Aminoácidos , Epitopos de Linfócito T/imunologia , Ensaios de Triagem em Larga Escala , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Técnicas Imunológicas , Processos Fotoquímicos , Ligação Proteica , Conformação Proteica , Linfócitos T Citotóxicos/imunologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...