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1.
Emerg Microbes Infect ; 9(1): 2157-2168, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32940547

RESUMO

This multicenter, retrospective study included 346 serum samples from 74 patients with coronavirus disease 2019 (COVID-19) and 194 serum samples from non-COVID-19 patients to evaluate the performance of five anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody tests, i.e. two chemiluminescence immunoassays (CLIAs): Roche Elecsys® Anti-SARS-CoV-2 Test (Roche Test) and Abbott SARS-CoV-2 IgG (Abbott Test), and three lateral flow immunoassays (LFIAs): Wondfo SARS-CoV-2 Antibody Test (Wondfo Test), ASK COVID-19 IgG/IgM Rapid Test (ASK Test), and Dynamiker 2019-nCoV IgG/IgM Rapid Test (Dynamiker Test). We found high diagnostic sensitivities (%, 95% confidence interval [CI]) for the Roche Test (97.4%, 93.4-99.0%), Abbott Test (94.0%, 89.1-96.8%), Wondfo Test (91.4%, 85.8-94.9%), ASK Test (97.4%, 93.4-99.0%), and Dynamiker Test (90.1%, 84.3-94.0%) after >21 days of symptom onset. Meanwhile, the diagnostic specificity was 99.0% (95% CI, 96.3-99.7%) for the Roche Test, 97.9% (95% CI, 94.8-99.2%) for the Abbott Test, and 100.0% (95% CI, 98.1-100.0%) for the three LFIAs. Cross-reactivity was observed in sera containing anti-cytomegalovirus (CMV) IgG/IgM antibodies and autoantibodies. No difference was observed in the time to seroconversion detection of the five serological tests. Specimens from patients with COVID-19 pneumonia demonstrated a shorter seroconversion time and higher chemiluminescent signal than those without pneumonia. Our data suggested that understanding the dynamic antibody response after COVID-19 infection and performance characteristics of different serological test are crucial for the appropriate interpretation of serological test result for the diagnosis and risk assessment of patient with COVID-19 infection.


Assuntos
Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/imunologia , Imunoensaio/métodos , Medições Luminescentes/métodos , Pneumonia Viral/diagnóstico , Pneumonia Viral/imunologia , Adulto , Idoso , Anticorpos Antivirais/sangue , Betacoronavirus/genética , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Reações Cruzadas/imunologia , Feminino , Humanos , Imunoensaio/normas , Medições Luminescentes/normas , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , Reprodutibilidade dos Testes , Soroconversão , Testes Sorológicos , Índice de Gravidade de Doença , Taiwan/epidemiologia
2.
Nat Commun ; 11(1): 4192, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826886

RESUMO

Bioluminescence imaging has been widely used in life sciences and biomedical applications. However, conventional bioluminescence imaging usually operates in the visible region, which hampers the high-performance in vivo optical imaging due to the strong tissue absorption and scattering. To address this challenge, here we present bioluminescence probes (BPs) with emission in the second near infrared (NIR-II) region at 1029 nm by employing bioluminescence resonance energy transfer (BRET) and two-step fluorescence resonance energy transfer (FRET) with a specially designed cyanine dye FD-1029. The biocompatible NIR-II-BPs are successfully applied to vessels and lymphatics imaging in mice, which gives ~5 times higher signal-to-noise ratios and ~1.5 times higher spatial resolution than those obtained by NIR-II fluorescence imaging and conventional bioluminescence imaging. Their capability of multiplexed imaging is also well displayed. Taking advantage of the ATP-responding character, the NIR-II-BPs are able to recognize tumor metastasis with a high tumor-to-normal tissue ratio at 83.4.


Assuntos
Trifosfato de Adenosina/metabolismo , Medições Luminescentes/métodos , Metástase Neoplásica/diagnóstico por imagem , Imagem Óptica/métodos , Animais , Técnicas Biossensoriais , Linhagem Celular Tumoral , Feminino , Transferência Ressonante de Energia de Fluorescência/instrumentação , Transferência Ressonante de Energia de Fluorescência/métodos , Xenoenxertos , Humanos , Medições Luminescentes/instrumentação , Camundongos , Imagem Óptica/instrumentação , Neoplasias Ovarianas/diagnóstico por imagem
3.
J Clin Virol ; 130: 104572, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32769024

RESUMO

BACKGROUND: The emergence of SARS-CoV-2 has led to the development of serological assays that could aid in an understanding of the burden of COVID-19 disease. Many available tests lack rigorous evaluation and therefore results may be misleading. OBJECTIVES: The aim of this study was to assess the performance of a novel multiplexed immunoassay for the simultaneous detection of antibodies against SARS-CoV-2 trimeric spike (S), spike receptor binding domain (RBD), spike N terminal domain and nucleocapsid antigen and a novel pseudo-neutralisation assay. METHODS: A multiplexed solid-phase chemiluminescence assay (Meso Scale Discovery) was evaluated for the simultaneous detection of IgG binding to four SARS-CoV-2 antigens and the quantification of antibody-induced ACE-2 binding inhibition (pseudo-neutralisation assay). Sensitivity was evaluated with a total of 196 COVID-19 serum samples (169 confirmed PCR positive and 27 anti-nucleocapsid IgG positive) from individuals with mild symptomatic or asymptomatic disease. Specificity was evaluated with 194 control serum samples collected from adults prior to December 2019. RESULTS: The specificity and sensitivity of the binding IgG assay was highest for S protein with a specificity of 97.4 % and sensitivity of 96.2 % for samples taken 14 days and 97.9 % for samples taken 21 days following the onset of symptoms. IgG concentration to S and RBD correlated strongly with percentage inhibition measured by the pseudo-neutralisation assay. CONCLUSION: Excellent sensitivity for IgG detection was obtained over 14 days since onset of symptoms for three SARS-CoV-2 antigens (S, RBD and N) in this multiplexed assay which can also measure antibody functionality.


Assuntos
Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Infecções por Coronavirus/diagnóstico , Imunoensaio/métodos , Imunoglobulina G/sangue , Pneumonia Viral/diagnóstico , Adulto , Betacoronavirus , Técnicas de Laboratório Clínico , Infecções por Coronavirus/imunologia , Feminino , Humanos , Medições Luminescentes/métodos , Masculino , Pessoa de Meia-Idade , Proteínas do Nucleocapsídeo/imunologia , Pandemias , Pneumonia Viral/imunologia , Sensibilidade e Especificidade , Glicoproteína da Espícula de Coronavírus/imunologia
4.
Yakugaku Zasshi ; 140(8): 969-977, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32741870

RESUMO

We developed a method of video-rate bioluminescence imaging to visualize proteins secreted from living cells. A protein of interest was fused to Gaussia luciferase (GLase), and the luminescence signals of secreted GLase with coelenterazine (luciferin) were visualized at a video-rate of 30-500 ms/frame by using a water-cooled EM-CCD camera. We established a subclonal rat INS-1E cell line, named iGL cells, stably expressing the fusion protein of insulin and GLase (Insulin-GLase). By stimulation with high glucose, 3D-cultured iGL cells showed synchronized oscillatory secretion of insulin for over 1 h, as similarly observed in an isolated rat pancreatic islet. In 2D-cultured iGL cells, the luminescence images indicated that synchronized insulin secretion was localized in intercellular spaces between cells. Further, the relative amount of insulin secretion from iGL cells was easily determined with a luminometer, and we demonstrated that cell-cell interaction of beta cells is fundamental to increase glucose-stimulated insulin secretion by synchronization. Thus, iGL cells would be valuable for studying oscillatory insulin secretion and evaluating anti-diabetic drugs. Our bioluminescence imaging method with GLase could be generally used for investigating protein secretion in 2D and 3D cell culture systems.


Assuntos
Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Medições Luminescentes/métodos , Imagem Molecular/métodos , Animais , Comunicação Celular , Linhagem Celular , Células Cultivadas , Humanos , Imidazóis , Luciferases , Pirazinas , Ratos
5.
Nat Commun ; 11(1): 4052, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792510

RESUMO

Turn-on fluorescence imaging is routinely studied; however, turn-on chemiluminescence has been rarely explored for in vivo imaging. Herein, we report the design and validation of chemiluminescence probe ADLumin-1 as a turn-on probe for amyloid beta (Aß) species. Two-photon imaging indicates that ADLumin-1 can efficiently cross the blood-brain barrier and provides excellent contrast for Aß plaques and cerebral amyloid angiopathy. In vivo brain imaging shows that the chemiluminescence signal of ADLumin-1 from 5-month-old transgenic 5xFAD mice is 1.80-fold higher than that from the age-matched wild-type mice. Moreover, we demonstrate that it is feasible to further dually-amplify signal via chemiluminescence resonance energy transfer (DAS-CRET) using two non-conjugated smart probes (ADLumin-1 and CRANAD-3) in solutions, brain homogenates, and in vivo whole brain imaging. Our results show that DAS-CRET can provide a 2.25-fold margin between 5-month-old 5xFAD mice and wild type mice. We believe that our strategy could be extended to other aggregating-prone proteins.


Assuntos
Peptídeos beta-Amiloides/química , Luminescência , Animais , Medições Luminescentes/métodos , Camundongos , Imagem Molecular/métodos , Imagem Óptica/métodos , Agregados Proteicos
6.
Faraday Discuss ; 222(0): 8-9, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32490453

RESUMO

This Faraday Discussion volume is unique in the hundred plus year history of the Faraday Discussion series, being produced at a time of unprecedented circumstances worldwide and without the preceding Faraday Discussion conference having taken place.


Assuntos
Infecções por Coronavirus/diagnóstico , Medições Luminescentes/métodos , Nanoestruturas/química , Pneumonia Viral/diagnóstico , Silício/química , Humanos , Imagem Óptica , Pandemias , Porosidade
7.
J Virol Methods ; 283: 113919, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32554043

RESUMO

The purpose of this study was to investigate the feasibility of serological total antibody tests combined with RT-PCR for detection of SARS-COV-2. We conducted a retrospective study in which 375 patients were enrolled during the outbreak of SARS-COV-2 from 25th January to 16th March 2020. Patients were divided into a COVID-19 group (n = 141) and a control group (n = 234). Serum samples and throat swabs were collected from 375 patients for total antibody testing against SARS-COV-2 and RT-PCR analysis, respectively. The results indicated that diagnostic sensitivity and specificity were 95.7 % and 98.7 %, 92.2 % and 100 % by total antibody tests and RT-PCR, respectively. The sensitivity and specificity of total antibody tests combined with RT-PCR were 98.6 % and 98.7 %. The sensitivity of the combined method was significantly higher than RT-PCR (X2 = 5.16, P < 0.05), and similar to that of total antibody tests (X2 = 1.15, P> 0.05). This study supported the advantage of the combined method for detection of SARS-COV-2 with a high degree of sensitivity and specificity, as a useful tool for accurate diagnosis and timely treatment of suspected patients, epidemiological investigation, as well as monitoring ongoing outbreaks of infections with SARS-COV-2.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Testes Sorológicos/métodos , Adolescente , Adulto , Idoso , Anticorpos Antivirais/imunologia , Estudos de Casos e Controles , China , Infecções por Coronavirus/imunologia , Feminino , Humanos , Imunoensaio/métodos , Medições Luminescentes/métodos , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/imunologia , Estudos Retrospectivos , Sensibilidade e Especificidade , Adulto Jovem
8.
Chemistry ; 26(34): 7583-7588, 2020 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-32428322

RESUMO

Co single-atom catalysts (SACs) with good aqueous solubility and abundant labelling functional groups were prepared in Co/Fe bimetallic metal-organic frameworks by a facile solvothermal method without high-temperature calcination. In contrast to traditional chemiluminescence (CL) catalysts, Co SACs accelerated decomposition of H2 O2 to produce a large amount of singlet oxygen (1 O2 ) rather than superoxide (O2 .- ) and hydroxyl radical (OH. ). They were found to dramatically enhance the CL emission of the luminol-H2 O2 reaction by 1349 times, and, therefore, were employed as very sensitive signal probes for conducting CL immunoassay of cardiac troponin I. The detection limit of the target analyte was as low as 3.3 pg mL-1 . It is the first time that employment of SACs for boosting CL reactions has been validated. The Co SACs can also be employed to trace other biorecognition events with high sensitivity.


Assuntos
Radical Hidroxila/química , Luminol/química , Oxigênio Singlete/química , Catálise , Limite de Detecção , Luminescência , Medições Luminescentes/métodos , Estruturas Metalorgânicas , Superóxidos
9.
Clin Chem Lab Med ; 58(8): 1357-1364, 2020 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-32447328

RESUMO

Objectives Faced with the COVID-19 pandemic and its impact on the availability and quality of both therapeutic and diagnostic methods, the Belgian authorities have decided to launch a procedure for additional evaluation of the performance of serological tests offered for sale on the national territory. This has been proposed with a double aim: (1) an in-depth verification of the analytical and clinical performances presented by the manufacturer and (2) an economy of scale in terms of centralized validation for all the laboratories using the tests subject to evaluation. Methods A retrospective validation study was conducted including the serum of 125 patients in order to determine the analytical and clinical performances of the LIAISON®SARS-CoV-2 from DiaSorin® detecting anti-SARS-CoV-2 IgG and to compare its clinical performance with the enzyme-linked immunosorbent assay (ELISA) test from Euroimmun®, one of the first commercially available tests allowing the detection of anti-SARS-CoV-2 IgA and IgG. Results The performances of the LIAISON®SARS-CoV-2 satisfied all the acceptance criteria and provided "real world" analytical and clinical performances very close to the ones reported by the manufacturer in its insert kit. Comparison between the LIAISON®SARS-CoV-2 and the ELISA method did not reveal any difference between the two techniques in terms of sensitivities and specificities regarding the determination of the IgG. Conclusions This study reports the validation of the LIAISON®SARS-CoV-2 allowing to detect IgG antibodies specifically directed against SARS-CoV-2. The analytical and clinical performances are excellent, and the automation of the test offers important rates, ideal for absorbing an extension of testing.


Assuntos
Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Imunoensaio/métodos , Pneumonia Viral/diagnóstico , Infecções por Coronavirus/imunologia , Humanos , Limite de Detecção , Luminescência , Medições Luminescentes/métodos , Pandemias , Pneumonia Viral/imunologia , Reprodutibilidade dos Testes , Estudos Retrospectivos
10.
Nat Commun ; 11(1): 2668, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32472057

RESUMO

Electrochemiluminescence (ECL) is a powerful transduction technique with a leading role in the biosensing field due to its high sensitivity and low background signal. Although the intrinsic analytical strength of ECL depends critically on the overall efficiency of the mechanisms of its generation, studies aimed at enhancing the ECL signal have mostly focused on the investigation of materials, either luminophores or coreactants, while fundamental mechanistic studies are relatively scarce. Here, we discover an unexpected but highly efficient mechanistic path for ECL generation close to the electrode surface (signal enhancement, 128%) using an innovative combination of ECL imaging techniques and electrochemical mapping of radical generation. Our findings, which are also supported by quantum chemical calculations and spin trapping methods, led to the identification of a family of alternative branched amine coreactants, which raises the analytical strength of ECL well beyond that of present state-of-the-art immunoassays, thus creating potential ECL applications in ultrasensitive bioanalysis.


Assuntos
Biomarcadores/análise , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Eletroquímica/métodos , Medições Luminescentes/métodos , Técnicas de Química Analítica , Físico-Química/métodos , Luminescência
11.
Exp Parasitol ; 214: 107905, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32387050

RESUMO

Non-invasive small animal in vivo imaging is an essential tool in a broad variety of biomedical sciences and enables continuous monitoring of disease progression in order to develop and improve diagnostic, therapeutic and preventive measures. Imaging parasites non-invasively in live animals allows efficient parasite distribution evaluation in the host organism and objective evaluation of parasitic diseases' burden and progression in individual animals. The aim of this systematic review was to summarize recent trends in small animal in vivo imaging and compare and discuss imaging of single-cell and multicellular eukaryotic parasites. A literature survey was performed using Web of Science and PubMed databases in research articles published between 1990 and 2018. The inclusion criteria were using any imaging method to visualize a range of protozoan and helminth parasites in laboratory animals in vivo. A total of 92 studies met our inclusion criteria. Protozoans and helminths were imaged in 88% and 12% of 92 studies, respectively. The most common parasite genus studied was the protozoan Plasmodium followed by Trypanosoma and Leishmania. The most frequent imaging method was bioluminescence. Among the helminths, Schistosoma and Echinococcus were the most studied organisms. In vivo imaging is applicable in both protozoans and helminths. In helminths, however, the use of in vivo imaging methods is limited to some extent. Imaging parasites in small animal models is a powerful tool in preclinical research aiming to develop novel therapeutic and preventive strategies for parasitic diseases of interest both in human and veterinary medicine.


Assuntos
Medições Luminescentes/métodos , Doenças Parasitárias em Animais/diagnóstico por imagem , Coelhos/parasitologia , Roedores/parasitologia , Animais
12.
Clin Chim Acta ; 507: 164-166, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: covidwho-125378

RESUMO

Validation studies of serological antibody tests must be properly designed for clinical, epidemiological and Public Health objectives such as confirmation of suspected COVID-19 cases, certification of seroconversion after infection, and epidemiological surveillance. We evaluated the kinetics of IgM, IgA and IgG SARS-CoV-2 antibodies in COVID-19 patients with confirmed (rRT-PCR) infection. We found that the IgA response appears and grows early, peaks at week 3, and it is stronger and more persistent than the IgM response. Further longitudinal investigations of virus-specific antibodies functions and of their protective efficacy over time are needed.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/sangue , Infecções por Coronavirus/diagnóstico , Glicoproteínas/sangue , Imunoglobulina A/sangue , Pneumonia Viral/sangue , Pneumonia Viral/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Estudos Longitudinais , Medições Luminescentes/métodos , Masculino , Pessoa de Meia-Idade , Pandemias , Reação em Cadeia da Polimerase em Tempo Real/métodos , Adulto Jovem
13.
Clin Chem Lab Med ; 58(7): 1081-1088, 2020 06 25.
Artigo em Inglês | MEDLINE | ID: covidwho-72353

RESUMO

Background Coronavirus disease 2019, abbreviated to COVID-19, represents an emerging health threat worldwide as, after initial reports in China, it has continued to spread rapidly. The clinical spectrum of the disease varies from mild to severe acute respiratory distress syndrome (ARDS). Moreover, many patients can be asymptomatic, thus increasing the uncertainty of the diagnostic work-up. Laboratory tests play a pivotal role in the diagnosis and management of COVID-19, the current gold standard being real-time reverse transcription polymerase chain reaction (rRT-PCR) on respiratory tract specimens. However, the diagnostic accuracy of rRT-PCR depends on many pre-analytical and analytical variables. The measurement of specific COVID-19 antibodies (both IgG and IgM) should serve as an additional, non-invasive tool for disease detection and management. Methods The imprecision of the MAGLUMI™ 2000 Plus 2019-nCov IgM and IgG assays (Snibe, Shenzhen, China) was assessed by adopting the Clinical and Laboratory Standards Institute (CLSI) EP15-A3 protocol. Linearity of dilution and recovery was evaluated by means of mixes of high-level pools and low-level pools of serum samples. Immunoglobulin time kinetics were evaluated using a series of serum samples, repeatedly collected from COVID-19-positive patients at different times, from <5 days up to 26-30 days. Results Findings at the analytical validation of the assay carried out according to the CLSI EP15-A3 guideline demonstrated that imprecision and repeatability were acceptable (repeatability was <4% and <6% for IgM and IgG, respectively, whilst intermediate imprecision was <6%). In addition, results of dilution and recovery studies were satisfactory. The kinetics of COVID-19 antibodies confirmed previously reported findings, showing a rapid increase of both IgM and IgG after 6-7 days from the symptom onset. IgG had 100% sensitivity on day 12, whilst 88% was the higher positive rate achieved for IgM after the same time interval. Conclusions The findings of this study demonstrate the validity of the MAGLUMI 2000 Plus CLIA assay for the measurement of specific IgM and IgG in sera of COVID-19 patients, and for obtaining valuable data on the kinetics of both (IgM and IgG) COVID-19 antibodies. These data represent a pre-requisite for the appropriate utilization of specific antibodies for the diagnosis and management of COVID-19 patients.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Técnicas Imunoenzimáticas/métodos , Pneumonia Viral/imunologia , Anticorpos Antivirais/sangue , China , Técnicas de Laboratório Clínico/métodos , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Cinética , Medições Luminescentes/métodos , Pandemias , Reação em Cadeia da Polimerase em Tempo Real
14.
Clin Chim Acta ; 507: 164-166, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32343948

RESUMO

Validation studies of serological antibody tests must be properly designed for clinical, epidemiological and Public Health objectives such as confirmation of suspected COVID-19 cases, certification of seroconversion after infection, and epidemiological surveillance. We evaluated the kinetics of IgM, IgA and IgG SARS-CoV-2 antibodies in COVID-19 patients with confirmed (rRT-PCR) infection. We found that the IgA response appears and grows early, peaks at week 3, and it is stronger and more persistent than the IgM response. Further longitudinal investigations of virus-specific antibodies functions and of their protective efficacy over time are needed.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/sangue , Infecções por Coronavirus/diagnóstico , Glicoproteínas/sangue , Imunoglobulina A/sangue , Pneumonia Viral/sangue , Pneumonia Viral/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Estudos Longitudinais , Medições Luminescentes/métodos , Masculino , Pessoa de Meia-Idade , Pandemias , Reação em Cadeia da Polimerase em Tempo Real/métodos , Adulto Jovem
15.
Clin Chem Lab Med ; 58(7): 1081-1088, 2020 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-32301749

RESUMO

Background Coronavirus disease 2019, abbreviated to COVID-19, represents an emerging health threat worldwide as, after initial reports in China, it has continued to spread rapidly. The clinical spectrum of the disease varies from mild to severe acute respiratory distress syndrome (ARDS). Moreover, many patients can be asymptomatic, thus increasing the uncertainty of the diagnostic work-up. Laboratory tests play a pivotal role in the diagnosis and management of COVID-19, the current gold standard being real-time reverse transcription polymerase chain reaction (rRT-PCR) on respiratory tract specimens. However, the diagnostic accuracy of rRT-PCR depends on many pre-analytical and analytical variables. The measurement of specific COVID-19 antibodies (both IgG and IgM) should serve as an additional, non-invasive tool for disease detection and management. Methods The imprecision of the MAGLUMI™ 2000 Plus 2019-nCov IgM and IgG assays (Snibe, Shenzhen, China) was assessed by adopting the Clinical and Laboratory Standards Institute (CLSI) EP15-A3 protocol. Linearity of dilution and recovery was evaluated by means of mixes of high-level pools and low-level pools of serum samples. Immunoglobulin time kinetics were evaluated using a series of serum samples, repeatedly collected from COVID-19-positive patients at different times, from <5 days up to 26-30 days. Results Findings at the analytical validation of the assay carried out according to the CLSI EP15-A3 guideline demonstrated that imprecision and repeatability were acceptable (repeatability was <4% and <6% for IgM and IgG, respectively, whilst intermediate imprecision was <6%). In addition, results of dilution and recovery studies were satisfactory. The kinetics of COVID-19 antibodies confirmed previously reported findings, showing a rapid increase of both IgM and IgG after 6-7 days from the symptom onset. IgG had 100% sensitivity on day 12, whilst 88% was the higher positive rate achieved for IgM after the same time interval. Conclusions The findings of this study demonstrate the validity of the MAGLUMI 2000 Plus CLIA assay for the measurement of specific IgM and IgG in sera of COVID-19 patients, and for obtaining valuable data on the kinetics of both (IgM and IgG) COVID-19 antibodies. These data represent a pre-requisite for the appropriate utilization of specific antibodies for the diagnosis and management of COVID-19 patients.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Técnicas Imunoenzimáticas/métodos , Pneumonia Viral/imunologia , Anticorpos Antivirais/sangue , China , Técnicas de Laboratório Clínico/métodos , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Cinética , Medições Luminescentes/métodos , Pandemias , Reação em Cadeia da Polimerase em Tempo Real
16.
Ecotoxicol Environ Saf ; 196: 110527, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32278138

RESUMO

Assessment of eco-toxicant using bioluminescent bacterial assay is a widely used and globally accepted method. In this work, a new luminescent bacterium was isolated from squid (Loligo duvauceli) and identified as Photobacterium leiognathi strain AK-MIE using 16S rRNA, phylogeny analysis. The predicted optimum conditions by RSM were 2.76% (w/v) NaCl, 2.28% (w/v) peptone, 0.34% (w/v) yeast extract, and pH 6.83 with 541,211.80 RLU of luminescent production whereas the predicted optimum conditions by ANN were 2.21% (w/v) NaCl, 2.27% (w/v) peptone, 0.39% (w/v) yeast extract, and pH 6.94 which produced 541,986.20 RLU. The validation analysis of both RSM and ANN show 0.60% and 0.69% deviation from the predicted results indicating that both models provided good quality predictions with ANN showing a superior data fitting capability for non-linear regression analysis. Toxicity tests show strain AK-MIE was sensitive to mercury (concentration causing 50% inhibition or IC50 of 0.00978 mgL-1), followed by cadmium (IC50 of 0.5288 mgL-1), copper IC50 of (0.8117 mgL-1), silver (IC50 of 1.109 mgL-1), and lead (IC50 of 10.71 mgL-1) which are more sensitive than previously isolated luminescent bacteria, suggesting that strain AK-MIE has the potential to be used in toxicity assessment of heavy metals in the environment. Based on the field trial results, several sediment samples from industrial areas in Bangi, Selangor managed to inhibit the bioluminescence of strain AK-MIE. Validation method carried out using ICP-MS proved the presence of several toxic heavy metal elements.


Assuntos
Monitoramento Ambiental/métodos , Poluentes Ambientais/análise , Substâncias Perigosas/análise , Medições Luminescentes/métodos , Metais Pesados/análise , Photobacterium/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Substâncias Perigosas/toxicidade , Concentração Inibidora 50 , Metais Pesados/toxicidade , Filogenia , RNA Ribossômico 16S , Testes de Toxicidade
17.
PLoS One ; 15(4): e0231634, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32298350

RESUMO

The WST-1 assay is the most common test to assess the in vitro cytotoxicity of chemicals. Tetrazolium-based assays can, however, be affected by the interference of tested chemicals, including carbon nanotubes or Mg particles. Here, we report a new interference of Mn materials with the WST-1 assay. Endothelial cells exposed to Mn particles (Mn alone or Fe-Mn alloy from 50 to 1600 µg/ml) were severely damaged according to the WST-1 assay, but not the ATP content assay. Subsequent experiments revealed that Mn particles interfere with the reduction of the tetrazolium salt to formazan. Therefore, the WST-1 assay is not suitable to evaluate the in vitro cytotoxicity of Mn-containing materials, and luminescence-based assays such as CellTiter-Glo® appear more appropriate.


Assuntos
Citotoxinas/toxicidade , Células Endoteliais/efeitos dos fármacos , Manganês/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Medições Luminescentes/métodos , Oxirredução , Sais de Tetrazólio/química , Testes de Toxicidade/métodos
18.
PLoS One ; 15(3): e0229672, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32214362

RESUMO

More than 170 types of human papilloma viruses (HPV) exist with many causing proliferative diseases linked to malignancy in indications such as cervical cancer and head and neck squamous cell carcinoma. Characterization of antibody levels toward HPV serology is challenging due to complex biology of oncoproteins, pre-existing titers to multiple HPV types, cross-reactivity, and low affinity, polyclonal responses. Using multiplex technology from MSD, we have developed an assay that simultaneously characterizes antibodies against E6 and E7 oncoproteins of HPV16 and 18, the primary drivers of HPV-associated oncogenesis. We fusion tagged our E6 and E7 proteins with MBP via two-step purification, spot-printed an optimized concentration of protein into wells of MSD 96-well plates, and assayed various cynomolgus monkey, human and HPV+ cervical cancer patient serum to validate the assay. The dynamic range of the assay covered 4-orders of magnitude and antibodies were detected in serum at a dilution up to 100,000-fold. The assay was very precise (n = 5 assay runs) with median CV of human serum samples ~ 5.3% and inter-run variability of 11.4%. The multiplex serology method has strong cross-reactivity between E6 oncoproteins from human serum samples as HPV18 E6 antigens neutralized 5 of 6 serum samples as strongly as HPV16 E6. Moderate concordance (Spearman's Rank = 0.775) was found between antibody responses against HPV16 E7 in the multiplex assay compared to standard ELISA serology methods. These results demonstrate the development of a high-throughput, multi-plex assay that requires lower sample quantity input with greater dynamic range to detect type-specific anti-HPV concentrations to E6 and E7 oncoproteins of HPV16 and 18.


Assuntos
Anticorpos Antivirais/sangue , Papillomavirus Humano 16/imunologia , Papillomavirus Humano 18/imunologia , Imunoensaio/métodos , Imunoglobulina G/sangue , Animais , Especificidade de Anticorpos , Reações Cruzadas , Proteínas de Ligação a DNA/imunologia , Técnicas Eletroquímicas , Ensaio de Imunoadsorção Enzimática , Feminino , Ensaios de Triagem em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/estatística & dados numéricos , Humanos , Imunoensaio/estatística & dados numéricos , Limite de Detecção , Medições Luminescentes/métodos , Medições Luminescentes/estatística & dados numéricos , Macaca fascicularis , Proteínas Oncogênicas Virais/imunologia , Proteínas E7 de Papillomavirus/imunologia , Proteínas Repressoras/imunologia , Neoplasias do Colo do Útero/imunologia , Neoplasias do Colo do Útero/virologia
19.
Acta Trop ; 206: 105444, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32173317

RESUMO

New drugs for the treatment of human leishmaniasis are urgently needed, considering the limitations of current available options. However, pre-clinical evaluation of drug candidates for leishmaniasis is challenging. The use of luciferase-expressing parasites for parasite load detection is a potentially powerful tool to accelerate the drug discovery process. We have previously described the use of Leishmania amazonensis mutants expressing firefly luciferase (Luc2) for drug testing. Here, we describe three new mutant L. amazonensis lines that express different variants of luciferases: NanoLuc, NanoLuc-PEST and RedLuc. These mutants were evaluated in drug screening protocols. NanoLuc-parasites, in spite of high bioluminescence intensity in vitro, were shown to be inadequate in discriminating between live and dead parasites. Bioluminescence detection from intracellular amastigotes expressing NanoLuc-PEST, RedLuc or Luc2 proved more reliable than microscopy to determine parasite killing. Increased sensitivity was observed in vivo with RedLuc-expressing parasites as compared to NanoLuc-expressing L. amazonensis. Our data indicates that NanoLuc is not suitable for in vivo parasite burden determination. Additionally, RedLuc and the conventional luciferase Luc2 demonstrated equivalent sensitivity in an in vivo model of cutaneous leishmaniasis.


Assuntos
Leishmaniose Cutânea/tratamento farmacológico , Luciferases/genética , Medições Luminescentes/métodos , Animais , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Leishmania mexicana/genética , Camundongos , Camundongos Endogâmicos BALB C
20.
Nat Commun ; 11(1): 1250, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32144257

RESUMO

Currently, there are no non-invasive tools to accurately diagnose wound and surgical site infections before they become systemic or cause significant anatomical damage. Fluorescence and photoacoustic imaging are cost-effective imaging modalities that can be used to noninvasively diagnose bacterial infections when paired with a molecularly targeted infection imaging agent. Here, we develop a fluorescent derivative of maltotriose (Cy7-1-maltotriose), which is shown to be taken up in a variety of gram-positive and gram-negative bacterial strains in vitro. In vivo fluorescence and photoacoustic imaging studies highlight the ability of this probe to detect infection, assess infection burden, and visualize the effectiveness of antibiotic treatment in E. coli-induced myositis and a clinically relevant S. aureus wound infection murine model. In addition, we show that maltotriose is an ideal scaffold for infection imaging agents encompassing better pharmacokinetic properties and in vivo stability than other maltodextrins (e.g. maltohexose).


Assuntos
Corantes Fluorescentes/administração & dosagem , Imagem Molecular/métodos , Miosite/diagnóstico por imagem , Infecção da Ferida Cirúrgica/diagnóstico por imagem , Trissacarídeos/administração & dosagem , Animais , Carbocianinas/administração & dosagem , Carbocianinas/química , Modelos Animais de Doenças , Estabilidade de Medicamentos , Escherichia coli/isolamento & purificação , Escherichia coli/metabolismo , Feminino , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Humanos , Injeções Intravenosas , Medições Luminescentes/métodos , Camundongos , Microscopia de Fluorescência/métodos , Sondas Moleculares/administração & dosagem , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Miosite/microbiologia , Técnicas Fotoacústicas/métodos , Ratos , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/metabolismo , Infecção da Ferida Cirúrgica/microbiologia , Trissacarídeos/química , Trissacarídeos/metabolismo
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