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1.
Methods Mol Biol ; 2451: 631-669, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35505039

RESUMO

The emergence of microbial resistance to antimicrobials among several common pathogenic microbial strains is an increasing problem worldwide. Thus, it is urgent to develop not only new antimicrobial therapeutics to fight microbial infections, but also new effective, rapid, and inexpensive methods to monitor the efficacy of these new therapeutics. Antimicrobial photodynamic therapy (aPDT) and antimicrobial blue light (aBL) therapy are receiving considerable attention for their antimicrobial potential and represent realistic alternatives to antibiotics. To monitor the photoinactivation process provided by aPDT and aBL, faster and more effective methods are required instead of laborious conventional plating and overnight incubation procedures. Bioluminescent microbial models are very interesting in this context. Light emission from bioluminescent microorganisms is a highly sensitive indication of their metabolic activity and can be used to monitor, in real time, the effects of antimicrobial agents and therapeutics. This chapter reviews the efforts of the scientific community concerning the development of in vitro, ex vivo, and in vivo bioluminescent bacterial models and their potential to evaluate the efficiency of aPDT and aBL in the inactivation of bacteria.


Assuntos
Anti-Infecciosos , Fotoquimioterapia , Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Bactérias , Testes Imunológicos , Fotoquimioterapia/métodos
2.
Biosensors (Basel) ; 12(4)2022 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-35448274

RESUMO

The detection of salivary cotinine is useful for convenient smoking tests in spite of the high background effect of saliva. For precise results, the conventional salivary cotinine analysis for smoking detection requires complex pretreatment processes. Hence, in this study, we developed a modified paper-based lateral flow immunoassay (LFIA), termed "gap-LFIA", for the direct application of saliva collected using cotton swabs for on-site detection. The gap-LFIA was constructed by modifying a conventional LFIA sensor, where the sample pad was divided to have a 3 mm gap. A saliva-collected cotton swab was inserted into the gap, and then, a buffer solution was added to the outer sample pad to dilute the saliva automatically. The gap-LFIA reduced the interference in salivary samples and showed improved signals, allowing for using the whole saliva directly without additional steps. Further, the deviation of results using a strip was less than that when the saliva was not diluted in a conventional cotinine kit, and it helped to distinguish between smokers and non-smokers more clearly in 15 min. This method of automatic dilution may apply to various clinical samples, including blood and serum, for direct application in future detections.


Assuntos
Cotinina , Saliva , Cotinina/análise , Imunoensaio/métodos , Testes Imunológicos , Saliva/química , Fumar
3.
Anal Chem ; 94(16): 6271-6280, 2022 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-35417142

RESUMO

Modulating the precise self-assembly of functional biomacromolecules is a critical challenge in biotechnology. Herein, functional biomacromolecule-assembled hierarchical hybrid nanoarchitectures in a spatially controlled fashion are synthesized, achieving the biorecognition behavior and signal amplification in the immunoassay simultaneously. Biomacromolecules with sequential assembly on the scaffold through the biomineralization process show significantly enhanced stability, bioactivity, and utilization efficiency, allowing tuning of their functions by modifying their size and composition. The hierarchically hybrid nanoarchitectures show great potential in construction of ultrasensitive immunoassay platforms, achieving a three order-of-magnitude increase in sensitivity. Notably, the well-designed HRP@Ab2 nanoarchitectures allow for optical immunoassays with a detection range from picogram mL-1 to microgram mL-1 on demand, providing great promise for quantitative analysis of both low-abundance and high-residue targets for biomedical applications.


Assuntos
Testes Imunológicos , Proteínas , Imunoensaio
5.
Methods Mol Biol ; 2491: 195-216, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35482192

RESUMO

Membrane proteins are favored drug targets and antibody therapeutics represent the fastest-growing category of pharmaceuticals. However, there remains a need for rapid and effective approaches for the discovery of antibodies that recognize membrane proteins to develop a robust clinical pipeline for targeted therapeutics. The challenges associated with recombinant expression of membrane proteins make whole cell screening techniques desirable, as these strategies allow presentation of the target membrane proteins in their native conformations. Here, we describe a workflow that employs both adherent cell-based and suspension cell-based whole cell panning methodologies to enrich for specific binders within a yeast-displayed antibody library. The first round of selection consists of an adherent cell-based approach, wherein a diverse library is panned over target-expressing mammalian cell monolayers in order to debulk the naïve library. Subsequent rounds involve the use of suspension cell-based approaches, facilitated with magnetic-activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS), to achieve further library enrichment. Finally, we describe a high-throughput approach to screen target binding and specificity of individual clones isolated from selection campaigns.


Assuntos
Proteínas de Membrana , Biblioteca de Peptídeos , Animais , Anticorpos , Citometria de Fluxo/métodos , Testes Imunológicos , Mamíferos , Proteínas de Membrana/genética , Suspensões
6.
Sci Rep ; 12(1): 5478, 2022 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-35361862

RESUMO

This paper presents a lateral flow assay (LFA) for the quantitative, fluorescence-based detection of the kidney biomarker cystatin C that features conjugates of capture antibodies and fusions of carbohydrate binding modules (CBM) with ZZ domains anchored on cellulose deposited over nitrocellulose (NC). The ZZ-CBM3 fusion provides a biomolecular interface between the cellulose layer and the Fc portion of the capture antibodies. By resorting to detection Fab fragments that lack the Fc portion we overcome the observed interference of full-length detection antibodies with the ZZ-CBM3 fusion at the test lines. Using the new LFA architecture, a linear concentration-response relationship was observed in the 0-10 ng/mL cystatin C concentration range, which is compatible with the clinically normal (5-120 ng/mL) and abnormal (> 250 ng/mL) levels of cystatin C, as long as proper dilutions are made. An inter assay CoV of 0.72% was obtained. Finally, mock urine samples characteristic of normal (100 ng/mL) and kidney tubular disease (4000 ng/mL) patients were successfully analyzed. Overall, we demonstrate an innovative LFA architecture that combines NC strips with layered cellulose, ZZ-CBM3 fusions and fluorescently labeled Fab fragments.


Assuntos
Celulose , Cistatina C , Celulose/química , Humanos , Imunoensaio , Fragmentos Fab das Imunoglobulinas , Testes Imunológicos
7.
J Extracell Vesicles ; 11(4): e12202, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35362268

RESUMO

With an exponential increase in extracellular vesicle (EV) studies in the past decade, focus has been placed on standardization of experimental design to ensure inter-study comparisons and validity of conclusions. In the case of in vitro assays, the composition of cell culture media is important to consider for EV studies. In particular, levels of lipoproteins, which are critical components of the interstitial fluid, should be taken into consideration. Results from this study reveal that lipoprotein levels in cell culture medium impact the effects that EVs have on recipient cells. Additionally, evidence of EV binding and fusion to lipoprotein-like structures in plasma is provided. However, it is unclear whether the impact of lipoproteins in cell culture is due to direct interactions with EVs, indirect effects, or a combination of both mechanisms. Taken together, cell culture studies performed in the absence of physiological levels of lipoproteins are unlikely to reflect interactions that occur between EVs and recipient cells in an in vivo environment.


Assuntos
Vesículas Extracelulares , Bioensaio , Técnicas de Cultura de Células , Vesículas Extracelulares/metabolismo , Testes Imunológicos , Lipoproteínas/metabolismo
8.
Sci Rep ; 12(1): 5617, 2022 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-35379866

RESUMO

Fragile X Syndrome (FXS) is caused by a trinucleotide expansion leading to silencing of the FMR1 gene and lack of expression of Fragile X Protein (FXP, formerly known as Fragile X Mental Retardation Protein, FMRP). Phenotypic presentation of FXS is highly variable, and the lack of reproducible, sensitive assays to detect FXP makes evaluation of peripheral FXP as a source of clinical variability challenging. We optimized a Luminex-based assay to detect FXP in dried blot spots for increased reproducibility and sensitivity by improving reagent concentrations and buffer conditions. The optimized assay was used to quantify FXP in 187 individuals. We show that the optimized assay is highly reproducible and detects a wide range of FXP levels. Mosaic individuals had, on average, higher FXP levels than fully methylated individuals, and trace amounts of FXP were consistently detectable in a subset of individuals with full mutation FXS. IQ scores were positively correlated with FXP levels in males and females with full mutation FXS demonstrating the clinical utility of this method. Our data suggest trace amounts of FXP detectable in dried blood spots of individuals with FXS could be clinically relevant and may be used to stratify individuals with FXS for optimized treatment.


Assuntos
Síndrome do Cromossomo X Frágil , Feminino , Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/diagnóstico , Síndrome do Cromossomo X Frágil/genética , Humanos , Imunoensaio , Testes Imunológicos , Masculino , Mutação , Reprodutibilidade dos Testes
9.
Parasit Vectors ; 15(1): 142, 2022 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-35461265

RESUMO

BACKGROUND: Strongyloidiasis, a nematode infection which is mainly caused by Strongyloides stercoralis in humans, can lead to a fatal syndrome in immunocompromised individuals. Its diagnosis is challenging due to the absence of a diagnostic gold standard. The infection is highly prevalent in migrants from endemic countries in tropical and subtropical areas, and a rapid diagnostic test would be helpful for screening purposes. The aim of this study was to estimate the accuracy of a novel immunochromatographic test (ICT) for the diagnosis of S. stercoralis infection. METHODS: A single-centre diagnostic accuracy study was undertaken using well-characterized frozen sera available from the biobank of a referral hospital for parasitic diseases in Italy. The included sera were from migrants from sub-Saharan Africa, and matching results were available for agar plate culture and/or polymerase chain reaction for S. stercoralis; moreover, the results of both a commercial enzyme-linked immunosorbent assay test and an in-house immunofluorescence test for strongyloidiasis were made available. Laboratory staff who read the ICT results were blinded as regards the results of the other tests. Two readers independently read the ICT, and a third one was involved when results were discrepant. The accuracy of the ICT was assessed both against the results of the panel of faecal tests and by latent class analysis (LCA). RESULTS: Agreement between the readers was excellent [Cohen's κ = 92.7%, 95% confidence interval (CI) 88.3-97.1%]. When assessed against the results of the faecal tests, the sensitivity and specificity of the ICT were 82.4% (95% CI 75.7-89.0%) and 73.8% (95% CI 66.8-80.9%), respectively. According to the LCA, the sensitivity and specificity were 86.3% (95% CI 80.1-92.5%) and 73.9% (95% CI 67.0-80.8%), respectively. CONCLUSIONS: The results of the ICT demonstrated ease of interpretation. The accuracy proved good, though the sensitivity might be further improved for screening purposes.


Assuntos
Strongyloides stercoralis , Estrongiloidíase , Migrantes , Animais , Ensaio de Imunoadsorção Enzimática/métodos , Fezes/parasitologia , Humanos , Testes Imunológicos , Sensibilidade e Especificidade , Estrongiloidíase/parasitologia
10.
MMW Fortschr Med ; 164(Suppl 1): 10, 2022 04.
Artigo em Alemão | MEDLINE | ID: mdl-35359279
12.
J Immunol Methods ; 504: 113262, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35341761

RESUMO

OBJECTIVES: Quantitative detection of interleukin-6 (IL-6) in serum and plasma can help monitor immune responses and the development of acute inflammation to guide patient management. We developed an IL-6 immunoassay for use with the automated ARCHITECT system for detecting an increase in the inflammatory response. METHODS: Immunized mouse sera were tested and selected B-cells were harvested for fusion with myeloma cells. A panel of monoclonal antibodies were produced, from which capture and detection monoclonal antibodies for the prototype IL-6 immunoassay were selected and screened on the ARCHITECT instrument. The antibody pair that most effectively captured and detected IL-6 was selected to develop a prototype IL-6 immunoassay. Calibrator and panel preparations using an internal recombinant IL-6 standard were compared to serum panels prepared with the IL-6 International Standard 89/548. Assay specificity and spike recovery were determined, and assay sensitivity was compared with the Roche EUA Elecsys IL-6 assay run on the cobas analyzer. RESULTS: Twenty-one antibodies in 441 antibody pairs were screened. The prototype IL-6 assay showed high sensitivity with an estimated limit of detection of 0.317 pg/mL and limit of quantitation of <1.27. Spike recovery was 90%-110% in serum and plasma. The internal recombinant human IL-6 calibrator showed excellent stability for 63 days at 2-8 °C. The prototype IL-6 immunoassay was specific for IL-6, exhibited no cross reactivity to related cytokines and interleukins, and was 10-fold more sensitive than the Elecsys IL-6 assay. CONCLUSIONS: The prototype ARCHITECT IL-6 automated immunoassay is a reliable and robust method for the quantitative determination of IL-6 in human serum and plasma.


Assuntos
Testes Imunológicos , Interleucina-6 , Animais , Anticorpos Monoclonais , Humanos , Imunoensaio/métodos , Fatores Imunológicos , Camundongos , Sensibilidade e Especificidade
14.
Methods Mol Biol ; 2442: 289-306, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35320532

RESUMO

Galectins are multifunctional glycan-binding proteins present in various tissues that participate in multiple physiological and pathological processes and are considered as not only biomarkers of human diseases but also molecular targets for treating cancer and inflammatory illnesses in many organs. In the glycobiology field, it is crucial to determine the pattern of galectin expression and location in cells and tissues. Confocal microscopy is a powerful imaging technology that represents a unique approach to investigate the expression and location of biomolecules in various tissues and cells. The confocal microscope acquires images of the specimen through the reflected or fluorescent light from the objective's focal plane, using laser light focused on a small spot inside the tissue or cell. This technique provides high-resolution and high-contrast images without artifacts generated by conventional microscopy and enables reconstruction of virtual tridimensional images by acquiring multiple sections from several focal planes, which makes it possible to obtain the precise spatial location of any cellular structure or molecule. Furthermore, confocal microscopy is a non-invasive tissue imaging strategy used in clinical practices. We describe herein the immunofluorescence confocal method for examining galectins in frozen tissue sections and mammalian cell culture.


Assuntos
Galectinas , Testes Imunológicos , Animais , Técnicas de Cultura de Células , Imunofluorescência , Humanos , Mamíferos , Microscopia Confocal/métodos
15.
J Hist Biol ; 55(1): 115-146, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35233686

RESUMO

This essay argues that the racialized geopolitics of the rhesus monkey trade conditioned the trajectory of tissue culture in polio research. Rhesus monkeys from north India were important experimental organisms in the American "war against polio" between the 1930s and 1950s. During this period, the National Foundation for Infantile Paralysis (NFIP) expended considerable effort to secure the nonhuman primate for researchers' changing experimental agendas. The NFIP drew on transnational networks to export hundreds of thousands of rhesus monkeys from colonial and later postcolonial India amid the geopolitical upheavals of World War II, the 1947 Partition, and the Cold War. In this essay, I trace how NFIP officials' anxieties about the geopolitics of the monkey trade configured research imperatives in the war against polio. I show how their anxieties more specifically shaped investment in tissue culture techniques as a possible means of obviating dependence on the market in monkeys. I do so by offering a genealogy of the contingent convergence between the use of rhesus monkeys and HeLa cell cultures in the 1954 Salk vaccine trial evaluation. Through this genealogy, I emphasize the geopolitical dimensions of the search for the "right" experimental organisms, tissues, and cells for the "job" of scientific research. The technical transformation of polio research, I argue, relied on the convergence of disparate, racialized biomedical economies.


Assuntos
Poliomielite , Animais , Células HeLa , Humanos , Testes Imunológicos , Macaca mulatta , Poliomielite/história , Poliomielite/prevenção & controle , Vacina Antipólio de Vírus Inativado , Estados Unidos
17.
Methods Mol Biol ; 2463: 221-233, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35344178

RESUMO

Cytotoxicity assays are important in vitro tools to measure the lysis of desired target cells via an effector immune cell of choice. Specific lysis of the target cells can be determined by labeling the target cells with a radioactive isotope or fluorescent molecule, co-incubating it with an effector cell, then measuring the release of the labeled molecule in the supernatant. Here, we describe and compare different cell cytotoxicity assays using a chromium-51 (51Cr) release and DELFIA EuTDA fluorescent assay using K562 as the target cells and peripheral blood mononuclear cell (PBMC) derived natural killer (NK) cells as the effector cells.


Assuntos
Células Matadoras Naturais , Leucócitos Mononucleares , Testes Imunológicos de Citotoxicidade , Citometria de Fluxo , Testes Imunológicos
18.
PLoS One ; 17(3): e0264929, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35271622

RESUMO

BACKGROUND: People experiencing homelessness who live in congregate shelters are at high risk of SARS-CoV2 transmission and severe COVID-19. Current screening and response protocols using rRT-PCR in homeless shelters are expensive, require specialized staff and have delays in returning results and implementing responses. METHODS: We piloted a program to offer frequent, rapid antigen-based tests (BinaxNOW) to residents and staff of congregate-living shelters in San Francisco, California, from January 15th to February 19th, 2021. We used the Reach-Effectiveness-Adoption-Implementation-Maintenance (RE-AIM) framework to evaluate the implementation. RESULTS: Reach: We offered testing at ten of twelve eligible shelters. Shelter residents and staff had variable participation across shelters; approximately half of eligible individuals tested at least once; few tested consistently during the study. Effectiveness: 2.2% of participants tested positive. We identified three outbreaks, but none exceeded 5 cases. All BinaxNOW-positive participants were isolated or left the shelters. Adoption: We offered testing to all eligible participants within weeks of the project's initiation. Implementation: Adaptations made to increase reach and improve consistency were promptly implemented. Maintenance: San Francisco Department of Public Health expanded and maintained testing with minimal support after the end of the pilot. CONCLUSION: Rapid and frequent antigen testing for SARS-CoV2 in homeless shelters is a viable alternative to rRT-PCR testing that can lead to immediate isolation of infectious individuals. Using the RE-AIM framework, we evaluated and adapted interventions to enable the expansion and maintenance of protocols.


Assuntos
Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , Pessoas em Situação de Rua/estatística & dados numéricos , COVID-19/imunologia , Teste para COVID-19/métodos , California , Surtos de Doenças/prevenção & controle , Habitação , Humanos , Testes Imunológicos/métodos , Programas de Rastreamento/métodos , Projetos Piloto , SARS-CoV-2/imunologia , SARS-CoV-2/patogenicidade , São Francisco
19.
Viruses ; 14(3)2022 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-35336875

RESUMO

Human transmission of SARS-CoV-2 and emergent variants of concern continue to occur globally, despite mass vaccination campaigns. Public health strategies to reduce virus spread should therefore rely, in part, on frequent screening with rapid, inexpensive, and sensitive tests. We evaluated two digitally integrated rapid tests and assessed their performance using stored nasal swab specimens collected from individuals with or without COVID-19. An isothermal amplification assay combined with a lateral flow test had a limit of detection of 10 RNA copies per reaction, and a positive percent agreement (PPA)/negative percent agreement (NPA) during the asymptomatic and symptomatic phases of 100%/100% and 95.83/100%, respectively. Comparatively, an antigen-based lateral flow test had a limit of detection of 30,000 copies and a PPA/NPA during the asymptomatic and symptomatic phases of 82.86%/98.68% and 91.67/100%, respectively. Both the isothermal amplification and antigen-based lateral flow tests had optimized detection of SARS-CoV-2 during the peak period of transmission; however, the antigen-based test had reduced sensitivity in clinical samples with qPCR Ct values greater than 29.8. Low-cost, high-throughput screening enabled by isothermal amplification or antigen-based techniques have value for outbreak control.


Assuntos
COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Humanos , Testes Imunológicos , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2/genética , Sensibilidade e Especificidade
20.
Viruses ; 14(3)2022 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-35337044

RESUMO

The developmental course of antibodies produced after a SARS-CoV-2 infection has been insufficiently investigated so far. Therefore, the aim of this study was to investigate the dynamics of SARS-CoV-2 antibody levels against the viral nucleocapsid- and spike-protein among Austrian blood donors as a representative group of a supposedly healthy population within the first year after a SARS-CoV-2 infection. The impact of age, sex, vaccination status, AB0-blood group and awareness about the infection was evaluated. Our study shows that the level of anti-N antibodies is declining, while anti-S antibody levels remain stable. Antibodies detected were functional in vitro. Age, sex and blood group do not influence antibody dynamics. However, blood group AB shows significantly lower antibody levels and in vitro functionality compared to other blood groups. Our data reveal that one out of five individuals was not aware of a previous SARS-CoV-2 infection and that the disease course neither affects the level of antibody production nor the in vitro functionality. We also found that 14% of participants show persisting COVID-19-related symptoms for up to nine months. Our results provide valuable insights into the dynamics of the immune response after a SARS-CoV-2 infection in a representative cohort of adult blood donors in Central Europe.


Assuntos
COVID-19 , SARS-CoV-2 , Adulto , Anticorpos Antivirais , Doadores de Sangue , Humanos , Testes Imunológicos
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