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1.
Sci Total Environ ; 855: 158897, 2023 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-36411601

RESUMO

Rapid detection technology of aquaculture fishery drug residues is needed to supplement large-scale instrument methods. To do this, the time-resolved fluorescence immunoassay (TRFIA) method and portable three-dimensional (3D) printing equipment platform were used, in combination with smartphones, to detect malachite green (MG) in pond sediments. The TRFIA was coupled to MG monoclonal antibodies (mAb) through lanthanide metal microspheres europium (Eu3+). The labeled antibody produced competitive immunity in the immune reaction system, and the specific fluorescence intensity in the product was determined by a portable 3D printing equipment platform to achieve quantitative analysis. To test this method, leucomalachite green (LMG) was converted to MG by oxidation of dicyanoquinone (DDQ), and a qualitative analysis was achieved. Methodological evaluation results were satisfactory, recoveries were 83 %-104 %, the limit of detection (LOD) was 0.3 ng/g, the limit of quantitation (LOQ) was 0.7 ng/g, and the coefficient of variation was 1.3 %-7.3 %. The linear equation y = -0.1496x + 0.5585 was in the range of 0-10 ng/g. The linear regression correlation coefficient was 99.2 %. The TRFIA was confirmed and positive samples were measured. Results were consistent with the standard method, which demonstrated that the TRFIA was feasible and that the detection results were reliable. Compared with the national standard method, the TRFIA saves time, is more convenient, and has high sensitivity. It provides an efficient technical method for the rapid screening of MG in the sediments of aquaculture environments.


Assuntos
Fluorimunoensaio , Impressão Tridimensional , Imunofluorescência , Microesferas
2.
BMC Biotechnol ; 22(1): 27, 2022 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-36180909

RESUMO

BACKGROUND: Lymphocytic leukemia (LL) is a primary malignant tumor of hematopoietic tissue, which seriously affects the health of children and the elderly. The study aims to establish a new detection method for screening acute/chronic LL using time-resolved fluorescence immunoassay (TRFIA) via quantitative detection of S100 calcium binding protein A8 (S100A8) and leucine-rich alpha-2-glycoprotein 1 (LRG1) in serum. METHODS: Here a sandwich TRFIA was optimized and established: Anti-S100A8/LRG1 caputre antibodies immobilized on 96-well plates captured S100A8/LRG1, and then banded together with the anti-S100A8/LRG1 detection antibodies labeled with Europium(III) (Eu3+)/samarium(III) (Sm3+) chelates. Finally time resolved fluorometry measured the fluorescence intensity. RESULTS: The sensitivity of S100A8 was 1.15 ng/mL(LogY = 3.4027 + 0.4091 × LogX, R2 = 0.9828, P < 0.001, dynamic range: 2.1-10,000 ng/mL), and 3.2 ng/mL for LRG1 (LogY = 3.3009 + 0.4082 × LogX, R2 = 0.9748, P < 0.001, dynamic range: 4.0-10,000 ng/mL). The intra-assay and inter-assay CVs were low, ranging from 5.75% to 8.23% for S100A8 and 5.30% to 9.45% for LRG1 with high specificity and affinity in serum samples. Bland-Altman plots indicated TRFIA and ELISA kits have good agreement in clinical serum samples. Additionally, the cutoff values for S100A8 and LRG1 were 1849.18 ng/mL and 588.08 ng/mL, respectively. CONCLUSION: The present TRFIA method could be used for the quantitative detection of S100A8 and LRG1 in serum, and it has high sensitivity, accuracy and specificity. Clinically, this TRFIA method could be suitable for screening of LL via the quantitative detection of S100A8 and LRG1.


Assuntos
Európio , Leucemia Linfocítica Crônica de Células B , Idoso , Proteínas de Ligação ao Cálcio , Criança , Fluorimunoensaio/métodos , Glicoproteínas , Humanos , Leucina , Leucemia Linfocítica Crônica de Células B/diagnóstico , Samário , Sensibilidade e Especificidade
3.
Anal Bioanal Chem ; 414(24): 7143-7151, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36006431

RESUMO

In recent years, more and more functional peptide ligands have been identified from phage display libraries and served the immunoassay of small molecules. After the identification, the phage particle instead limits further application of peptide ligands, so it is of great significance to explore the peptide ligand as an independent detection reagent. In this work, the identified peptidomimetic of benzothiostrobin was synthesized and labelled with biotin, which was combined with Eu3+-labelled streptavidin to develop the peptide-based time-resolved fluoroimmunoassay (P-TRFIA). Under the optimal conditions, the half-maximum inhibitory concentration (IC50) of proposed P-TRFIA is 3.63 ng mL-1, which is similar to the TRFIA using phage-borne peptidomimetic and Eu3+-labelled anti-phage antibody (IC50: 4.55 ng mL-1), also more sensitive than previously reported immunoassays for benzothiostrobin. In addition, the proposed P-TRFIA shows excellent specificity and accuracy for analysis of spiked samples, and its detection results shows good consistency with high-performance liquid chromatography for the detection of environment and agro-products samples with unknown benzothiostrobin concentrations.


Assuntos
Biotina , Peptidomiméticos , Acrilatos , Benzotiazóis , Fluorimunoensaio/métodos , Ligantes , Peptídeos/química , Sensibilidade e Especificidade , Estreptavidina
4.
J Clin Lab Anal ; 36(9): e24603, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35870181

RESUMO

AIM: To establish a highly sensitive time-resolved fluorescence immunoassay (TRFIA) of kidney injury molecule-1 (Kim-1) and evaluate its clinical value in acute kidney injury (AKI). METHODS: The Kim-1-TRFIA was established by the double-antibody sandwich method, and the method was evaluated. The established Kim-1-TRFIA was used to detect the concentration of Kim-1 in the serum of healthy controls and patients with AKI. RESULTS: The optimal coating antibody concentration and optimal Eu3+ -labeled antibody dilution ratio for Kim-1-TRFIA are 1 µg/ml and 1:140, respectively. The linear range is 42.71-4666.69 pg/ml. The intra- and inter-assay coefficients of variation are <10%. The specificity of our Kim-1-TRFIA is acceptable. The recovery is between 95.14% and 102.84%. The concentration of Kim-1 in the serum of patients with AKI is 126.50 ± 67.99 pg/ml, which is significantly higher than that in the serum of healthy controls (49.72 ± 16.40 pg/ml, p < 0.001). Staging patients with AKI by glomerular filtration rate shows that the serum concentration of Kim-1 increases significantly with increasing disease severity (p < 0.05). CONCLUSION: A highly sensitive Kim-1-TRFIA was established. With this immunoassay, a good differential diagnosis can be made, and healthy people and AKI patients can be differentiated by detecting the concentration of Kim-1 in the serum. Moreover, the severity of AKI patients can be determined.


Assuntos
Injúria Renal Aguda , Injúria Renal Aguda/diagnóstico , Biomarcadores , Fluorimunoensaio/métodos , Receptor Celular 1 do Vírus da Hepatite A , Humanos , Imunoensaio/métodos , Testes Imunológicos , Soro
5.
Biosensors (Basel) ; 12(5)2022 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-35624639

RESUMO

As a common herbicide in farmland, there has been wide concern over quinclorac residue because of its potential risks to the environment and human health. For the detection and monitoring of quinclorac residue in the environment, enzyme-linked immunoassay (ELISA) and time-resolved fluoroimmunoassay (TRFIA) were established. The half-maximal inhibition concentrations (IC50) of ELISA and TRFIA were 0.169 mg/L and 0.087 mg/L with a linear range (IC20-IC80) of 0.020-1.389 mg/L and 0.004-1.861 mg/L, respectively. Compared with ELISA, the limit of detection (LOD, IC20) and IC50 of TRFIA improved approximately 5-fold and 2-fold. The cross-reaction rates for the quinclorac analogs were less than 2%. The average recoveries of quinclorac in river water, paddy water, paddy soil, and brown rice samples were 77.3-106.1%, with RSDs of 1.7-12.5%. More importantly, the results of the two methods were consistent with that of the referenced method of UPLC-MS/MS (R2 > 0.98). ELISA and TRFIA both showed good detection performance and could meet the requirements of the quantitative determination of quinclorac. Therefore, the proposed ELISA and TRFIA could be applied to the rapid and sensitive detection and monitoring of quinclorac residue in the environment.


Assuntos
Fluorimunoensaio , Espectrometria de Massas em Tandem , Cromatografia Líquida , Fluorimunoensaio/métodos , Humanos , Quinolinas , Água/química
6.
J Fluoresc ; 32(4): 1501-1507, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35511384

RESUMO

To establish a rapid and highly sensitive assay for tumor-associated trypsinogen-2 (TAT-2) based on the time-resolved fluorescence immunoassay (TRFIA) and evaluate its potential clinical value in patients with lung cancer. The double-antibody sandwich method was used in detecting TAT-2 antigen concentrations, and two types of TAT-2 antibodies (coating antibodies and Eu3+ labeled antibodies) were used. A TAT-2-TRFIA method was then established, evaluated, and used in detecting the serum TAT-2 levels of healthy subjects and patients with lung cancer. The linear range of the TAT-2-TRFIA method was 1.53-300 ng/mL, the intra-assay coefficient of variation (CV) were between 1.67% and 8.42%, and the inter-assay CV were between 4.29% and 11.44%. The recovery rates of TAT-2-TRFIA were between 99.17% and 107.06%. The cross-reactivities of trypsin and T-cell immunoglobulin mucin 3 were 0.02% and 0.82%, respectively. The serum TAT-2 levels of patients with lung cancer were higher than those of healthy subjects (P < 0.001). Combined with TAT-2, the sensitivity and specificity of CEA and CA-125 for lung cancer improved significantly. Conclusion: We successfully established a highly sensitive TAT-2-TRFIA method, which was able to facilitate the timely diagnosis of lung cancer.


Assuntos
Neoplasias Pulmonares , Tripsinogênio , Fluorimunoensaio/métodos , Humanos , Neoplasias Pulmonares/diagnóstico , Sensibilidade e Especificidade , Tripsina
7.
Anal Biochem ; 648: 114674, 2022 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-35351395

RESUMO

AIM: This study aimed to establish a highly sensitive time-resolved fluorescence immunoassay (TRFIA) for the detection of serum lipoprotein-associated phospholipase A2 (Lp-PLA2) and evaluate the clinical application value of Lp-PLA2 in patients with breast cancer. METHODS: The level of Lp-PLA2 was detected using the double-antibody sandwich method. First, the Lp-PLA2-TRFIA method was established, and the method was evaluated on the basis of linearity, sensitivity, precision, specificity, and recovery rate. Then, the fluorescence counts in serum of healthy subjects and patients with breast cancer were detected by Lp-PLA2-TRFIA, and the levels of Lp-PLA2 were calculated using a standard curve. RESULTS: Lp-PLA2-TRFIA had a wide linear range (43.48-2000 ng/mL). The intra-assay precisions of Lp-PLA2-TRFIA ranged from 2.66% to 4.84% (<10%), and the inter-assay precisions were between 5.39% and 6.95% (<15%). No cross-reaction was observed among Lp-PLA2, Tumor-associated trypsinogen-2, and T-cell immunoglobulin mucin 3. In addition, the recovery rates were between 90% and 100%. The serum Lp-PLA2 levels of patients with breast cancer were significantly higher than those of healthy subjects. CONCLUSIONS: We successfully established a highly sensitive Lp-PLA2-TRFIA method, and found serum Lp-PLA2 may be associated with dyslipidemia in breast cancer and could be used for auxiliary diagnose.


Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase , Neoplasias da Mama , Biomarcadores , Neoplasias da Mama/diagnóstico , Feminino , Fluorimunoensaio , Humanos
8.
J Agric Food Chem ; 70(13): 4102-4111, 2022 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-35333506

RESUMO

A simple and sensitive fluoroimmunoassay (FIA) based on a heavy-chain antibody (VHH) for rapid detection of fenitrothion was developed. A VHH library was constructed from an immunized alpaca, and one clone recognizing fenitrothion (namely, VHHjd8) was achieved after careful biopanning. It was biotinylated by fusing with the Avi tag and biotin ligase to obtain a fusion protein (VHHjd8-BT), showing both binding capacity to fenitrothion and the streptavidin poly-horseradish peroxidase conjugate (SA-polyHRP). Based on a competitive assay format, the absorbance spectrum of oxidized 3,3',5,5'-tetramethylbenzidine generated by SA-polyHRP overlapped the emission spectrum of carbon dots, which resulted in quenching of signals due to the inner-filter effect. The developed FIA showed an IC50 value of 1.4 ng/mL and a limit of detection of 0.03 ng/mL, which exhibited 15-fold improvement compared with conventional enzyme-linked immunosorbent assay. The recovery test of FIA was validated by standard GC-MS/MS, and the results showed good consistency, indicating that the assay is an ideal tool for rapid screening of fenitrothion in bulk food samples.


Assuntos
Fenitrotion , Anticorpos de Domínio Único , Ensaio de Imunoadsorção Enzimática/métodos , Fluorimunoensaio/métodos , Anticorpos de Domínio Único/química , Estreptavidina/química , Espectrometria de Massas em Tandem
9.
Sci Rep ; 12(1): 1951, 2022 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-35121780

RESUMO

Serum protein electrophoresis (SPE) separates serum proteins into bands whose shape and amplitude can alert clinicians to a range of disorders. This is followed by more specific immunoassays to quantify important antigens and confirm a diagnosis. Here we develop a high-speed capillary electrophoresis (HSCE) platform capable of simultaneous SPE and immunoassay measurements. A single laser excitation source is focused into the detection zone of the capillary to measure both refractive index (SPE) and fluorescence signals (immunoassays). The refractive index signal measures characteristic SPE profiles for human serum separated in 100 mM boric acid (pH 10), 100 mM arginine (pH 11), and 20 mM CHES (pH 10). For the immunoassay, the fluorescence electropherograms reveal that CHES provides the optimal buffer for measuring the immunocomplex and separating it from the free antigen. Immunoassays in CHES yield a LOD of 23 nM and a LOQ of 70 nM for the detection of fluorescein. The high pH reduces protein adsorption but reduces antibody affinity. Preliminary studies carried out in 50 mM barbital at pH 8 show improved stability of the immunocomplex and better separation for immunoassay quantification. Further optimization will open new capabilities for measuring orthogonal diagnostic signals in seconds with HSCE.


Assuntos
Eletroforese das Proteínas Sanguíneas , Proteínas Sanguíneas/análise , Eletroforese Capilar , Fluorimunoensaio , Afinidade de Anticorpos , Complexo Antígeno-Anticorpo , Biomarcadores/sangue , Humanos , Concentração de Íons de Hidrogênio , Medições Luminescentes , Microscopia de Fluorescência , Valor Preditivo dos Testes , Estabilidade Proteica
10.
Sci Rep ; 12(1): 1263, 2022 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-35075142

RESUMO

In a viral pandemic, a few important tests are required for successful containment of the virus and reduction in severity of the infection. Among those tests, a test for the neutralizing ability of an antibody is crucial for assessment of population immunity gained through vaccination, and to test therapeutic value of antibodies made to counter the infections. Here, we report a sensitive technique to detect the relative neutralizing strength of various antibodies against the SARS-CoV-2 virus. We used bright, photostable, background-free, fluorescent upconversion nanoparticles conjugated with SARS-CoV-2 receptor binding domain as a phantom virion. A glass bottom plate coated with angiotensin-converting enzyme 2 (ACE-2) protein imitates the target cells. When no neutralizing IgG antibody was present in the sample, the particles would bind to the ACE-2 with high affinity. In contrast, a neutralizing antibody can prevent particle attachment to the ACE-2-coated substrate. A prototype system consisting of a custom-made confocal microscope was used to quantify particle attachment to the substrate. The sensitivity of this assay can reach 4.0 ng/ml and the dynamic range is from 1.0 ng/ml to 3.2 [Formula: see text]g/ml. This is to be compared to 19 ng/ml sensitivity of commercially available kits.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Teste Sorológico para COVID-19 , COVID-19/imunologia , Nanopartículas/química , SARS-CoV-2/imunologia , Enzima de Conversão de Angiotensina 2/química , Fluorimunoensaio , Humanos , Testes de Neutralização
11.
BMC Nephrol ; 23(1): 30, 2022 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-35031018

RESUMO

BACKGROUND: AKI is related to severe adverse outcomes and mortality with Coronavirus Disease 2019 (COVID-19) patients, that early diagnosed and intervened is imperative. Neutrophil gelatinase-associated lipocalin (NGAL) is one of the most promising biomarkers for detection of acute kidney injury (AKI), but current detection methods are inadequacy, so more rapid, convenient and accuracy methods are needed to detect NGAL for early diagnosis of AKI. Herein, we established a rapid, reliable and accuracy lateral flow immunoassay (LFIA) based on europium nanoparticles (EU-NPS) for the detection of NGAL in human urine specimens. METHODS: A double-antibody sandwich immunofluorescent assay using europium doped nanoparticles was employed and the NGAL monoclonal antibodies (MAbs) conjugate as labels were generated by optimizing electric fusion parameters. Eighty-three urine samples were used to evaluate the clinical application efficiency of this method. RESULTS: The quantitative detection range of NGAL in AKI was 1-3000 ng/mL, and the detection sensitization was 0.36 ng/mL. The coefficient of variation (CV) of intra-assay and inter-assay were 2.57-4.98 % and 4.11-7.83 %, respectively. Meanwhile, the correlation coefficient between europium nanoparticles-based lateral fluorescence immunoassays (EU-NPS-LFIA) and ARCHITECT analyzer was significant (R2 = 0.9829, n = 83, p < 0.01). CONCLUSIONS: Thus, a faster and easier operation quantitative assay of NGAL for AKI has been established, which is very important and meaningful to diagnose the early AKI, suggesting that the assay can provide an early warning of final outcome of disease.


Assuntos
Injúria Renal Aguda/diagnóstico , Európio , Fluorimunoensaio/métodos , Lipocalina-2/urina , Nanopartículas Metálicas , Injúria Renal Aguda/virologia , Animais , Anticorpos Monoclonais/isolamento & purificação , COVID-19/complicações , Ensaio de Imunoadsorção Enzimática , Humanos , Lipocalina-2/imunologia , Camundongos , Proteínas Recombinantes/isolamento & purificação , Reprodutibilidade dos Testes , SARS-CoV-2
12.
J Fluoresc ; 32(2): 419-426, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35025016

RESUMO

This study aimed to establish a Europium label time-resolved fluorescence immunoassay (TRFIA) to detect the chronic kidney disease (CKD) biomarker Cystatin-C. An Europium based Time resolved fluorescence immunoassay was developed to detect the concentration of Cystatin-C in a urine sample to increase the sensitivity with captured anti-Cystatin-C antibodies immobilized on nitrocellulose membrane and then bonded with detection anti-Cystatin-C labelled with CM-EU, followed by fluorescence measurement using time-resolved fluorometry in 15 min. The performance of this TRFIA was evaluated using the clinical urine serum and compared with the ELISA assays. The linear calibration range was 0.015-32 µg/ml, and the limit of detection (LOD) quantified was 0.0001 µg/ml. This current work has improved the LOD of our previous work from 0.013 µg/ml to 0.001 µg/ml. These results indicated that the CM-EU nanoparticle-based LFIA is rapid, more sensitive, reliable, and reproducible for point-of-care testing of Cys-C concentrations in urine.


Assuntos
Cistatina C/urina , Európio , Fluorimunoensaio/métodos , Insuficiência Renal Crônica/diagnóstico , Anticorpos/urina , Biomarcadores/urina , Cistatina C/imunologia , Humanos , Limite de Detecção , Nanopartículas
13.
J Fluoresc ; 32(2): 629-636, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35025017

RESUMO

The aim of this study was to establish a time-resolved fluorescent immunoassay (TRFIA) for the detection of serum Galectin-3 (Gal-3) and apply this method to evaluate the clinical significance of serum Gal-3 in predicting Idiopathic Membranous Nephropathy (IMN) progression. The Gal-3-TRFIA was established using the double antibody sandwich method, with the capture antibodies coated on a 96-well microplate and the detection antibodies chelated with Europium (III) (Eu3+). Serum Gal-3 was detected in 81 patients with IMN and 123 healthy controls to further evaluate the value of the Gal-3 in staging of IMN. The sensitivity of the Gal-3-TRFIA assay was 0.85 ng/mL, and the detection range was 0.85-1000 ng/mL. The Gal-3 intra-batch and inter-batch coefficients of variation were 3.45% and 5.12%, respectively. The correlation coefficient (R) between the Gal-3-TRFIA assay and commercially available enzyme-linked immunosorbent assay kits was 0.83. The serum Gal-3 concentration was higher in patients with IMN (65.57 ± 55.90 ng/mL) compared to healthy controls (16.29 ± 9.91 ng/mL, P < 0.0001). In this study, a wide detection range Gal-3-TRFIA assay was developed using lanthanide (Eu3+) chelates for the detection of Gal-3 concentrations in serum. Gal-3 concentration is elevated in patients with IMN.


Assuntos
Fluorimunoensaio/métodos , Galectina 3/sangue , Glomerulonefrite Membranosa/sangue , Glomerulonefrite Membranosa/diagnóstico , Anticorpos/sangue , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Galectina 3/imunologia , Humanos , Estudos Prospectivos , Sensibilidade e Especificidade , Fatores de Tempo
14.
Sci Rep ; 12(1): 105, 2022 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-34996935

RESUMO

Soluble immune checkpoint molecules are emerging novel mediators of immune regulation. However, it is unclear whether soluble immune checkpoint proteins affect the development of hepatocellular carcinoma (HCC) during nucleos(t)ide analogue (NA) treatment in patients with chronic hepatitis B virus infection. This study included 122 NA-naïve patients who received NA therapy. We assessed the associations of clinical factors, including soluble immune checkpoint proteins, with HCC development during NA treatment. The baseline serum concentrations of 16 soluble immune checkpoint proteins were measured using multiplexed fluorescent bead-based immunoassay. In total, 13 patients developed HCC during the follow-up period (median duration, 4.3 years). Of the 16 proteins, soluble inducible T-cell co-stimulator (≥ 164.71 pg/mL; p = 0.014), soluble programmed cell death-1 (sPD-1) (≤ 447.27 pg/mL; p = 0.031), soluble CD40 (≤ 493.68 pg/mL; p = 0.032), and soluble herpes virus entry mediator (≤ 2470.83 pg/mL; p = 0.038) were significantly associated with HCC development (log-rank test). In multivariate analysis, an sPD-1 level ≤ 447.27 pg/mL (p = 0.014; hazard ratio [HR], 4.537) and α-fetoprotein level ≥ 6.4 ng/mL (p = 0.040; HR, 5.524) were independently and significantly associated with HCC development. Pre-treatment sPD-1 is a novel predictive biomarker for HCC development during NA treatment.


Assuntos
Antivirais/uso terapêutico , Carcinoma Hepatocelular/prevenção & controle , Guanina/análogos & derivados , Hepatite B Crônica/tratamento farmacológico , Neoplasias Hepáticas/prevenção & controle , Nucleosídeos/uso terapêutico , Receptor de Morte Celular Programada 1/sangue , Adulto , Idoso , Biomarcadores/sangue , Carcinoma Hepatocelular/diagnóstico por imagem , Carcinoma Hepatocelular/virologia , Feminino , Fluorimunoensaio , Guanina/efeitos adversos , Guanina/uso terapêutico , Hepatite B Crônica/sangue , Hepatite B Crônica/diagnóstico , Hepatite B Crônica/virologia , Humanos , Neoplasias Hepáticas/diagnóstico por imagem , Neoplasias Hepáticas/virologia , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Fatores de Tempo , Resultado do Tratamento , Adulto Jovem
15.
J Hazard Mater ; 426: 127845, 2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-34865894

RESUMO

Pollution of N-methyl carbamate (NMC) pesticides is threatening the non-target organisms' survival. Thus, broad-specific antibodies and class-selective immunoassays are demanding for multiple NMCs determination. In this study, we employed a molecular docking-based virtual screening strategy to fast profile antibody spectrum, based on a designed chemical pool containing 17 compounds. A monoclonal antibody (mAb)-6G against carbofuran was used as the objective. The recombinant full-length IgG was successfully expressed to validate the antibody sequences for homology modeling. After docking, we manually categorized the antibody-chemical binding strength into three groups. Non-competitive surface plasmon resonance (SPR) demonstrated the mAb-6G affinitive binding toward five NMCs (carbofuran, isoprocarb, propoxur, carbaryl and carbosulfan), which were classified into strong and moderate binding categories. Antibody binding properties were confirmed again by ic-ELISA and lateral flow immunochromatographic strip. Subsequently, an ultrasensitive indirect competitive fluoromicrosphere-based immunoassay (ic-FMIA) was established with the IC50 (half-maximal inhibitory concentration) values of 0.08-3.37 ng/mL. This portable assay presented a 30-230-fold improved sensitivity than traditional ic-ELISA and was applied in European surface water analysis. Overall, our work provides an efficient platform integrating in-silico and experimental methodologies to accelerate the characterization of hapten-specific antibody binding properties and the development of high-sensitive immunoassays for multi-pollutants monitoring.


Assuntos
Praguicidas , Carbamatos , Computadores , Ensaio de Imunoadsorção Enzimática , Fluorimunoensaio , Imunoensaio , Simulação de Acoplamento Molecular
16.
J Appl Microbiol ; 132(2): 1250-1259, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34312955

RESUMO

AIMS: Alternaria longipes is a causal agent of brown spot of tobacco, which remains a serious threat to tobacco production. Herein, we established a detection method for A. longipes in tobacco samples based on the principle of time-resolved fluoroimmunoassay, in order to fulfil the requirement of rapid, sensitive and accurate detection in situ. METHODS AND RESULTS: A monoclonal antibody against A. longipes was generated, and its purity and titration were assessed using western blot and ELISA. The size of europium (III) nanospheres was measured to confirm successful antibody conjugation. The method described here can detect A. longipes protein lysates as low as 0.78 ng ml-1 , with recovery rates ranging from 85.96% to 99.67% in spiked tobacco. The specificity was also confirmed using a panel of microorganisms. CONCLUSIONS: The fluorescent strips allow rapid and sensitive onsite detection of A. longipes in tobacco samples, with high accuracy, specificity, and repeatability. SIGNIFICANCE AND IMPACT OF THE STUDY: This novel detection method provides convenience of using crude samples without complex procedures, and therefore allows rapid onsite detection by end users and quick responses towards A. longipes, which is critical for disease control and elimination of phytopathogens.


Assuntos
Alternaria , Tabaco , Ensaio de Imunoadsorção Enzimática , Fluorimunoensaio
17.
J Hazard Mater ; 424(Pt C): 127411, 2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-34629198

RESUMO

The excessive use of carbaryl has resulted in the risk of its exposure. In this study, we isolated six nanobodies (Nbs) from a camelid phage display library against the biomarker of carbaryl, 1-naphthol (1-NAP). Owing to its characteristics of easy genetic modifications, we produced a nanobody-alkaline phosphatase (Nb-CC4-ALP) fusion protein with good stability. A dual-emission system based ratiometric fluoroimmunoassay (RFIA) for quick and highly sensitive determination of 1-NAP was developed. Silicon nanoparticles (SiNPs) was used as an internal reference and for aggregation-induced emission enhancement (AIEE) of gold nanoclusters (AuNCs), while AuNCs could be quenched by MnO2 via oxidation. In the presence of ALP, ascorbic acid phosphate (AAP) can be transformed into ascorbic acid (AA), the later can etch MnO2 to recover the fluorescence of the AuNCs. Based on optimal conditions, the proposed assay showed 220-fold sensitivity improvement in comparison with conventional monoclonal antibody-based ELISA. The recovery test of urine samples and the validation by standard HPLC-FLD demonstrated the proposed assay was an ideal tool for screening 1-NAP and provided technical support for the monitoring of carbaryl exposure.


Assuntos
Nanopartículas Metálicas , Praguicidas , Fosfatase Alcalina/genética , Carbaril/toxicidade , Fluorimunoensaio , Limite de Detecção , Compostos de Manganês , Nanopartículas Metálicas/toxicidade , Naftóis , Óxidos , Fosfatos
18.
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
19.
J Immunol Methods ; 499: 113179, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34728217

RESUMO

OBJECTIVE: In this study, a novel, simple, and rapid immunoassay for the determination of gastrin-17 (G-17) in human serum was established by combining immunomagnetic beads with time-resolved fluorescence immunoassay (TRFIA). METHODS: Immunomagnetic beads were coated with anti-G-17 M01 antibody, anti-G-17 M02 antibody was labeled with Eu3+ chelates. The concentration of G-17 in the serum was detected with the double-antibody sandwich method. RESULTS: The limit of background(LOB), limit of detection (LOD), and limit of quantification (LOQ) were 0.09, 0.104, and 0.39 pmol/L, respectively. The detection range of G-17-TRFIA was 0.39-100 pmol/L. The average intra- and inter-assay coefficients of variation (CV) were 5.95%-9.07% and 6.09%-8.14%, respectively. The recoveries for the serum samples ranged from 94.70% to 100.95%. The specificity of our G-17-TRFIA was acceptable. The correlation coefficient between G-17-TRFIA and commercial G-17-ELISA methods was R2 = 0.9092. CONCLUSIONS: A novel G-17-TRFIA detection method was successfully established to provide a reference for the early diagnosis of patients with atrophic gastritis in clinical research.


Assuntos
Ensaio de Imunoadsorção Enzimática , Fluorimunoensaio , Gastrinas/sangue , Separação Imunomagnética , Anticorpos/química , Anticorpos/imunologia , Gastrinas/imunologia , Humanos , Fatores de Tempo
20.
J Fluoresc ; 31(6): 1771-1777, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34495467

RESUMO

Currently, atherosclerosis accounts for the majority of cardiovascular morbidity and mortality worldwide, and predicting the stability of atherosclerotic plaque is the main method to prevent atherosclerotic death. This study aims to establish a dual-label time-resolved fluorescence immunoassay (TRFIA) of matrix metalloprotein-9 (MMP-9) and lipoprotein-associated phospholipaseA2 (Lp-PLA2) to predict atherosclerotic plaque stability. A dual-label TRFIA was introduced for the simultaneous quantification of MMP-9 and Lp-PLA2 using fluorescent lanthanide (Eu3+ and Sm3+) chelates. The performance (sensitivity, specificity, accuracy, precision and reference intervals in different subjects) of this TRFIA was evaluated and compared with commercial kit. The sensitivity of the TRFIA for MMP-9 was 0.85 ng/mL and for Lp-PLA2 was 0.68 ng/mL with high affinity and specificity. The average recoveries were 94.58% to 109.82%, and 104.32% to 109.26%, respectively. All intra- and inter-assay CVs ranged from 3.10% to 5.46%. For the normal subjects, the cutoff value was 160.70 ng/mL for MMP-9 and 183.73 ng/mL for LP-PLA2; for the subjects with stable plaque, the cutoff value was 181.98~309.22 ng/mL for MMP-9 and 194.73~337.89 ng/mL for LP-PLA2; for the subjects with unstable plaque, the cutoff value was 330.43 ng/mL for MMP-9 and 343.23 ng/mL for LP-PLA2. This TRFIA detection results agreed well with the results of commercial kit (R2=0.9567 and R2=0.9771, respectively) in clinical serum samples. The TRFIA developed has a wide detection range and good sensitivity for the high-throughput simultaneous detection of MMP-9 and Lp-PLA2 in serum, which provides a new method for predicting the stability of atherosclerotic plaque.


Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/sangue , Fluorimunoensaio , Metaloproteinase 9 da Matriz/sangue , 1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Európio/química , Humanos , Metaloproteinase 9 da Matriz/metabolismo , Samário/química , Fatores de Tempo
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