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1.
Sci Adv ; 10(28): eadn5698, 2024 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-38985882

RESUMO

Gold nanoparticle-based lateral flow immunoassays (AuNP LFIAs) are widely used point-of-care (POC) sensors for in vitro diagnostics. However, the sensitivity limitation of conventional AuNP LFIAs impedes the detection of trace biomarkers. Several studies have explored the size and shape factors of AuNPs and derivative nanohybrids, showing limited improvements or enhanced sensitivity at the cost of convenience and affordability. Here, we investigated surface chemistry on the sensitivity of AuNP LFIAs. By modifying surface ligands, a surface chemistry strategy involving weakly ionized AuNPs enables ultrasensitive naked-eye LFIAs (~100-fold enhanced sensitivity). We demonstrated how this surface chemistry-amplified immunoassay approach modulates nanointerfacial bindings to promote antibody adsorption and higher activity of adsorbed antibodies. This surface chemistry design eliminates complex nanosynthesis, auxiliary devices, or additional reagents while efficiently improving sensitivity with advantages: simplified fabrication process, excellent reproducibility and reliability, and ultrasensitivity toward various biomarkers. The surface chemistry using weakly ionized AuNPs represents a versatile approach for sensitizing POC sensors.


Assuntos
Ouro , Nanopartículas Metálicas , Sistemas Automatizados de Assistência Junto ao Leito , Ouro/química , Nanopartículas Metálicas/química , Imunoensaio/métodos , Humanos , Técnicas Biossensoriais/métodos , Reprodutibilidade dos Testes , Biomarcadores/análise
2.
J Vis Exp ; (208)2024 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-39007561

RESUMO

Quantum dots, also known as semiconductor nanocrystals, are novel fluorescent labels for biological imaging and sensing. However, quantum dot-antibody conjugates with small dimensions (~10 nm), prepared through laborious purification procedures, exhibit limited sensitivity in detecting certain trace disease markers using lateral flow immunoassay strips. Herein, we present a method for the preparation of quantum dot nanobeads (QDNB) using a one-step emulsion evaporation method. Using the as-prepared QDNB, a fluorescent lateral flow immunoassay was fabricated to detect disease biomarkers using C-reactive protein (CRP) as an example. Unlike single quantum dot nanoparticles, quantum dot nanobead-antibody conjugates are more sensitive as immunoassay labels due to signal amplification by encapsulating hundreds of quantum dots in one polymer composite nanobead. Moreover, the larger size of QDNBs facilitates easier centrifugation separation when conjugating QDNBs with antibodies. The fluorescent lateral flow immunoassay based on QDNBs was fabricated, and the CRP concentration in the sample was measured in 15 min. The test results can be qualitatively assessed under UV light illumination and quantitatively measured using a fluorescent reader within 15 min.


Assuntos
Proteína C-Reativa , Pontos Quânticos , Pontos Quânticos/química , Imunoensaio/métodos , Imunoensaio/instrumentação , Proteína C-Reativa/análise , Humanos , Corantes Fluorescentes/química
3.
Sensors (Basel) ; 24(13)2024 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-39001098

RESUMO

The quartz tuning fork (QTF) is a promising instrument for biosensor applications due to its advanced properties such as high sensitivity to physical quantities, cost-effectiveness, frequency stability, and high-quality factor. Nevertheless, the fork's small size and difficulty in modifying the prongs' surfaces limit its wide use in experimental research. Our study presents the development of a QTF immunosensor composed of three active layers: biocompatible natural melanin nanoparticles (MNPs), glutaraldehyde (GLU), and anti-IgG layers, for the detection of immunoglobulin G (IgG). Frequency shifts of QTFs after MNP functionalization, GLU activation, and anti-IgG immobilization were measured with an Asensis QTF F-master device. Using QTF immunosensors that had been modified under optimum conditions, the performance of QTF immunosensors for IgG detection was evaluated. Accordingly, a finite element method (FEM)-based model was produced using the COMSOL Multiphysics software program (COMSOL License No. 2102058) to simulate the effect of deposited layers on the QTF resonance frequency. The experimental results, which demonstrated shifts in frequency with each layer during QTF surface functionalization, corroborated the simulation model predictions. A modelling error of 0.05% was observed for the MNP-functionalized QTF biosensor compared to experimental findings. This study validated a simulation model that demonstrates the advantages of a simulation-based approach to optimize QTF biosensors, thereby reducing the need for extensive laboratory work.


Assuntos
Técnicas Biossensoriais , Imunoglobulina G , Melaninas , Nanopartículas , Quartzo , Imunoglobulina G/química , Imunoglobulina G/imunologia , Técnicas Biossensoriais/métodos , Técnicas Biossensoriais/instrumentação , Nanopartículas/química , Melaninas/química , Quartzo/química , Imunoensaio/métodos , Imunoensaio/instrumentação , Simulação por Computador , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Anti-Idiotípicos/química , Humanos
4.
Methods Mol Biol ; 2829: 277-286, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38951344

RESUMO

Quantitative immunoassays, such as the traditional enzyme-linked immunosorbent assay (ELISA), are used to determine concentrations of an antigen in a matrix of unknown antigen concentration. Magnetic immunoassays, such as the Luminex xMAP technology, allow for the simultaneous detection of multiple analytes and offer heightened sensitivity, specificity, low sample volume requirements, and high-throughput capabilities. Here, we describe a quantitative immunoassay using the Luminex MAGPIX® System to determine the antigen concentration from liquid samples with unknown concentrations. In detail, we describe a newly developed assay for determining production yields of Drosophila S2-produced Marburg virus (MARV) glycoprotein in insect-cell-culture-derived supernatant. The potential applications of this assay could extend to the quantification of viral antigens in fluids derived from both in vitro and in vivo models infected with live MARV, thereby providing additional applications for virological research.


Assuntos
Antígenos Virais , Microesferas , Animais , Imunoensaio/métodos , Antígenos Virais/imunologia , Antígenos Virais/análise , Marburgvirus/imunologia , Marburgvirus/isolamento & purificação , Drosophila , Técnicas de Cultura de Células/métodos , Linhagem Celular , Ensaio de Imunoadsorção Enzimática/métodos
5.
Pan Afr Med J ; 48: 10, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38946743

RESUMO

Introduction: the utility of glycated haemoglobin (HbA1c) for the diagnosis and monitoring of diabetes in sub-Saharan Africa is uncertain due to limited data on the performance of the available HbA1c assay methods in this population, which has a high prevalence of haemoglobin variants. We aimed to compare the diagnostic accuracy of the major HbA1c methodologies (Boronate Affinity, Capillary Electrophoresis, High Performance Liquid Chromatography, Immunoassay) in an African population, and assess the impact of the common haemoglobin variant HbAS (sickle cell trait). Methods: whole blood samples were obtained from 182 individuals living with type 2 diabetes in Uganda. HbA1c values for each method were compared to average glucose measured over 14 days by continuous glucose monitoring (CGM). To determine concordance, the three HbA1c assay methods were compared to the capillary electrophoresis method. Results: there was a strong correlation between CGM average glucose levels and all four HbA1c methodologies (r=0.81-0.89) which did not differ in those with and without HbAS (present in 37/182 participants). The presence of HbAS did not alter the relationship between HbA1c and CGM glucose for any assay (p for interaction >0.2 for all methods). Diagnostic accuracy for CGM average glucose thresholds of 7 and 10mmol/L was similar across methods (area under the receiver operating characteristic curve 0.80-0.84 and 0.76-0.84 respectively). The maximum bias between the HbA1c assay methodologies was 2 mmol/mol (2.07%). Conclusion: all major HbA1c technologies offer accurate and comparable HbA1c measurement even in this population with high prevalence of haemoglobin variants.


Assuntos
Glicemia , Diabetes Mellitus Tipo 2 , Eletroforese Capilar , Hemoglobinas Glicadas , Sensibilidade e Especificidade , Humanos , Hemoglobinas Glicadas/análise , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/sangue , Eletroforese Capilar/métodos , Feminino , Glicemia/análise , Masculino , Pessoa de Meia-Idade , Cromatografia Líquida de Alta Pressão/métodos , Uganda , Adulto , Imunoensaio/métodos , Imunoensaio/normas , Automonitorização da Glicemia/métodos , Idoso , Hemoglobinas Anormais/análise
6.
Lab Chip ; 24(14): 3536-3545, 2024 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-38946347

RESUMO

Early-stage diagnosis of prostatic carcinoma is essential for successful treatment and, thus, significant prognosis improvement. In laboratory practice, the standard non-invasive diagnostic approach is the immunochemical detection of the associated biomarker, prostate-specific antigen (PSA). Ultrasensitive detection of PSA is essential for both diagnostic and recurrence monitoring purposes. To achieve exceptional sensitivity, we have developed a microfluidic device with a flow-through cell for single-molecule analysis using photon-upconversion nanoparticles (UCNPs) as a detection label. For this purpose, magnetic microparticles (MBs) were first optimized for the capture and preconcentration of PSA and then used to implement a bead-based upconversion-linked immunoassay (ULISA) in the microfluidic device. The digital readout based on counting single nanoparticle-labeled PSA molecules on MBs enabled a detection limit of 1.04 pg mL-1 (36 fM) in 50% fetal bovine serum, which is an 11-fold improvement over the respective analog MB-based ULISA. The microfluidic technique conferred several other advantages, such as easy implementation and the potential for achieving high-throughput analysis. Finally, it was proven that the microfluidic setup is suitable for clinical sample analysis, showing a good correlation with a reference electrochemiluminescence assay (recovery rates between 97% and 105%).


Assuntos
Antígeno Prostático Específico , Antígeno Prostático Específico/análise , Antígeno Prostático Específico/sangue , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Masculino , Nanopartículas/química , Imunoensaio/instrumentação , Imunoensaio/métodos , Limite de Detecção , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/sangue
7.
Sex Transm Dis ; 51(8): 545-547, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-38989902

RESUMO

ABSTRACT: At our medical center, HIV nucleic acid tests are recommended when the HIV antigen-antibody screening immunoassay and antibody differentiation tests are discordant, but not done reflexively. A retrospective chart review found that 35% of discordant test results did not have HIV nucleic acid test completed as recommended.


Assuntos
Algoritmos , Infecções por HIV , Técnicas de Amplificação de Ácido Nucleico , Humanos , Infecções por HIV/diagnóstico , Estudos Retrospectivos , Masculino , Feminino , Adulto , Teste de HIV , RNA Viral , Programas de Rastreamento/métodos , Pessoa de Meia-Idade , HIV-1/isolamento & purificação , HIV-1/imunologia , Anticorpos Anti-HIV/sangue , Imunoensaio/métodos
8.
Clin Chim Acta ; 561: 119839, 2024 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-38964570

RESUMO

Immunoassays are important tools in diagnosing giardiasis, though there are several controversies inherent in the existing methods. We conducted a systematic review and meta-analysis to assess the pooled diagnostic accuracy of immunoassays in detecting the gastrointestinal disease-causing parasite Giardia lamblia. Our comprehensive search, which included PubMed, Scopus, and ScienceDirect from 2000 up until 2023, resulted in 34 studies reporting the performance of 24 different immunoassays. The overall pooled sensitivity and specificity of immunoassays and subgroup analyses were determined. Notably, ImmunoCardSTAT® and RIDASCREEN® Giardia were the most used assays (n = 6 studies each). They exhibited sensitivity and specificity of 84 % and 99 % and 93 % and 99 %, respectively. Sub-group analysis on the type of immunoassays (without the case-control studies) showed that commercial ELISA had higher sensitivity (96 %) compared to a commercial immunochromatographic (88 %), which justifies the difference of sensitivity between ImmunoCardSTAT® and RIDASCREEN® Giardia. However, the applicability between these two in clinical settings, replacing the gold standard, should be considered including the time, equipment requirement, and budget. Samples from symptomatic patients showed higher sensitivity (92 %) compared to asymptomatic patients (79 %). Overall, immunoassays can be a practical replacement for the current gold standard, but more information should be gathered regarding the cost of providing more conclusive suggestions on these findings.


Assuntos
Giardia lamblia , Giardíase , Giardia lamblia/imunologia , Giardia lamblia/isolamento & purificação , Imunoensaio/métodos , Humanos , Giardíase/diagnóstico , Giardíase/imunologia
9.
Molecules ; 29(13)2024 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-38998952

RESUMO

The sensitivity of immunoassays is generally limited by the low signal reporter/recognition element ratio. Nanomaterials serving as the carriers can enhance the loading number of signal reporters, thus improving the detection sensitivity. However, the general immobilization strategies, including direct physical adsorption and covalent coupling, may cause the random orientation and conformational change in proteins, partially or completely suppressing the enzymatic activity and the molecular recognition ability. In this work, we proposed a strategy to load recognition elements of antibodies and enzyme labels using boronic acid-modified metal-organic frameworks (MOFs) as the nanocarriers for signal amplification. The conjugation strategy was proposed based on the boronate ester interactions between the carbohydrate moieties in antibodies and enzymes and the boronic acid moieties on MOFs. Both enzymes and MOFs could catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by H2O2, therefore achieving dual signal amplification. To indicate the feasibility and sensitivity of the strategy, colorimetric immunoassays of prostate specific antigen (PSA) were performed with boronic acid-modified Cu-MOFs as peroxidase mimics to catalyze TMB oxidation and nanocarriers to load antibody and enzyme (horseradish peroxidase, HRP). According to the change in the absorbance intensity of the oxidized TMB (oxTMB), PSA at the concentration range of 1~250 pg/mL could be readily determined. In addition, this work presented a site-specific and oriented conjugation strategy for the modification of nanolabels with recognition elements and signal reporters, which should be valuable for the design of novel biosensors with high sensitivity and selectivity.


Assuntos
Ácidos Borônicos , Colorimetria , Estruturas Metalorgânicas , Estruturas Metalorgânicas/química , Colorimetria/métodos , Ácidos Borônicos/química , Imunoensaio/métodos , Humanos , Benzidinas/química , Oxirredução , Antígeno Prostático Específico/análise , Peróxido de Hidrogênio/química , Anticorpos/química , Técnicas Biossensoriais/métodos , Peroxidase do Rábano Silvestre/química , Peroxidase do Rábano Silvestre/metabolismo
10.
World J Microbiol Biotechnol ; 40(9): 269, 2024 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-39009934

RESUMO

Gold Nanoparticles (AuNPs) have gained significant attention in biosensor development due to their unique physical, chemical, and optical properties. When incorporated into biosensors, AuNPs offer several advantages, including a high surface area-to-volume ratio, excellent biocompatibility, ease of functionalization, and tunable optical properties. These properties make them ideal for the detection of various biomolecules, including proteins, nucleic acids, and bacterial and viral biomarkers. Traditional methods for detecting bacteria and viruses, such as RT-PCR and ELISA, often suffer from complexities, time consumption, and labor intensiveness. Consequently, researchers are continuously exploring novel devices to address these limitations and effectively detect a diverse array of infectious pathogenic microorganisms. In light of these challenges, nanotechnology has been instrumental in refining the architecture and performance of biosensors. By leveraging advancements in nanomaterials and strategies of biosensor fabrication the sensitivity and specificity of biosensors can be enhanced, enabling more precise detection of pathogenic bacteria and viruses. This review explores the versatility of AuNPs in detecting a variety of biomolecules, including proteins, nucleic acids, and bacterial and viral biomarkers. Furthermore, it evaluates recent advancements in AuNPs-based biosensors for the detection of pathogens, utilizing techniques such as optical biosensors, lateral flow immunoassays, colorimetric immunosensors, electrochemical biosensors, and fluorescence nanobiosensors. Additionally, the study discusses the existing challenges in the field and proposes future directions to improve AuNPs-based biosensors, with a focus on enhancing sensitivity, selectivity, and their utility in clinical and diagnostic applications.


Assuntos
Bactérias , Técnicas Biossensoriais , Ouro , Nanopartículas Metálicas , Vírus , Técnicas Biossensoriais/métodos , Ouro/química , Nanopartículas Metálicas/química , Vírus/isolamento & purificação , Bactérias/isolamento & purificação , Nanotecnologia/métodos , Humanos , Biomarcadores/análise , Viroses/diagnóstico , Imunoensaio/métodos
11.
Anal Chim Acta ; 1316: 342813, 2024 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-38969419

RESUMO

In the immunoassay process, for fulfilling the need to identify multiple analytes in a small amount of complex sample matrix, it is desirable to develop highly efficient and specific multiplex suspension array technology. Raman coding strategy offers an attractive solution to code the suspension arrays by simply combing narrow spectral bands with stable signal intensities through solid-phase synthesis on the resin beads. Based on this strategy, we report the bead-based spontaneous Raman codes for multiplex immunoassay. The study resulted in superior selectivity of the Raman-encoded beads for binding with single and multiple analytes, respectively. With the use of mixed types of Raman-encoded immunoassay beads, multiple targets in small amounts of samples were identified rapidly and accurately. By confirming the feasibility of bead-based spontaneous Raman codes for multiplex immunoassay, we anticipate this novel technology to be widely applied in the near future.


Assuntos
Análise Espectral Raman , Análise Espectral Raman/métodos , Imunoensaio/métodos , Humanos
12.
Vet Med Sci ; 10(4): e1532, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38952277

RESUMO

BACKGROUND: Antibodies have been proven effective as diagnostic agents for detecting zoonotic diseases. The variable domain of camel heavy chain antibody (VHH), as an antibody derivative, may be used as an alternative for traditional antibodies in existing immunodiagnostic reagents for detecting rapidly spreading infectious diseases. OBJECTIVES: To expedite the isolation of specific antibodies for diagnostic purposes, we constructed a semi-synthetic camel single domain antibody library based on the phage display technique platform (PDT) and verified the validity of this study. METHODS: The semi-synthetic single domain antibody sequences consist of two parts: one is the FR1-FR3 region amplified by RT-PCR from healthy camel peripheral blood lymphocytes (PBLs), and the other part is the CDR3-FR4 region synthesised as an oligonucleotide containing CDR3 randomised region. The two parts were fused by overlapping PCR, resulting in the rearranged variable domain of heavy-chain antibodies (VHHs). Y. pestis low-calcium response V protein (LcrV) is an optional biomarker to detect the Y. pestis infection. The semi-synthetic library herein was screened using recombinant (LcrV) as a target antigen. RESULTS: After four cycles of panning the library, four VHH binders targeting 1-270 aa residues of LcrV were isolated. The four VHH genes with unique sequences were recloned into an expression vector and expressed as VHH-hFc chimeric antibodies. The purified antibodies were identified and used to develop a lateral flow immunoassay (LFA) test strip using latex microspheres (LM) for the rapid and visual detection of Y. pestis infection. CONCLUSIONS: These data demonstrate the great potential of the semi-synthetic library for use in isolation of antigen-specific nanobodies and the isolated specific VHHs can be used in antigen-capture immunoassays.


Assuntos
Antígenos de Bactérias , Camelus , Anticorpos de Domínio Único , Yersinia pestis , Animais , Yersinia pestis/imunologia , Anticorpos de Domínio Único/imunologia , Antígenos de Bactérias/imunologia , Peste/diagnóstico , Peste/veterinária , Peste/imunologia , Imunoensaio/métodos , Imunoensaio/veterinária , Anticorpos Antibacterianos/imunologia
13.
J Agric Food Chem ; 72(26): 14967-14974, 2024 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-38957086

RESUMO

Nanobodies (Nbs) serve as powerful tools in immunoassays. However, their small size and monovalent properties pose challenges for practical application. Multimerization emerges as a significant strategy to address these limitations, enhancing the utilization of nanobodies in immunoassays. Herein, we report the construction of a Salmonella-specific fenobody (Fb) through the fusion of a nanobody to ferritin, resulting in a self-assembled 24-valent nanocage-like structure. The fenobody exhibits a 35-fold increase in avidity compared to the conventional nanobody while retaining good thermostability and specificity. Leveraging this advancement, three ELISA modes were designed using Fb as the capture antibody, along with unmodified Nb422 (FbNb-ELISA), biotinylated Nb422 (FbBio-ELISA), and phage-displayed Nb422 (FbP-ELISA) as the detection antibody, respectively. Notably, the FbNb-ELISA demonstrates a detection limit (LOD) of 3.56 × 104 CFU/mL, which is 16-fold lower than that of FbBio-ELISA and similar to FbP-ELISA. Moreover, a fenobody and nanobody sandwich chemiluminescent enzyme immunoassay (FbNb-CLISA) was developed by replacing the TMB chromogenic substrate with luminal, resulting in a 12-fold reduction in the LOD. Overall, the ferritin-displayed technology represents a promising methodology for enhancing the detection performance of nanobody-based sandwich ELISAs, thereby expanding the applicability of Nbs in food detection and other fields requiring multivalent modification.


Assuntos
Ensaio de Imunoadsorção Enzimática , Ferritinas , Salmonella , Anticorpos de Domínio Único , Ferritinas/imunologia , Ferritinas/química , Ferritinas/genética , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/imunologia , Salmonella/imunologia , Salmonella/genética , Ensaio de Imunoadsorção Enzimática/métodos , Limite de Detecção , Afinidade de Anticorpos , Anticorpos Antibacterianos/imunologia , Imunoensaio/métodos
14.
Mikrochim Acta ; 191(8): 453, 2024 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-38970675

RESUMO

An electrochemical biosensor has been developed for detection of Escherichia coli O157 by integrating lateral flow with screen-printed electrodes. The screen-printed electrodes were attached under the lateral flow detection line, and organic-inorganic nanoflowers prepared from E. coli O157-specific antibodies as an organic component were attached to the lateral flow detection line. In the presence of E. coli O157, an organic-inorganic nanoflower-E. coli O157-antimicrobial peptide-labelled ferrocene sandwich structure is formed on the lateral flow detection line. Differential pulse voltammetry is applied using a smartphone-based device to monitor ferrocene on the detection line. The resulting electrochemical biosensor could specifically detect E. coli O157 with a limit of detection of 25 colony-forming units mL-1. Through substitution of antibodies of organic components in organic-inorganic nanoflowers, biosensors have great potential for the detection of other pathogens in biomedical research and clinical diagnosis.


Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas , Escherichia coli O157 , Escherichia coli O157/isolamento & purificação , Escherichia coli O157/imunologia , Técnicas Biossensoriais/métodos , Imunoensaio/métodos , Imunoensaio/instrumentação , Técnicas Eletroquímicas/métodos , Técnicas Eletroquímicas/instrumentação , Limite de Detecção , Nanoestruturas/química , Eletrodos , Compostos Ferrosos/química , Anticorpos Imobilizados/imunologia , Metalocenos/química , Anticorpos Antibacterianos/química , Anticorpos Antibacterianos/imunologia , Peptídeos Antimicrobianos/química
15.
Mikrochim Acta ; 191(7): 434, 2024 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-38951317

RESUMO

An enhanced lateral flow assay (LFA) is presented for rapid and highly sensitive detection of acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antigens with gold nanoflowers (Au NFs) as signaling markers and gold enhancement to amplify the signal intensities. First, the effect of the morphology of gold nanomaterials on the sensitivity of LFA detection was investigated. The results showed that Au NFs prepared by the seed growth method showed a 5-fold higher detection sensitivity than gold nanoparticles (Au NPs) of the same particle size, which may benefit from the higher extinction coefficient and larger specific surface area of Au NFs. Under the optimized experimental conditions, the Au NFs-based LFA exhibited a detection limit (LOD) of 25 pg mL-1 for N protein using 135 nm Au NFs as the signaling probes. The signal was further amplified by using a gold enhancement strategy, and the LOD for the detection of N protein achieved was 5 pg mL-1. The established LFA also exhibited good repeatability and stability and showed applicability in the diagnosis of SARS-CoV-2 infection.


Assuntos
Antígenos Virais , Proteínas do Nucleocapsídeo de Coronavírus , Ouro , Limite de Detecção , Nanopartículas Metálicas , SARS-CoV-2 , Ouro/química , SARS-CoV-2/imunologia , Nanopartículas Metálicas/química , Humanos , Antígenos Virais/análise , Antígenos Virais/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/análise , Fosfoproteínas/imunologia , Fosfoproteínas/análise , Fosfoproteínas/química , COVID-19/diagnóstico , COVID-19/virologia , Imunoensaio/métodos , Teste Sorológico para COVID-19/métodos
16.
Front Endocrinol (Lausanne) ; 15: 1289923, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38978630

RESUMO

Objective: It is well known that macro-thyroid-stimulating hormone (macro-TSH) could interfere with the detection of TSH. The anti-TSH autoantibody is an essential component of macro-TSH. However, the epidemiological characteristics and the clinical interference of the anti-TSH autoantibody are unclear. Methods: In this study, the radioimmunoprecipitation technique was used to detect the anti-TSH autoantibody. Platforms with different detection mechanisms were applied to measure the TSH in patients with the anti-TSH autoantibody. Polyethylene glycol (PEG) precipitation was used to determine the immunoassay interference. Results: The prevalence of the anti-TSH autoantibody in patients with mild subclinical hypothyroidism (SCH) and autoimmune thyroiditis, but normal thyroid function, was 4.78%. All 10 patients with anti-TSH antibodies had autoimmune diseases, with five of them having significant clinical test interference. Conclusion: The appearance of the anti-TSH antibody is not associated with thyroid autoantibodies. The presence of the anti-TSH autoantibody can interfere with the detection of TSH and can affect clinical diagnosis and treatment.


Assuntos
Autoanticorpos , Hipotireoidismo , Tireotropina , Humanos , Autoanticorpos/sangue , Autoanticorpos/imunologia , Tireotropina/sangue , Tireotropina/imunologia , Feminino , Masculino , Adulto , Pessoa de Meia-Idade , Hipotireoidismo/diagnóstico , Hipotireoidismo/imunologia , Hipotireoidismo/sangue , Tireoidite Autoimune/imunologia , Tireoidite Autoimune/sangue , Tireoidite Autoimune/diagnóstico , Testes de Função Tireóidea , Idoso , Imunoensaio/métodos , Ensaio de Radioimunoprecipitação
17.
ACS Appl Bio Mater ; 7(7): 4702-4709, 2024 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-38910532

RESUMO

A label-free electrochemical immunosensor was developed for the rapid and sensitive detection of neuron-specific enolase (NSE). The electropolymerization of dopamine in conjunction with highly conductive carbon nanotubes offers a simple and quick platform for the direct anchoring of antibodies without the assistance of any coupling agent as well as a blocking agent. The developed immunosensor exhibited a wider detection range from 120 pM (9 ng mL-1) to 3 nM (200 ng mL-1) for NSE with a high sensitivity of 3.9 µA pM-1 cm-2 in 0.1 M phosphate-buffered saline (PBS) at physiological pH (7.4). Moreover, the short recognition time (15 min) for the antigen enabled the detection to be fast and less invasive. Additionally, the evaluation of a rate constant at various concentrations of NSE via feedback mode of scanning electrochemical microscopy (SECM) explained the profound effect of antigen concentration on the rate of flow of electrons. Therefore, the proposed immunosensor can be a promising tool for the early detection of small cell lung cancer in a very short period of time with consistent accuracy.


Assuntos
Materiais Biocompatíveis , Técnicas Biossensoriais , Indóis , Nanotubos de Carbono , Fosfopiruvato Hidratase , Polímeros , Nanotubos de Carbono/química , Fosfopiruvato Hidratase/imunologia , Fosfopiruvato Hidratase/metabolismo , Fosfopiruvato Hidratase/análise , Polímeros/química , Indóis/química , Humanos , Imunoensaio/métodos , Materiais Biocompatíveis/química , Teste de Materiais , Tamanho da Partícula , Técnicas Eletroquímicas
18.
Expert Rev Mol Diagn ; 24(6): 533-540, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38879820

RESUMO

BACKGROUND: Cryptococcosis is a global invasive mycosis associated with significant morbidity and mortality. Cryptococcal antigen (CrAg) testing from serum and cerebrospinal fluid (CSF) has been regarded as a gold standard for early diagnosis. This study aimed to develop and validate a rapid and sensitive sandwich chemiluminescent magnetic microparticle immunoassay (CMIA) for quantitative detection of CrAg in sera. RESEARCH DESIGN AND METHODS: CMIA is based on magnetic beads modified with capture antibodies and biotinylated antibodies and Streptavidin-polyHRP, where biotinylated antibodies functioned as the recognition element and Streptavidin-polyHRP as the signal component. Assay parameters were first optimized, and then assay performances were evaluated. RESULTS: Under optimized conditions, the total runtime of the CMIA was 22 min. The assay had a wide linear range (2 -10,000 ng/mL) and high analytical sensitivity (0.24 ng/mL), together with acceptable reproducibility, accuracy, and stability. Besides, it exhibited no cross-reactivity with other pathogens. Importantly, the assay showed 92.91% (95% CI, 80.97-93.02%) overall qualitative agreement with a commercial ELISA kit in a retrospective cohort of 55 cases with confirmed cryptococcal infection, and 72 controls without evidence of invasive fungal disease (IFD). CONCLUSION: These results demonstrated that the present study paved a novel strategy for reliable quantitative detection of CrAg in sera.


Assuntos
Antígenos de Fungos , Criptococose , Medições Luminescentes , Humanos , Antígenos de Fungos/sangue , Antígenos de Fungos/imunologia , Medições Luminescentes/métodos , Imunoensaio/métodos , Criptococose/diagnóstico , Criptococose/sangue , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Feminino , Masculino , Pessoa de Meia-Idade , Adulto , Cryptococcus/imunologia , Estudos Retrospectivos
19.
J Immunol Methods ; 531: 113699, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38823575

RESUMO

Bead array assays, such as those sold by Luminex, BD Biosciences, Sartorius, Abcam and other companies, are a well-established platform for multiplexed quantification of cytokines and other biomarkers in both clinical and discovery research environments. In 2011, the National Institute of Allergy and Infectious Diseases (NIAID)-funded External Quality Assurance Program Oversight Laboratory (EQAPOL) established a proficiency assessment program to monitor participating laboratories performing multiplex cytokine measurements using Luminex bead array technology. During every assessment cycle, each site was sent an assay kit, a protocol, and blinded samples of human sera spiked with recombinant cytokines. Site results were then evaluated for performance relative to peer laboratories. After over a decade of biannual assessments, the cumulative dataset contained over 15,500 bead array observations collected at more than forty laboratories in twelve countries. These data were evaluated alongside post-assessment survey results to empirically test factors that may contribute to variability and accuracy in Luminex bead-based cytokine assays. Bead material, individual technical ability, analyte, analyte concentration, and assay kit vendor were identified as significant contributors to assay performance. In contrast, the bead reader instrument model and the use of automated plate washers were found not to contribute to variability or accuracy, and sample results were found to be highly-consistent between assay kit-manufacturing lots and over time. In addition to these statistical analyses, subjective evaluations identified technical ability, instrument failure, protocol adherence, and data transcription errors as the most common causes of poor performance in the proficiency program. The findings from the EQAPOL multiplex program were then used to develop recommended best practices for bead array monitoring of human cytokines. These included collecting samples to assay as a single batch, centralizing analysis, participating in a quality assurance program, and testing samples using paramagnetic-bead kits from a single manufacturer using a standardized protocol.


Assuntos
Citocinas , Ensaio de Proficiência Laboratorial , Humanos , Citocinas/sangue , Reprodutibilidade dos Testes , Controle de Qualidade , Imunoensaio/métodos , Imunoensaio/normas , Estados Unidos , Biomarcadores/sangue
20.
Anal Chem ; 96(25): 10116-10120, 2024 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-38858219

RESUMO

In this letter, a sensitive microfluidic immunosensor chip was developed using tetrakis(4-aminophenyl)ethene (TPE)-derived covalent organic frameworks (T-COF) as aggregation-induced electrochemiluminescence (AIECL) emitters and nanobodies as efficient immune recognition units for the detection of thymic stromal lymphopoietin (TSLP), a novel target of asthma. The internal rotation and vibration of TPE molecules were constrained within the framework structure, forcing nonradiative relaxation to convert into pronounced radiative transitions. A camel-derived nanobody exhibited superior specificity, higher residual activity and epitope recognition postcuring compared to monoclonal antibodies. Benefiting from the affinity between silver ions (Ag+) and cytosine (C), a double-stranded DNA (dsDNA) embedded with Ag+ was modified onto the surface of TSLP. A positive correlation was obtained between the TSLP concentration (1.00 pg/mL to 4.00 ng/mL) and ECL intensity, as Ag+ was confirmed to be an excellent accelerator of the generation of free radical species. We propose that utilizing COF to constrain luminescent molecules and trigger the AIECL phenomenon is another promising method for preparing signal tags to detect low-abundance disease-related markers.


Assuntos
Citocinas , Técnicas Eletroquímicas , Medições Luminescentes , Estilbenos , Linfopoietina do Estroma do Timo , Citocinas/análise , Citocinas/metabolismo , Estilbenos/química , Humanos , Estruturas Metalorgânicas/química , Técnicas Biossensoriais , Imunoensaio/métodos , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia , Técnicas Analíticas Microfluídicas/instrumentação
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