Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 12.323
Filtrar
1.
Biochem Med (Zagreb) ; 29(3): 030709, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31624462

RESUMO

Introduction: Our aim was to compare analytical specifications of two assays (monoclonal vs. polyclonal) for free light chains (FLCs) quantification optimized for two different analytical platforms, nephelometer ProSpec (Siemens, Erlangen, Germany) and turbidimetric analyser Optilite (The Binding Site, Birmingham, UK). Materials and methods: The evaluation included verification of the precision, repeatability and reproducibility, estimation of accuracy and method comparison study with 37 serum samples of haematological patients. Kappa and lambda FLC were measured in each sample by both methods and kappa/lambda ratio was calculated. Results: Results show satisfactory precision of both methods with coefficients of variation for ProSpec of CVwr = 2.20% and CVbr = 3.44%, and for Optilite CVwr = 2.82% and CVbr = 4.15%. Estimated bias for FLC lambda was higher on the ProSpec analyser, but bias for FLC kappa was higher on the Optilite analyser. Correlation coefficients were 0.98; P < 0.001 for FLC kappa and 0.97; P < 0.001 for FLC lambda. Considering normal/pathological FLC ratio moderate agreement within assays was detected (κ = 0.621). When the results were categorized according to criteria for progressive disease, 4/37 (0.10) cases were differently classified. Lambda FLC values by Optilite in three samples with monoclonal FLC lambda were more than twelve times higher than by ProSpec. A 25% difference in FLC ratio was detected in 16/37 (0.43) and 50% difference in 13/37 (0.35) patients. Conclusions: All manufacturers' precision claims could not be achieved in the verification study. The comparison of results to biological variations data showed that coefficients of variations are acceptable for both assays. The assays should not be used interchangeably in haematological patients.


Assuntos
Imunoensaio/métodos , Humanos , Paraproteinemias/metabolismo
2.
J Agric Food Chem ; 67(41): 11481-11488, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31545895

RESUMO

Dry tea matrix contains an abundance of caffeine and polyphenols which are different from the food matrix (e.g., protein, lipid, and carbohydrates), and only a few studies have tried aflatoxins determination with tea samples. Here, a specific, accurate, and sensitive method was developed and validated for the simultaneous determination of aflatoxin B1, B2, G1, and G2 in dark teas. Aflatoxins were extracted by acetonitrile/water, press-filtered, and cleaned by multifunctional purification column (MFC) and immunoaffinity column (IAC) in tandem. The cleaned extract was analyzed by liquid chromatography tandem mass spectrometry. The matrix interference was effectively reduced by MFC-IAC cleaning method. Recoveries at the spiking concentrations of 5-60 µg/kg ranged from 77.5 to 93%, with relative standard deviations <11.0%. The correlation coefficients of aflatoxins standard were >0.998. The limits of detection were 0.024-0.21 µg/kg and the limits of quantification were 0.08-0.74 µg/kg. The intra- and interday accuracy ranged from 74 to 87%, and the intra- and interday precisions ranged from 0.4 to 3.1%. After the method validation, the aflatoxins contaminations in 100 collected dark teas were detected, and the results were compared with those of other methods.


Assuntos
Aflatoxinas/química , Cromatografia Líquida de Alta Pressão/métodos , Contaminação de Alimentos/análise , Imunoensaio/métodos , Espectrometria de Massas em Tandem/métodos , Chá/química , Camellia sinensis/química , Folhas de Planta/química
3.
Analyst ; 144(17): 5108-5116, 2019 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-31373337

RESUMO

We report here the influence of antibody immobilization strategy for protein immunosensors on screen printed carbon electrode arrays in terms of antibody binding activity, analytical sensitivity, limit of detection, and stability. Horseradish peroxidase (HRP) was the model analyte with anti-HRP immobilized on the sensors, and HRP activity was used for detection. Covalently immobilized anti-HRP antibodies on electrodes coated with chitosan, electrochemically reduced graphene oxide (rGO), and dense gold nanoparticle (AuNP) films had only 20-30% of the total immobilized antibodies active for binding. Active antibodies increased to 60% with passively adsorbed antibodies on bare electrodes, to 85% with oriented antibodies using protein A covalently immobilized on AuNP-coated carbon electrode, and to 98% when attached to protein A passively adsorbed onto bare electrodes. Passively adsorbed antibodies on bare electrodes lost activity in 1-2 days, but antibodies immobilized using other strategies remained relatively stable after 5 days. Covalent immobilization gave limits of detection (LOD) of 40 fg mL-1, while passively adsorbed antibodies or protein A on carbon electrodes had LODs 4-8 fg mL-1, but were unstable. Sensitivity was highest for antibodies covalently attached to AuNP electrodes (2.40 nA per log pg per mL) that also had highest antibody coverage, and decreased slightly when protein A on AuNP was used to orient antibodies. Passively adsorbed antibodies and oriented antibodies on protein A gave slightly lower sensitivities. Immobilization strategy or antibody orientation did not have a significant effect on LOD, but dynamic range increased as the number of active antibodies on sensor surfaces increased.


Assuntos
Anticorpos Imobilizados/química , Carbono/química , Técnicas Biossensoriais/métodos , Quitosana/química , Técnicas Eletroquímicas/métodos , Eletrodos , Grafite/química , Peroxidase do Rábano Silvestre/química , Imunoensaio/métodos , Limite de Detecção , Oxirredução , Propriedades de Superfície
4.
Chem Commun (Camb) ; 55(69): 10312-10315, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31397446

RESUMO

Herein, we report a novel magnet-mediated antibody-boronate sandwich-typed assay (ABSTA) strategy for the ultrasensitive, specific, rapid, and enzyme-free detection of glycoproteins in complex samples. The proposed ABSTA method exhibited ultrahigh sensitivity for HCG with a detection limit of 0.19 mIU mL-1, which is approximately 40-fold lower than that of conventional sandwich enzyme immunoassay.


Assuntos
Anticorpos Imobilizados/química , Ácidos Borônicos/química , Gonadotropina Coriônica/sangue , Corantes Fluorescentes/química , Imãs/química , Anticorpos Monoclonais/química , Gonadotropina Coriônica/análise , Fluorescência , Humanos , Imunoensaio/métodos , Limite de Detecção
5.
Int J Nanomedicine ; 14: 4293-4307, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31354261

RESUMO

Purpose: Antibodies are key reagents in the development of immunoassay. We attempted to develop high-performance CPP immunoassays using high-affinity monoclonal antibodies prepared via cytokine-assisted immunization. Methods: We used fetal liver tyrosine kinase 3 ligand (Flt3L), CC subtype chemokine ligand 20 (CCL20), and granulocyte-macrophage colony-stimulating factor (GM-CSF) to assist traditional subcutaneous immunization of preparing high-affinity monoclonal antibodies, and further to develop high-performance immunoassay methods for CPP. Results: This novel immune strategy significantly enhanced immune response against CPP. Six anti-CPP monoclonal antibodies (mAbs) with high affinity were successfully screened and selected for application in a fully automated magnetic chemiluminescence immunoassay (CLIA). This robust and rapid assay can efficiently detect CPP in the range of 1.2-1250 pmol L-1 with a detection limit of 6.25 pmol L-1. Significantly, the whole incubation process can be completed in 30 min as compared to about 4.5 hr for the control ELISA kit. Furthermore, this assay exhibited high sensitivity and specificity, low intra-assay and inter-assay coefficients of variation (CVs < 15%). The developed assay was applied in the detection of CPP in 115 random serum samples and results showed a high correlation with data obtained using a commercially available ELISA kit (correlation coefficient, 0.9737). Conclusion: Our assay could be applied in the point-of-care testing of CPP in the serum samples, and also the method developed in this study could be adopted to explore the detection and diagnosis of other biomarkers for various diseases.


Assuntos
Anticorpos Monoclonais/imunologia , Citocinas/metabolismo , Glicopeptídeos/sangue , Imunização , Imunoensaio/métodos , Medições Luminescentes/métodos , Testes Imediatos , Animais , Anticorpos Monoclonais/biossíntese , Feminino , Humanos , Concentração de Íons de Hidrogênio , Limite de Detecção , Nanopartículas de Magnetita/química , Camundongos Endogâmicos BALB C , Sensibilidade e Especificidade
6.
J Agric Food Chem ; 67(32): 9022-9031, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31339724

RESUMO

The quantitative multiplex immunochromatographic assay (mICA) has received an increasing amount of attention in multitarget detection. However, the quantitative results in the reported mICAs were obtained by recording the signals on the test lines that with which various analyte-independent factors readily interfere, resulting in inaccurate quantitation. The ratiometric strategy using the T/C value (ratios of signals on the test line to those of the control line) for signal correction can effectively circumvent these issues to enable more accurate detection. Herein, we present for the first time a novel ratiometric mICA strip with multiple T lines for the simultaneous quantitative detection of aflatoxin B1 (AFB1), fumonisin B1 (FB1), and ochratoxin A (OTA) using highly luminescent quantum dot nanobeads (QBs) as enhanced signal reporters. To achieve reliable ratiometric signal output, a biotin-streptavidin system was introduced to replace the conventional anti-mouse IgG antibody for reliable reference signals on the control line that are completely independent of the signal probe and analyte. By using stable T/C values as quantitative signals, our proposed QB-mICA method can successfully detect three mycotoxins with concentrations as low as 1.65 pg/mL for AFB1, 1.58 ng/mL for FB1, and 0.059 ng/mL for OTA. The detection performance of the developed QB-mICA strip, including precision, specificity, and reliability, was further evaluated using artificially contaminated cereal samples. The results demonstrate the improved accuracy and reliability of quantitative determination by comparison with the anti-mouse IgG antibody. Thus, this work provides a promising strategy for developing a ratiometric mICA method for accurately quantifying multiple analytes using the biotin-SA system, opening up a new direction in quantitative mICAs.


Assuntos
Aflatoxina B1/análise , Fumonisinas/análise , Imunoensaio/métodos , Ocratoxinas/análise , Animais , Biotina/química , Grão Comestível/química , Contaminação de Alimentos/análise , Imunoensaio/instrumentação , Luminescência , Camundongos , Micotoxinas , Pontos Quânticos , Estreptavidina/química
7.
J Agric Food Chem ; 67(32): 9096-9103, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31356079

RESUMO

A monoclonal antibody (mAb) was raised against tebuconazole (TEB) using a hapten where the p-chloro substituent of the TEB molecule was replaced with a long-chain carboxylic acid. The resulting mAb showed high sensitivity and specificity against TEB characterized by ELISA with a half-maximal inhibitory concentration (IC50) of 0.19 ng mL-1 and with cross-reactivity (CR) values below 0.01% to several analogues of triazole fungicides. On the basis of the mAb produced, a quantum dot beads-based fluorescence immunochromatographic test strip assay (QBs-FITSA) was developed for rapid and sensitive detection of TEB in agricultural product samples. The QBs-FITSA exhibited a linear detection range from 0.02 to 1.25 ng mL-1 with a limit of detection (LOD) of 0.02 ng mL-1. Furthermore, using produced mAb, multiple high-throughput rapid immunoassay formats could be achieved as a convenient monitoring tool for evaluation of human and environmental exposure to TEB.


Assuntos
Brassica/química , Cucumis sativus/química , Fungicidas Industriais/análise , Imunoensaio/métodos , Triazóis/análise , Triticum/química , Anticorpos Monoclonais/análise , Contaminação de Alimentos/análise , Fungicidas Industriais/isolamento & purificação , Imunoensaio/instrumentação , Limite de Detecção , Pontos Quânticos/química , Triazóis/isolamento & purificação
8.
Chem Commun (Camb) ; 55(68): 10060-10063, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31328750
9.
Anal Bioanal Chem ; 411(23): 6067-6080, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31273413

RESUMO

Rapid detection of trace Salmonella is urgently needed to ensure food safety. We present an innovative pretreatment strategy, based on a two-step enrichment culture and immunomagnetic separation, combined with a chemiluminescence microparticle immunoassay to detect at least one proliferative Salmonella cell in 25 mL (25 g) food. The capture performance of immunomagnetic beads (IMBs) of sizes for Salmonella was investigated, and the IMBs of size 2.8 µm showed a high capture efficiency of 60.7% in 25 mL milk and 74.5% in 25 mL chicken culture filtrate, which ensured the successful capture of trace Salmonella after 2.5 h in situ enrichment even from only one Salmonella cell. The separated Salmonella cells, reaching an amount of 103 colony-forming units (CFU) by a secondary enrichment for 3 h, were detected by a horseradish peroxidase chemiluminescence reaction with 4-(1-imidazolyl)phenol as an enhancer, which evidenced a linear response for Salmonella concentrations ranging from 2.3 × 102 to 7.8 × 104 CFU/mL. The entire detection process was completed within 8 h, with a very low detection limit of 1 CFU/25 mL (25 g), which was verified by colony counting, and a small degree of interference of 0.17-1.06%. Trace Salmonella from five different serovars in milk and chicken was successfully detected without false negative or false positive results. Furthermore, this study provides a basis to develop a fully automated instrument based on IMBs that includes all steps from sample preparation to chemiluminescence microparticle immunoassay for high-throughput screening of foodborne pathogens. Graphical abstract.


Assuntos
Contaminação de Alimentos/análise , Medições Luminescentes/métodos , Leite/microbiologia , Aves Domésticas/microbiologia , Salmonella/isolamento & purificação , Animais , Galinhas/microbiologia , Contaminação de Alimentos/economia , Inocuidade dos Alimentos/métodos , Imunoensaio/economia , Imunoensaio/métodos , Separação Imunomagnética/economia , Separação Imunomagnética/métodos , Limite de Detecção , Medições Luminescentes/economia , Infecções por Salmonella/microbiologia , Fatores de Tempo
10.
Anal Bioanal Chem ; 411(23): 6057-6066, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31278555

RESUMO

In this study, we report a direct surface plasmon resonance (SPR) biosensor based on an oriented assembly of antibody for the rapid detection of chlorpyrifos residue in agricultural samples. In this covalent-orientated strategy, staphylococcal protein A (SPA) was first covalently bound to the surface for monitoring chlorpyrifos residue, with subsequent binding of the antibody in an orientated fashion via its fragment crystallizable (Fc) region. Consequently, the SPA-modified biosensor exhibited a satisfactory specificity and a low detection limit of 0.056 ng mL-1 for chlorpyrifos, with a linear detection range of 0.25-50.0 ng mL-1. Under optimal conditions, the sensor chip could be regenerated for at least 210 cycles. The results presented here indicate that the SPA-modified sensor chip can successfully improve the sensitivity and obviating the need of the modification of the antibody. The developed SPR biosensor method has the great potential for rapid, sensitive, and specific detection with broad applications in areas of environmental monitoring and food safety. Graphical abstract.


Assuntos
Clorpirifos/análise , Análise de Perigos e Pontos Críticos de Controle/métodos , Inseticidas/análise , Proteína Estafilocócica A/química , Ressonância de Plasmônio de Superfície/métodos , Anticorpos Imobilizados/química , Brassica/química , Contaminação de Alimentos/análise , Imunoensaio/métodos , Limite de Detecção , Malus/química , Zea mays/química
11.
Anal Chim Acta ; 1078: 151-160, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31358213

RESUMO

Herein, we report a new signal amplification scheme for quantitative biochemical analysis based on gold nanoparticle (GNPs) catalyzed polymerization of transparent silane solution to milky white and turbid siloxane. Using immunoassay as a proof of concept, GNP labeled immunoprobe was used to bind captured antigen and catalyse the polymerization reaction allowing sensitive biochemical investigation. The polymerization reaction was optimized for standard 96 well polystyrene microtiter plates and we discovered that sodium lactate acts as an enhancer in the polymerization reaction as it reduces detection time to merely 30 min. The sensing strategy was applied to detection and quantification of Salmonella Typhimurium in water and egg samples and the platform showed excellent visibly quantifiable analytical response up to 100 cells mL-1. Furthermore, clinical utility and potential of the method was validated by detecting Vi capsular polysaccharide (Vi antigen) responsible for typhoidal Salmonellosis in human serum in sandwich format with a detection limit of 1 ng mL-1. The method serves as the first report towards nanoparticle triggered polymerization for development of rapid and low cost quantitative biochemical assay.


Assuntos
Ouro/química , Imunoensaio/métodos , Nanopartículas Metálicas/química , Polissacarídeos Bacterianos/sangue , Salmonella typhimurium/isolamento & purificação , Siloxanas/síntese química , Animais , Anticorpos/imunologia , Galinhas , Água Potável/microbiologia , Ovos/microbiologia , Contaminação de Alimentos/análise , Humanos , Limite de Detecção , Tamanho da Partícula , Polimerização , Polissacarídeos Bacterianos/imunologia , Estudo de Prova de Conceito , Salmonella typhimurium/imunologia , Silanos/química , Temperatura Ambiente
12.
Tumour Biol ; 41(7): 1010428319860728, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31264534

RESUMO

Colon cancer represents one of the most common cancers in the world. Despite improved treatment, mortality remains high. In order to improve the assessment of prognosis for colon cancer patients, identifying new prognostic markers remains necessary. We analyzed preoperative serum samples from 148 colon cancer patients surgically treated at Helsinki University Hospital from 1998 through 2002 using a multiplex proximity extension assay (Oncology II panel, Olink Bioscience, Uppsala, Sweden), a panel constituting 92 immunological and oncological markers. We performed univariate and multivariate analyses on these patients and calculated the disease-specific survival among patients using the log-rank test for Kaplan-Meier estimates. In the univariate survival analysis of 92 biomarkers, 26 resulted in p < 0.1. Among these, eight biomarkers emerged as statistically significant (p < 0.05). Patients with low levels of kallikrein 13 had a poor prognosis. Moreover, patients with high levels of amphiregulin, carcinoembryonic antigen-related adhesion molecule 5, interleukin 6, mucin 16, syndecan 1, transforming growth factor alpha, and vimentin also had a poor prognosis. In the multivariate analysis, kallikrein 13 and mucin 16 emerged as independent prognostic markers. The role of kallikrein 13, a member of the serine protease kallikrein biomarker family, in tumorigenesis remains unclear. Mucin 16 is also known as carbohydrate antigen 125, a well-known ovarian cancer biomarker. Patients with low levels of kallikrein 13 (hazard ratio: 0.36; 95% confidence interval: 0.14-0.92; p = 0.033) and high levels of mucin 16 (hazard ratio: 3.15; 95% confidence interval: 1.68-5.93; p < 0.005) had a poor prognosis. Mucin 16 and kallikrein 13 represent independent prognostic markers for colon cancer. Furthermore, the clinical utility of mucin 16 and kallikrein 13 serum tests warrants additional investigation.


Assuntos
Antígeno Ca-125/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Calicreínas/metabolismo , Proteínas de Membrana/metabolismo , Idoso , Biomarcadores Tumorais/metabolismo , Feminino , Humanos , Imunoensaio/métodos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Análise de Sobrevida
13.
APMIS ; 127(9): 635-641, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31237033

RESUMO

In this study, several innate immunological adjuvants and related compounds were compared with respect to complement activation in serum and induction of cytokine release in whole blood samples using immunoassays. As found, simple lipids had no effect on the complement system or on cytokine release, whereas lipopolysaccharides induced prominent release of pro-inflammatory cytokines (IL1ß, TNF and IFNγ) without affecting the complement system, except for one, which activated the lectin pathway (LP). Moreover, saponin induced IL1ß and MCP1 release and did not affect the complement system. The polysaccharide inulin exhausted the alternative pathway (AP) completely without affecting the LP and the classical pathway (CP), whereas zymosan exhausted the AP and had a major effect on the LP and CP as well. Peptidoglycans mainly affected the LP. Inulin, agarose and cellulose induced IL1ß and MCP1 release, while dextran had no effect on cytokine secretion. Zymosan mainly induced IL1ß release. The inorganic compound aluminium hydroxide, Al(OH)3 , activated the complement system very efficiently (all three pathways) but only induced MCP1 release. Other compounds tested had minor/individual effects. Collectively, well-known adjuvants, such as aluminum hydroxide, activated the complement system and/or induced pro-inflammatory cytokine release. Since complement activation generates anaphylactic peptides, a simple definition of an (innate) immunological adjuvant can be inferred: it activates the (innate) immune system by complement activation and/or release of cytokines so as to attract cells of the adaptive immune system.


Assuntos
Adjuvantes Imunológicos/sangue , Adjuvantes Imunológicos/farmacologia , Ativação do Complemento/efeitos dos fármacos , Ativação do Complemento/imunologia , Proteínas do Sistema Complemento/imunologia , Citocinas/imunologia , Humanos , Imunoensaio/métodos , Soro/imunologia
14.
Food Chem ; 293: 41-48, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31151629

RESUMO

Milk by-products such as whey and caseinate are widely used as ingredients or processing aids in food industry. However, since they could cause allergic reactions they are included in Allergen Control Plans. ß-Lactoglobulin is the major whey protein and caseins are main proteins in milk. Selection of a unique target to analyze the presence of milk in foods could be insufficient when the source of milk proteins is unknown. A new test based on lateral flow immunocromatography that combines the simultaneous and independent detection of both proteins (ß-lactoglobulin and casein) in one rapid test was developed. The assay was validated according to AOAC guidelines being able to detect ß-lactoglobulin (0.5 ppm), casein (2 ppm), whey and powder milk (1-5 ppm). No cross-reactivity was found with a panel of 38 food commodities. The method is a rapid and suitable tool to identify milk proteins in processed food, ingredients, and rinsing water.


Assuntos
Caseínas/análise , Análise de Alimentos/métodos , Imunoensaio/métodos , Lactoglobulinas/análise , Animais , Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Caseínas/imunologia , Lactoglobulinas/imunologia , Leite/metabolismo , Proteínas do Soro do Leite/metabolismo
15.
Mem Inst Oswaldo Cruz ; 114: e190047, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31166422

RESUMO

OBJECTIVES: We tested a rapid and specific immunochromatographic assay (that detects human blood in forensic samples) to determine if human blood was present in triatomines and their fecal excreta. METHODS: We fed Triatoma rubida human blood (positive control) or mouse blood (negative control) and performed the assay on the abdominal contents and fecal excreta. Triatomine field specimens collected in and around human habitations and excreta were also tested. FINDINGS: The assay was positive in triatomines fed human blood (N = 5/5) and fecal excreta from bugs known to have ingested human blood (N = 5/5). Bugs feeding on mice (N = 15/15) and their fecal excreta (N = 8/8) were negative for human blood. Human blood was detected in 47% (N = 23/49) triatomines, representing six different species, collected in the field. MAIN CONCLUSIONS: The pilot study shows that this rapid and specific test may have applications in triatomine research. Further study is needed to determine the sensitivity of this assay compared to other well-established techniques, such as DNA- and proteomics-based methodologies and the assay's application in the field.


Assuntos
Sangue , Fezes/química , Imunoensaio/métodos , Triatominae , Animais , Doença de Chagas/transmissão , Humanos , Camundongos , Projetos Piloto , Padrões de Referência , Valores de Referência , Reprodutibilidade dos Testes , Fatores de Tempo
16.
Food Chem ; 297: 124965, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253343

RESUMO

Considering the health risks of E. coli O157:H7 presence in food and water, an affordable and highly sensitive detection method is crucial. Herein, we report the first use of a single antibody-based fluorescent lateral flow immunoassay (FLFIA) depending on non-radiative energy transfer between graphene oxide and quantum dots for determination of E. coli O157:H7 in beef and river water. FLFIA showed a high sensitivity rate thousand-fold better than the conventional lateral flow (LF). In inoculated minced beef and river water samples, the limits of detection were 178 and 133 CFU g-1 or mL-1, respectively. Besides, it presented a high selectivity in the presence of other possible interfering bacteria. The single antibody approach reduced the assay cost to 60% less than the conventional LF. Alongside, the results could be read by portable LF readers or smartphones. These advantages offer FLFIA as a promising technology for pathogen detection in food and water.


Assuntos
Escherichia coli O157 , Microbiologia de Alimentos/instrumentação , Microbiologia de Alimentos/métodos , Imunoensaio/métodos , Carne Vermelha/microbiologia , Animais , Anticorpos , Bovinos , Desenho de Equipamento , Escherichia coli O157/imunologia , Corantes Fluorescentes/química , Microbiologia de Alimentos/economia , Grafite , Imunoensaio/economia , Imunoensaio/instrumentação , Óxidos , Pontos Quânticos , Rios/microbiologia , Sensibilidade e Especificidade , Smartphone , Microbiologia da Água
17.
Environ Sci Pollut Res Int ; 26(23): 23471-23479, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31197672

RESUMO

A rapid and sensitive immunoassay for the simultaneous detection of imidacloprid and thiacloprid was developed by using magnetic nanoparticles (MNPs) and upconversion nanoparticles (UCNPs). The UCNPs of NaYF4:Yb, Er and NaYF4:Yb, Tm were synthesized and conjugated with anti-imidacloprid monoclonal antibody (mAb) and anti-thiacloprid mAb as signal labels, while the MNPs were conjugated with antigens of thiacloprid and imidacloprid as separation elements. The fluorescence intensities of Yb/Er- and Yb/Tm-doped UCNPs were detected simultaneously in 544 nm and 477 nm under the excitation of NIR light (980 nm). The amounts of mAb-conjugated UCNPs that were separated by antigen-conjugated MNPs were determined based on competitive immunoassays. Under the optimal conditions, the 50% inhibiting concentration (IC50) and limit of detection (LOD, IC10) were 5.80 and 0.32 ng/mL for imidacloprid and 6.45 and 0.61 ng/mL for thiacloprid, respectively. The immunoassay exhibited negligible cross-reactivity with analogs of imidacloprid and thiacloprid except imidaclothiz (86.2%). The average recoveries of imidacloprid and thiacloprid in environmental and agricultural samples, including paddy water, soil, pears, oranges, cucumbers, and wheat, ranged from 78.4 to 105.9% with relative standard deviations (RSDs) of 2.1-11.9% for imidacloprid and ranged from 82.5 to 102.3% with RSDs of 1.0-16.5% for thiacloprid. In addition, the results of the immunoassay correlated well with high-performance liquid chromatography for the detection of the authentic samples.


Assuntos
Imunoensaio/métodos , Nanopartículas de Magnetita/química , Neonicotinoides/análise , Nitrocompostos/análise , Tiazinas/análise , Fluorescência , Magnetismo , Nanopartículas/química , Tiazóis
18.
Int Arch Allergy Immunol ; 180(1): 28-36, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31189157

RESUMO

BACKGROUND: Clinically meaningful specific IgE determination is an important step in the diagnosis of allergic diseases. While patient's history and skin prick tests are available during the medical visit, most IgE immunoassays require hours to several days to be available. Recent developments in the field of nanofluidic technology open new horizons for point-of-care management of this unmet medical need. OBJECTIVE: This study aimed to compare IgE diagnostic agreement between a nanofluidic assay (abioSCOPE®) and a laboratory reference method (Phadia Laboratory System®) in a real-world clinical setting. METHODS: Sera from 105 patients whose routine allergy diagnostic workup required a blood sampling were used to compare the novel nanofluidic IgE assay to a reference method in a blind manner for a panel of five respiratory allergens. To assess the agreement between methods, patient records were reviewed by four independent experts to establish the final diagnosis. Experts were blinded to the IgE serological method used, but had access to patient history, skin prick tests, and blood test results. RESULTS: Analytic agreement between the two methods was 81% for the tested panel of allergens (ranging from 77 to 89%). The overall agreement in clinical diagnosis decision taken by the expert panel was 94.6% with the nanofluidic IgE assay when compared to the reference method. CONCLUSION: The nanofluidic IgE assay, as determined through an evaluation based on clinical history, skin prick tests, and IgE measurement, is a valuable tool for allergy experts to identify patients' sensitization patterns at the point of care, and for routine IgE diagnostic workup.


Assuntos
Imunoensaio/métodos , Imunoensaio/normas , Imunoglobulina E/imunologia , Dispositivos Lab-On-A-Chip , Nanotecnologia/métodos , Adolescente , Adulto , Idoso , Alérgenos/imunologia , Feminino , Humanos , Hipersensibilidade/diagnóstico , Hipersensibilidade/imunologia , Imunoglobulina E/sangue , Masculino , Pessoa de Meia-Idade , Padrões de Referência , Valores de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Testes Cutâneos , Adulto Jovem
19.
J Med Microbiol ; 68(7): 1021-1032, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31188094

RESUMO

INTRODUCTION: The spread of carbapenem-resistant Acinetobacter baumannii has led to a worldwide healthcare problem. Carbapenem resistance in A. baumannii is mainly mediated by the acquisition of the carbapenem-hydrolyzing oxacillinase OXA-23. The phenotypic detection of carbapenem-producing A. baumannii is challenging and time-consuming. Hence, there is an unmet medical need for reliable and rapid diagnostic tools to detect OXA-23-producing Acinetobacter isolates to enable successful patient management. AIM: Development of an immunochromatographic lateral flow test (ICT) for the rapid and reliable detection of OXA-23-producing carbapenem-resistant Acinetobacter isolates. METHODOLOGY: For the development of an antibody-based ICT, we generated anti-OXA-23 monoclonal antibodies (MoAbs) and screened them sequentially for their ability to bind native OXA-23. Selected OXA-23-specific MoAbs were tested in different combinations for their capacity to capture and detect OXA-23His6 by sandwich enzyme-linked immunosorbent assay (ELISA) and ICT. A well-characterized collection of carbapenem-resistant Acinetobacter isolates with defined carbapenem resistance mechanisms were used to evaluate the specificity of the final OXA-23 ICT prototype. RESULTS: The antibody pairs best suited for the sandwich ELISA format did not match the best pairs in the ICT format selected during the development process of the final prototype OXA-23 ICT. This prototype was able to differentiate between OXA-23 subfamily-mediated carbapenem resistance and carbapenem-resistant Acinetobacter isolates overexpressing other OXAs with 100  % specificity and a turnaround time of 20 min from culture plate to result. CONCLUSION: With this rapid detection assay one can save 12-48 h of diagnostic time, which could help avoid inappropriate use of carbapenems and enable earlier intervention to control the transmission of OXA-23-producing carbapenem-resistant Acinetobacter isolates to other patients and healthcare workers.


Assuntos
Acinetobacter baumannii/isolamento & purificação , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Farmacorresistência Bacteriana Múltipla , Imunoensaio/métodos , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/enzimologia , Acinetobacter baumannii/imunologia , Animais , Antibacterianos/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Proteínas de Bactérias , Carbapenêmicos/metabolismo , Clonagem Molecular , Feminino , Regulação Bacteriana da Expressão Gênica , Camundongos , Testes de Sensibilidade Microbiana , beta-Lactamases/genética , beta-Lactamases/imunologia , beta-Lactamases/metabolismo
20.
Anal Bioanal Chem ; 411(17): 3789-3800, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31161320

RESUMO

MicroRNAs (miRNAs) in a blood sample are usually measured by quantitative reverse transcription PCR (qRT-PCR), microarray, and next-generation sequencing (NGS) which requires time-consuming pre-treatment, manual operation, and a stand-alone instrument. To overcome these disadvantages, miRNA testing has been developed using the automated analyzers routinely used in clinical laboratories. An isothermal DNA amplification reaction was adapted to a fully automated immunoassay analyzer that conducts extraction, amplification, and detection processes at 37 °C in 44 min. In a reaction vessel, a pre-designed single-stranded signal DNA was amplified in the presence of miRNA, using DNA templates, DNA polymerase, and nicking endonuclease. Then, the amplified signal DNA was hybridized by one DNA probe attached to a magnetic particle and another DNA probe labeled with acridinium ester. After the chemiluminescence reaction, luminescence intensity was automatically measured. The automated assays of cancer-related miRNAs were implemented on the analyzer with throughput of 66 tests per hour. In the assays with one-step amplification, three miRNAs (miR-21-5p, miR-18a-5p, and miR-500a-3p) at concentrations lower than 100 fM were automatically detected and the cross reactivity for miR-21-5p with fifteen similar miRNAs was not higher than 0.02%. In the assay with two-step amplification, detection sensitivity and amplification rate for miR-21-5p were 3 fM and 103-fold, respectively. The coefficient of variations (CVs) in the measurement at the target concentrations from 5 fM to 1000 pM were less than 8%. Furthermore, we also achieved automated nucleic acid detection in human serum. The proposed fully automated miRNA assays showed high sensitivity, low cross reactivity, and reproducibility suitable for clinical use. Graphical abstract.


Assuntos
Imunoensaio/métodos , MicroRNAs/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , Automação , Humanos , Luminescência
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA