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1.
Food Chem ; 366: 130594, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34303207

RESUMO

In this work, a single-emission, dual-enzyme immunofluorometric magnetosensor was fabricated to simultaneously detect three illegal colorants in chili seasoning. Specifically, two enzymatic reactions catalyzed by horse radish peroxidase-labeled Rhodamine (RhB) antibody and glucose oxidase-labeled Sudan dyes (SuDs) antibody were performed within a functional microfluidic chip, leading to production of strongly fluorescent Resorufin. In addition, a compact analyzer assisted by a smartphone was developed to quantify signals. Compared with the available multiplex optical biosensors, this work demonstrated four superiorities: 1) Simple optical structure. Only single wavelength excitation/emission module was needed; 2) High multiplexing capacity through spatial resolution and signal resolution; 3) Precise determination by discriminant analysis; 4) Easy-operated and high-throughput parallel detection on 16-channel chips. Ultralow detection limits for RhB (0.0072 ng/mL), Sudan I (0.0040 ng/mL) and Sudan II (0.0260 ng/mL) were obtained by this magnetosensor, which opens a new approach in field detection of multiplex illegal dyes in food system.


Assuntos
Corantes , Fluorimunoensaio
2.
Anal Chim Acta ; 1179: 338820, 2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34535251

RESUMO

The number of CD8+ T lymphocytes (CD8 cells) in peripheral blood can directly reflect the immune status of the body and is widely used for auxiliary diagnosis and prognostic evaluation of diseases. There is an urgent need to develop a simple CD8 cell-counting platform to meet clinical needs. Our group designed a paper-based cell-counting method based on a blocking competition strategy. In addition, we developed a time-resolved fluorescence-blocking competitive lateral flow immunoassay (TRF-BCLFIA) for point-of-care CD8 cell counting that functions by measuring europium nanoparticle (EuNP)-labeled CD8 antibody probes that are not captured by CD8 cells, and we indirectly calculated the concentration of CD8 cells in samples. Within 30 min, four operation steps can provide an accurate CD8 cell count for a 75-µL whole-blood sample, and this approach can be implemented on a handheld device. The TRF-BCLFIA reliably quantified CD8 cells in whole-blood samples, in which the assay exhibited a linear correlation (R2 = 0.989) readout for CD8 cell concentrations ranging from 137 to 821 cells/µL. To validate this approach, our newly developed CD8 cell-counting tool was used to assess 33 tumor patient blood samples. The results showed a high consistency with a flow cytometry-based absolute count. This analysis approach is a promising alternative for the costly standard flow cytometry-based tools for CD8 cell counting in tumor patients in community clinics, small hospitals, and low medical resource regions. This technology would deliver simple diagnostics to patients anywhere in the world, regardless of geography or socioeconomic status.


Assuntos
Európio , Nanopartículas Metálicas , Linfócitos T CD8-Positivos , Citometria de Fluxo , Fluorimunoensaio , Humanos
3.
Molecules ; 26(14)2021 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-34299518

RESUMO

To monitor the illegal used of furaltadone, a highly sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and fluorescence-linked immunosorbent assay (FLISA) based on a monoclonal antibody (mAb) were developed for the detection of 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ), the major metabolite of furaltadone in animal tissues. The highly specific mAb, which was very sensitive to a nitrophenyl derivative of AMOZ (2-NP-AMOZ) with IC50 values of 0.11 and 0.09 ng/mL for ic-ELISA and FLISA, respectively, was selected for the development of immunoassays. For both the ic-ELISA and FLISA for AMOZ-spiked experiments, acceptable recovery rates of 81.1-105.3% and coefficients of variation of 4.7-9.8% were obtained. In addition, results from both ic-ELISA and FLISA methods for spiked samples' data showed excellent correlation coefficients ranging from 0.9652 to 0.9927. Meanwhile, the proposed ic-ELISA and FLISA for thirty spiked samples were confirmed by standard LC-MS/MS with high correlation coefficients of 0.9911 and 0.9921, respectively. These results suggest that the developed ic-ELISA and FLISA are valid and cost-effective tools for high-throughput monitoring methods for AMOZ residues in animal tissues.


Assuntos
Anticorpos Monoclonais/imunologia , Morfolinos/análise , Morfolinos/imunologia , Animais , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Fluorimunoensaio/métodos , Contaminação de Alimentos/análise , Imunoadsorventes/química , Camundongos Endogâmicos BALB C , Modelos Moleculares
4.
Clin Biochem ; 96: 49-55, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34252448

RESUMO

BACKGROUND: The renal biopsy is an accurate and reliable gold standard for membranous nephropathy (MN) diagnosis. However, it is an invasive procedure involving the risk of hemorrhage or infection. Thus, an alternative approach that can facilitate the effective diagnosis and treatment monitoring of idiopathic membranous nephropathy (IMN) is urgently needed. METHODS: We established a dual-labeled time-resolved fluoroimmunoassay (TRFIA) to simultaneously detect phospholipase A2 receptor (PLA2R)-IgG4 and PLA2R-IgG antibodies. Utilizing this assay, we determined the ratio of autoantibodies in the serum of patients with different kidney diseases and normal controls. RESULTS: The sensitivity of TRFIA for detecting anti-PLA2R-IgG and anti-PLA2R-IgG4 was 0.12 µg/mL and 0.001 µg/mL, respectively. Human IgA did not interfere with the assay. Compared to anti-PLA2R-IgG alone, the positive rate of IMN could be increased from 86.5 % to 91.7 % through the combined use of anti-PLA2R-IgG4 and the PLA2R-IgG4/IgG ratio. In contrast, the false-positive rates for the detection of IgA nephropathy, lupus nephropathy, diabetic nephropathy, and minimal change nephropathy decreased from 25 to 50 % to 0 %. CONCLUSIONS: The dual-labeled PLA2R-IgG4/IgG-TRFIA for simultaneous detection of anti-PLA2R-IgG4 and anti-PLA2R-IgG will contribute to improved accuracy of IMN diagnosis.


Assuntos
Autoanticorpos/sangue , Glomerulonefrite Membranosa/sangue , Imunoglobulina G/sangue , Receptores da Fosfolipase A2/sangue , Fluorimunoensaio , Glomerulonefrite Membranosa/diagnóstico , Humanos , Sensibilidade e Especificidade
5.
Methods Mol Biol ; 2341: 89-94, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34264464

RESUMO

Oxygen consumption is a fundamental characteristic of staphylococcal physiology reflecting the energy and metabolic state of the bacterial cell. During aerobic growth, oxygen consumption rates (OCR) depend on nutrient availability and vary at different growth stages. The measurement of oxygen consumption rates provides a versatile tool to characterize the impact of various mutations, environmental cues, and antibiotics on bacterial growth and fitness. In this chapter, we describe a MitoXpress® Xtra-based oxygen consumption assay for fast and reliable determination of respiration rates in Staphylococcus aureus. This highly reproducible and simple method requires a minimal set of reagents and allows rapid screening of multiple samples through real-time determination of the OCR with an oxygen-sensing probe and fluorescence plate reader.


Assuntos
Antibacterianos/farmacologia , Oxigênio/análise , Staphylococcus aureus/crescimento & desenvolvimento , Técnicas Biossensoriais , Fluorimunoensaio , Aptidão Genética , Mutação , Consumo de Oxigênio , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética
6.
Anal Methods ; 13(27): 3017-3023, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34164636

RESUMO

BACKGROUND: the level of serum antibodies against the M-type phospholipase A2 receptor (anti-PLA2R-IgG) is closely related to the disease activity of idiopathic membranous nephropathy (IMN). Therefore, the establishment of a sensitive and rapid method for detecting anti-PLA2R-IgG will be beneficial for the differential diagnosis of IMN. METHODS: magnetic microspheres coupled with the PLA2R antigen were used to capture anti-PLA2R-IgG in serum samples, and europium-labeled goat anti-human IgG antibodies were used for tracking. An anti-PLA2R-IgG-time-resolved fluoroimmunoassay (TRFIA) based on magnetic microspheres using an indirect method was established and analyzed. Various indicators of this method were evaluated. RESULTS: the sensitivity of the anti-PLA2R-IgG-TRFIA based on magnetic microspheres was 0.51 RU mL-1, and the linear detection range was 0.51-1000 RU mL-1. The average intra- and inter-assay coefficients of variation (CVs) were 3.62% and 4.45%, respectively, and the average recovery was 95.60%. No cross-reactivity with IgA was observed. The median (interquartile range) concentration of anti-PLA2R-IgG in patients with IMN was 40.37 RU mL-1 (11.33 to 83.05 RU mL-1). The cut-off values of the anti-PLA2R-IgG concentration for healthy volunteers and those with other kidney diseases were determined to be 8.06 RU mL-1 and 13.23 RU mL-1, respectively. Additionally, the positive rates of anti-PLA2R-IgG in patients with IMN corresponding to the above cut-off values were 91.07% and 71.32%, respectively. The correlation coefficient between the magnetic microsphere-based anti-PLA2R-TRFIA and the PLA2R-ELISA kit for detecting anti-PLA2R-IgG was 0.944. CONCLUSION: a highly sensitive and rapid magnetic microsphere-based anti-PLA2R-IgG-TRFIA was successfully established to detect the concentrations of anti-PLA2R-IgG in the sera of patients with IMN.


Assuntos
Autoanticorpos , Receptores da Fosfolipase A2 , Fluorimunoensaio , Humanos , Fenômenos Magnéticos , Microesferas
8.
Ann Clin Biochem ; 58(5): 505-519, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34006121

RESUMO

BACKGROUND: Retinol-binding protein4 (RBP) assays using polyclonal antibodies (pRBP) have major problems of non-linearity of dilution and a very small useable dynamic range. Our objective was to develop a specific assay with a wider dynamic range to detect tubular proteinuria. METHODS: mRBP (monoclonal capture and second antibody with colorimetric detection) and fluoroimmunoassays for RBP (fRBP) (polyclonal capture and monoclonal second antibody with fluorescence detection) were developed and compared with pRBP. Four hundred and eighty-eight patient samples were collected; 290 samples were analysed by mRBP and 198 samples with fRBP and compared with pRBP. RESULTS: mRBP assay has the advantages of better linearity on dilution and wider analytical range over pRBP. It is limited by poor signal in the patients with albuminuria and glomerular proteinuria and inferior discrimination between patient groups. fRBP had an intra-assay and inter-assay CV of <6% and <8%, respectively, and analytical range was 2.3-599 µg/L. fRBP was linear on dilution within the analytical range. Correlation (r) was 0.8722 (95% CI 0.7621 to 0.9333, P< 0.0001); Mann-Whitney test revealed no significant difference (U = 18,877, n = 198, P = 0.5244) asserting that the medians of the two samples were identical. Bland-Altman test between pRBP and fRBP showed a mean negative bias of 16.43 (CI -994 to 1027) µg/mmol. CONCLUSIONS: The combination assay with fluorescence detection (fRBP) proved more discriminatory than a purely monoclonal system especially in patients with significant proteinuria and has advantages of better linearity on dilution and wider analytical range than the existing pRBP assay and compared extremely well with pRBP.


Assuntos
Albuminúria/urina , Anticorpos Monoclonais/química , Nefropatias/urina , Proteínas Plasmáticas de Ligação ao Retinol/urina , Adulto , Idoso , Ensaio de Imunoadsorção Enzimática , Feminino , Fluorimunoensaio , Humanos , Masculino , Pessoa de Meia-Idade
9.
Methods Mol Biol ; 2227: 43-49, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33847929

RESUMO

Detection of complement activation products can be carried out in a number of ways, and different methods are used in different laboratories. No international standard for measuring complement activation in the clinical setting has been agreed upon.Here we describe a modified assay for measuring C3dg. The assay is simple, inexpensive and stable. The estimation of C3dg directly reflects complement turnover independently of activation pathway.


Assuntos
Precipitação Química , Complemento C3b/análise , Imunofluorescência/métodos , Fragmentos de Peptídeos/análise , Animais , Biomarcadores/análise , Biomarcadores/sangue , Eletroforese das Proteínas Sanguíneas , Ativação do Complemento/fisiologia , Complemento C3b/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/métodos , Fluorimunoensaio/métodos , Humanos , Imunoeletroforese/métodos , Inflamação/sangue , Inflamação/diagnóstico , Mediadores da Inflamação/análise , Mediadores da Inflamação/sangue , Fragmentos de Peptídeos/isolamento & purificação , Polietilenoglicóis/química , Coelhos
10.
J Pharm Biomed Anal ; 200: 114071, 2021 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-33866295

RESUMO

Brucellosis is a worldwide infectious zoonotic disease, posing severe threats to human health and social-economic development. By comparing with time-consuming, low sensitive and non-quantitative conventional serological methods, herein, protein G (prG) coupled with europium nanospheres (EuNPs) (detection probe) and highly purified Brucella lipopolysaccharide (LPS) (capture antigen) were used to develop a novel time-resolved fluorescence lateral flow immunoassay (TF-LFIA) for detecting anti-Brucella IgG antibody in human plasmas. The entire testing took 15 min. With a satisfactory purity, the purified LPS weakly cross-reacted with Y. enterocolitica O9 diagnostic antibody; however, none reacted with sera from patients with other Gram-negative bacterial infections. Following coefficient of determination (R2 = 0.9961), 0.3 IU/mL was reported as the limit of detection (LOD), much lower than those of Serological Agglutination Test (SAT), Rose-Bengal Plate Agglutination Test (RBPT) and colloidal gold LFIA (CG-LFIA). Intra-day and inter-day precisions (CV, coefficient variation) of TF-LFIA varied less than 8% or 12 %, while intra-day and inter-day accuracies were 94-106 % or 93-107 %, respectively. The correlation coefficient (R2) of TF-LFIA measurement to the different concentrations of spiked Brucella antibody was 0.9967, suggesting TF-LFIA had high reliability and reproducibility. TF-LFIA was demonstrated for 100 % specificity, 98.57 % sensitivity and 99.63 % accuracy in detection of Brucella antibody from clinical samples, respectively, significantly higher compared to SAT and RBPT. In conclusion, the established TF-LFIA is a simple, rapid and quantitative immunoassay for early diagnosis or epidemiological surveillance of Brucella infection in humans.


Assuntos
Brucelose , Anticorpos Antibacterianos , Brucelose/diagnóstico , Fluorimunoensaio , Humanos , Imunoensaio , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Testes Sorológicos
11.
J Clin Lab Anal ; 35(5): e23758, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33720453

RESUMO

BACKGROUND: To establish a time-resolved fluorescence immunoassay of interleukin (IL)-18 (IL-18-TRFIA) and detect its concentration in different liver disease serum samples. METHODS: The IL-18 coating antibody and the Eu3+ -labeled detection antibody were used for the IL-18-TRFIA to detect serum IL-18 concentration in patients with liver cancer, hepatitis B, hepatitis C, autoimmune hepatitis, fatty liver disease, and healthy controls. The double-antibody sandwich method was used and methodological evaluation was performed. RESULTS: The average intra- and inter-assay coefficient of variation for IL-18-TRFIA was 4.80% and 5.90%, respectively. The average recovery rate was 106.19 ± 3.44%. The sensitivity (10.96 pg/mL) was higher than that obtained using the ELISA method (62.5 pg/mL). The detection range was 10.96-1000 pg/mL. IL-6 and galectin-3 did not cross-react with IL-18-TRFIA. The serum concentration of IL-18 was (776.99; 653.48-952.39 pg/mL) in hepatitis C, (911; 775.55-1130.03 pg/mL) in fatty liver, (1048.88; 730.04-1185.10 pg/mL) in liver cancer, and (949.12; 723.70-1160.28 pg/mL) in hepatitis B. Moreover, IL-18 serum levels were significantly higher in patients than the healthy controls (483.09; 402.52-599.70/mL) (p < 0.0001). Autoimmune hepatitis with a serum IL-18 concentration of 571.62; 502.47-730.31 pg/mL was not significantly different from the healthy controls (p > 0.05). CONCLUSION: We established a highly sensitive IL-18-TRFIA method that successfully detected serum IL-18 concentrations in different liver diseases. Furthermore, IL-18 serum concentration was higher in patients with liver cancer, hepatitis C, hepatitis B, and fatty liver disease compared to healthy controls.


Assuntos
Fluorimunoensaio/métodos , Interleucina-18/sangue , Hepatopatias/sangue , Estudos de Casos e Controles , Fluorescência , Humanos , Limite de Detecção , Neoplasias Hepáticas/sangue , Curva ROC , Padrões de Referência , Sensibilidade e Especificidade , Fatores de Tempo
12.
ACS Appl Mater Interfaces ; 13(9): 11414-11423, 2021 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-33620204

RESUMO

Plasmon-enhanced fluorescence (PEF) is a simple and highly effective approach for improving the signal-to-noise ratio and sensitivity of various fluorescence-based bioanalytical techniques. Here, we show that the fluorescence enhancement efficacy of gold nanorods (AuNRs), which are widely employed for PEF, is highly dependent on their absolute dimensions (i.e., length and diameter). Notably, an increase in the dimensions (length × diameter) of the AuNRs from 46 × 14 to 120 × 38 nm2 while holding the aspect ratio constant leads to nearly 300% improvement in fluorescence enhancement efficiency. Further increase in the AuNR size leads to a decrease of the fluorescence enhancement efficiency. Through finite-difference time-domain (FDTD) simulation, we reveal that the size-dependent fluorescence enhancement efficiency of AuNR stems from the size-dependent electromagnetic field around the plasmonic nanostructures. AuNRs with optimal dimensions resulted in a nearly 120-fold enhancement in the ensemble fluorescence emission from molecular fluorophores bound to the surface. These plasmonic nanostructures with optimal dimensions also resulted in a nearly 30-fold improvement in the limit of detection of human interleukin-6 (IL-6) compared to AuNRs with smaller size, which are routinely employed in PEF.


Assuntos
Corantes Fluorescentes/química , Interleucina-6/análise , Nanotubos/química , Anticorpos Imobilizados/imunologia , Fluorescência , Fluorimunoensaio/métodos , Ouro/química , Humanos , Interleucina-6/imunologia , Tamanho da Partícula , Ressonância de Plasmônio de Superfície
13.
Mikrochim Acta ; 188(1): 11, 2021 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-33389211

RESUMO

An ultrasensitive and rapid fluorescent immunoassay based on a broad-spectrum monoclonal antibody (mAb) was developed to detect pyrrolizidine alkaloids (PAs) in honey samples. First, Discovery Studio software was used to analyze and predict the target hapten, and retrorsine (RTS) was selected to react with succinic anhydride (HS) for hapten synthesization. A sensitive and broad-spectrum monoclonal antibody (mAb 13E1) was obtained for nine PAs. Then, fluorescent gold nanoclusters (AuNCs) were conjugated with mAb as a label probe and used in establishing a qualitative and quantitative lateral flow immunoassay (AuNCs-LFIA) for the determination of four PAs (retrorsine, platyphylline, senecionine, integerrimine) in honey within 14 min. The limits of detection (LOD) were 0.083 µg/kg. The recovery in spiked honey samples were 87.98-119.57%, with coefficients of variation of ≤ 11.5%. A total of 45 commercial import honey samples from nine different countries were tested through AuNCs-LFIA and UPLC-MS/MS method, and satisfactory consistency (R2 = 0.995) was obtained. The rates of positive samples were 55.56% (25/45), and the average concentrations of four PAs were 3.24-46.47 µg/kg. This ultrasensitive multi-PA method provides an alternative analytical tool for evaluating the human risk posed by the consumption of PA-contaminated honey.


Assuntos
Corantes Fluorescentes/química , Fluorimunoensaio/métodos , Nanopartículas Metálicas/química , Alcaloides de Pirrolizidina/análise , Animais , Anticorpos Monoclonais Murinos/imunologia , Feminino , Contaminação de Alimentos/análise , Ouro/química , Haptenos/imunologia , Mel/análise , Limite de Detecção , Camundongos Endogâmicos BALB C , Alcaloides de Pirrolizidina/imunologia , Software
14.
Methods Mol Biol ; 2261: 247-262, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33420994

RESUMO

The comprehensive analysis of serum cytokine levels can be challenging due to low sample volumes and time consuming when using single-target methods like enzyme-linked immunosorbent assay (ELISA). Bead-based detection systems allow the simultaneous detection of multiple analytes using minimal sample volumes. Here we describe the use of a multiplex cytokine, chemokine, and growth factor assay for mouse cytokines in a 96-well format. This assay is based on antibody-coupled fluorescent magnetic beads combined with biotinylated secondary detection antibody followed by fluorescent-tagged streptavidin in a sandwich-like composition. Final assay readout provides the concentrations of 23 different cytokines, chemokines, and growth factors in up to 76 samples.


Assuntos
Quimiocinas/sangue , Citocinas/sangue , Endotoxemia/sangue , Técnica Indireta de Fluorescência para Anticorpo , Fluorimunoensaio , Peptídeos e Proteínas de Sinalização Intercelular/sangue , Proteômica , Animais , Biotinilação , Modelos Animais de Doenças , Endotoxemia/induzido quimicamente , Endotoxemia/genética , Lipopolissacarídeos , Camundongos Knockout , Receptores de Glucocorticoides/deficiência , Receptores de Glucocorticoides/genética
15.
Methods Mol Biol ; 2261: 263-276, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33420995

RESUMO

Multiplex immunoassays enable the measurement of multiple proteins in a small volume of sample in a single well, enhancing discovery and profiling of multiple biomarkers when compared to singleplex immunoassays. In order to ensure optimal results are generated, it is important to know what critical actions and assay steps can adversely affect results. This chapter covers best practices for sample collection and storage, to important considerations during the assay run, and ways to ensure optimal data generation and efficient data analysis. When these practices are applied, the potential to generate actionable and quality results accelerates the research and discovery process.


Assuntos
Fluorimunoensaio , Proteínas/análise , Proteômica , Métodos Analíticos de Preparação de Amostras , Animais , Biotinilação , Humanos , Fluxo de Trabalho
16.
J Clin Microbiol ; 59(2)2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33139422

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the challenges inherent to the serological detection of a novel pathogen such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Serological tests can be used diagnostically and for surveillance, but their usefulness depends on their throughput, sensitivity, and specificity. Here, we describe a multiplex fluorescent microsphere-based assay, 3Flex, that can detect antibodies to three major SARS-CoV-2 antigens-spike (S) protein, the spike ACE2 receptor-binding domain (RBD), and nucleocapsid (NP). Specificity was assessed using 213 prepandemic samples. Sensitivity was measured and compared to that of the Abbott Architect SARS-CoV-2 IgG assay using serum samples from 125 unique patients equally binned (n = 25) into 5 time intervals (≤5, 6 to 10, 11 to 15, 16 to 20, and ≥21 days from symptom onset). With samples obtained at ≤5 days from symptom onset, the 3Flex assay was more sensitive (48.0% versus 32.0%), but the two assays performed comparably using serum obtained ≥21 days from symptom onset. A larger collection (n = 534) of discarded sera was profiled from patients (n = 140) whose COVID-19 course was characterized through chart review. This revealed the relative rise, peak (S, 23.8; RBD, 23.6; NP, 16.7 [in days from symptom onset]), and decline of the antibody response. Considerable interperson variation was observed with a subset of extensively sampled intensive care unit (ICU) patients. Using soluble ACE2, inhibition of antibody binding was demonstrated for S and RBD, and not for NP. Taking the data together, this study described the performance of an assay built on a flexible and high-throughput serological platform that proved adaptable to the emergence of a novel infectious agent.


Assuntos
Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , Microesferas , Peptidil Dipeptidase A/metabolismo , SARS-CoV-2/isolamento & purificação , Idoso , Idoso de 80 Anos ou mais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , COVID-19/sangue , COVID-19/patologia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Feminino , Fluorimunoensaio , Humanos , Imunoglobulina G/sangue , Cinética , Masculino , Pessoa de Meia-Idade , Fosfoproteínas/imunologia , SARS-CoV-2/imunologia , Sensibilidade e Especificidade , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo
17.
Biotechnol Appl Biochem ; 68(3): 597-602, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32533780

RESUMO

As a highly contagious and potentially fatal disease of dogs, canine parvovirus type 2 (CPV-2) usually causes severe myocarditis and gastroenteritis, while vaccine injection has greatly reduced the incidence of CPV-2 diseases. However, there is currently a lack of simple and effective method for quantitative detection of CPV-2 in vaccine. Therefore, this study aims to prepare an accurate method to determine the CPV-2 antigen (CPV-2-Ag) in vaccine. Here, a sandwich time-resolved fluorescence immunoassay (TRFIA) was established and optimized. Anti-CPV-2 antibodies were immobilized on 96-well plates to capture CPV-2-Ag, and then bound together with the detection antibodies labeled with Europium(III) (Eu3+ ) chelates; finally, time-resolved fluorometry was employed to measure the fluorescence intensity. Vaccination was performed to evaluate the relationship between CPV-2-Ag concentration and antibody titer. The sensitivity is 1.15 mEU/mL (LogY = 1.524 + 0.8667 × LogX, R2  = 0.9933), and the average recovery is among 91.00% to 106.39% without cross-reactions with the other canine viral antigen. The correlation between ELISA assay and this method is up to 0.9861. And, there is high correlation between the CPV-2-Ag concentration and antibody titers (R2  = 0.9234). This immunoassay established has high sensitivity, accuracy, and specificity, which indicate that this method could be suitable for quantitative detection of CPV-2-Ag in vaccine evaluation.


Assuntos
Antígenos Virais/análise , Fluorimunoensaio , Parvovirus Canino/isolamento & purificação , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Cães , Ensaio de Imunoadsorção Enzimática , Parvovirus Canino/imunologia , Vacinação
18.
Biotechnol Appl Biochem ; 68(1): 157-164, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32180269

RESUMO

Neonatal infectious diseases are a serious threat to the health of newborns. The aim was to establish a new detection method for the simultaneous measurement of (1,3)-ß-d-glucan and procalcitonin in serum for the early screening and efficacy testing of neonatal infectious diseases. We established a sandwich dual-label time-resolved fluorescence immunoassay (TRFIA): anti-(1,3)-ß-d-glucan/procalcitonin antibodies immobilized on 96-well plates captured (1,3)-ß-d-glucan/procalcitonin antigens and then banded together with the detection antibodies labeled with europium(III) (Eu3+ )/samarium(III) (Sm3+ ) chelates. Finally, time-resolved fluorometry was used to measure the fluorescence intensity. The linear correlation coefficient (R2 ) of the (1,3)-ß-d-glucan standard curve was 0.9913, and the R2 of the procalcitonin standard curve was 0.9911. The detection sensitivity for (1,3)-ß-d-glucan was 0.4 pg/mL (dynamic range: 0.6-90 pg/mL), and the average recovery was 101.55%. The detection sensitivity for procalcitonin was 0.02 ng/mL (dynamic range: 0.05-95 ng/mL), and the average recovery was 104.61%. There was a high R2 between the present TRFIA method and a commercially available assay (R2  = 0.9829 for (1,3)-ß-d-glucan and R2  = 0.9704 for procalcitonin). Additionally, the cutoff values for (1,3)-ß-d-glucan and procalcitonin were 23.95 pg/mL and 0.055 ng/mL, respectively. The present TRFIA method has high sensitivity, accuracy, and specificity and is an effective method for early screening and efficient testing of neonatal invasive fungal infection.


Assuntos
Anticorpos/química , Európio/química , Polissacarídeos Fúngicos/análise , Pró-Calcitonina/análise , Proteoglicanas/análise , Fluorimunoensaio , Humanos , Samário
19.
J Clin Microbiol ; 59(2)2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33239376

RESUMO

The Quidel Sofia severe acute respiratory syndrome (SARS) fluorescent immunoassay (FIA) test (SOFIA) is a rapid antigen immunoassay for the detection of SARS coronavirus 2 (SARS-CoV-2) proteins from nasal or nasopharyngeal swab specimens. The purpose of this study was to compare the results of the SOFIA test to those of the Hologic Aptima SARS-CoV-2 TMA test (APTIMA TMA), a high-throughput molecular diagnostic test that uses transcription-mediated amplification (TMA) for the detection of SARS-CoV-2 nucleic acid from upper respiratory tract specimens. Three hundred forty-seven symptomatic patients from an urgent care center in an area with a high prevalence of SARS-CoV-2 infections were tested in parallel using nasal swabs for the SOFIA test and nasopharyngeal swabs for the APTIMA TMA test. The SOFIA test demonstrated a positive percent agreement (PPA) of 82.0% with the APTIMA TMA test for symptomatic patients tested ≤5 days from symptom onset and a PPA of 54.5% for symptomatic patients >5 days from symptom onset. The Cepheid Xpert Xpress SARS-CoV-2 reverse transcription-PCR (RT-PCR) test was used to determine the cycle threshold (CT ) value for any specimens that were discrepant between the SOFIA and APTIMA TMA tests. Using a CT value of ≤35 as a surrogate for SARS-CoV-2 culture positivity, we estimate that the SOFIA test detected 87.2% of symptomatic patients tested ≤5 days from symptom onset who were likely to be culture positive.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/isolamento & purificação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Instituições de Assistência Ambulatorial , Antígenos Virais/análise , COVID-19/patologia , Criança , Pré-Escolar , Testes Diagnósticos de Rotina , Fluorimunoensaio , Humanos , Lactente , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular , Nasofaringe/virologia , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Sensibilidade e Especificidade , Adulto Jovem
20.
Anal Biochem ; 612: 114012, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33189703

RESUMO

A sandwich-type electrochemiluminescence (ECL) immunosensor based on the resonance energy transfer (RET) was proposed for ultrasensitive detection of cardiac troponin I (cTnI). The RET behavior could be generated between graphite carbon nitride nanosheets (m-CNNS) as donor and copper oxide@graphene oxide (CuO@GO) as acceptor, achieving the quenching effect of CuO@GO on m-CNNS for cTnI detection. The m-CNNS synthesized by mechanical grinding of the graphite carbon nitride (CN) not only has better dispersion and higher specific surface area, but also has high luminous efficiency and stable chemical properties. Therefore, m-CNNS was used as the matrix material and luminophore. As the acceptor, CuO@GO prepared by in-situ chemical synthesis of CuO NPs onto GO sheets also has a high specific surface area, which could be used as a label of secondary antibody (Ab2). Under optimal conditions, cTnI could be determined within the linear range of 0.1 pg mL-1 to 100 ng mL-1 and had a low detection limit (0.028 pg mL-1, S/N = 3). Meanwhile, the prepared ECL immunosensor possessed great stability, specificity and reproducibility, providing a new method for detecting cTnI and other biomarkers.


Assuntos
Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Fluorimunoensaio/métodos , Troponina I/análise , Troponina I/sangue , Anticorpos Imobilizados/química , Cobre/química , Grafite/síntese química , Grafite/química , Humanos , Limite de Detecção , Nanoestruturas/química , Compostos de Nitrogênio/química , Reprodutibilidade dos Testes
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