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Rapid and sensitive dengue non-structural protein 1 (NS1) detection assay is essential for the treatment of disease and currently releases high medical cost burdens. To address the limitations of conventional LFIA strips, we have developed an improved Sup35NM-Z-based LFIA that immobilizes antibodies on cellulose membranes in an orientated manner to increase the sensitivity of LFIA strips. A dual-functional Sup35NM nanofibril was fabricated by fusion with the antibody binding domain; resultant nanofibril from the amyloid Sup35NM was sprayed on the T-line to orientate the capture antibody and produces fluorescence signals. Antibody binding analysis showed that self-assembly of the Sup35NM monomer does not affect the binding activity of the Z-domain with the antibody. The NS1 for DENV-2 infection was chosen as a model target antigen to assess the feasibility of the Sup35NM-Z-domain-based LFIA platform. Under optimal conditions, the Sup35NM-Z-domain-based LFIA detected NS1 within 15 min with a detection limit of 1.29 ng/ml, while the detection limit of traditional LFIA with the same concentration of anti-NS1-Ab1 on the T-line by conventional physical adsorption was 2.20 ng/ml, 1.7 times higher than that of Sup35NM-Z-domain-based LFIA. As compared to traditional LFIAs, the Sup35NM-Z-based LFIA had a wide detection range of 1.29-625 ng/mL. The LFIA's clinical performance in identifying NS1 was also assessed using 15 clinical samples. The LFIA accurately recognized positive and negative samples, equal to 86.7% accuracy. The developed Sup35NM-Z-domain-based LFIA in this study offers great potential for the identification of target markers because of its greatly improved sensitivity and wider detection range.
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Anticuerpos , Dengue , Humanos , Inmunoensayo , Dengue/diagnósticoRESUMEN
The ongoing coronavirus disease 2019 (COVID-19) epidemic has made a huge impact on health, economies, and societies all over the world. Although reverse transcription-polymerase chain reaction (RT-PCR)-based nucleic acid detection has been primarily used in the diagnosis of COVID-19, it is time-consuming with limited application scenarios and must be operated by qualified personnel. Antibody test, particularly point-of-care antibody testing, is a suitable complement to nucleic acid test as it provides rapid, portable, and cost-effective detection of infections. In this study, a Rapid Antibody Test Kit was developed based on fluorescence immunochromatography for the sensitive, accurate, and automated detection of immunoglobulin M (IgM) and IgG antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human serum, plasma, and whole blood samples within 10 min. The sensitivity, specificity, precision, and stability of the test kit were of good performance. No cross-activity and no interference was observed. In the multiple-center parallel study, 223 samples from hospitalized patients were used to evaluate the clinical specificity of the test. Both SARS-CoV-2 IgM and IgG achieved a clinical specificity of 98.21%. The clinical sensitivities of SARS-CoV-2 IgM and IgG were 79.54% and 87.45%, respectively, among 733 reverse transcription-polymerase chain reaction (RT-PCR) confirmed SARS-CoV-2 samples. For the combined IgM and IgG assays, the sensitivity and specificity were 89.22% and 96.86%, respectively. Our results demonstrate that the combined use of IgM and IgG could serve as a more suitable alternative detection method for patients with COVID-19, and the developed kit is of great public health significance for the prevention and control of the COVID-19 pandemic.
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Anticuerpos Antivirales/sangre , Prueba de COVID-19/métodos , COVID-19/diagnóstico , Técnica del Anticuerpo Fluorescente/métodos , Inmunoensayo/métodos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Juego de Reactivos para Diagnóstico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , COVID-19/inmunología , Niño , Preescolar , Femenino , Fluorescencia , Humanos , Masculino , Ratones , Persona de Mediana Edad , Pruebas en el Punto de Atención , Proteínas Recombinantes , SARS-CoV-2/inmunología , SARS-CoV-2/aislamiento & purificación , Sensibilidad y Especificidad , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Adulto JovenRESUMEN
We have developed a reliable and rapid immunoassay based on a facile synthesis of fluorescent nanoparticles integrated in immunochromatography technique to quantitatively detect C-reactive protein (CRP). The method is based on a sandwich immunoassay using the Nile-red doped nanoparticles/CRP monoclonal antibody conjugate. The method is simple and fast, with a detection limit of 0.091â¯mg/L. It provides quantitative analysis in the range of 0.1-160â¯mg/L, which is adequate for detecting CRP of acute inflammatory or cardiovascular disease. This strategy displayed a good reproducibility and stability to straightforwardly analyze the plasma samples without complicated washing steps, thereby reducing the operating procedures for non-professionals and promoting the detection efficiency and the whole detection process can be completed in 3â¯min. This approach for carrying out immunoassays can be applied to the detection of CRP in the point-of-care tests.
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Anticuerpos Monoclonales/química , Proteína C-Reactiva/análisis , Nanopartículas/química , Oxazinas/química , Cromatografía de Afinidad/métodos , Humanos , Inmunoensayo/métodos , Límite de Detección , Reproducibilidad de los ResultadosRESUMEN
A reliable lateral flow immunoassay (LFIA) based on a facile one-step synthesis of single microspheres in combining with immunochromatography technique was developed to establish a new point-of-care test (POCT) for the rapid and early detection of cardiac troponin I (cTnI), a kind of cardiac specific biomarker for acute myocardial infarction (AMI). The double layered microspheres with clear core-shell structures were produced using soap-free emulsion polymerization method with inexpensive compounds (styrene and acrylic acid). The synthetic process was simple, rapid and easy to control due to one-step synthesis without any complicated procedures. The microspheres are nanostructure with high surface area, which have numerous carboxyl groups on the out layer, resulting in high-efficiency coupling between the carrier and antibody via amide bond. Meanwhile, the red fluorescent dye, Nile-red (NR), was wrapped inside the microspheres to improve its stability, as well to reduce the background noise, because of its higher emission wavelength than interference from real plasma samples. The core-shell structures provided different functional areas to separate antibody and dyes, so the immunoassay has highly sensitive, wide working curves in the range of 0â»40 ng/mL, low limits of detection (LOD) at 0.016 ng/mL, and limits of quantification (LOQ) at 0.087 ng/mL with coefficient of variations (CV) of 10%. This strategy suggested an outstanding platform for LFIA, with good reproducibility and stability to straightforwardly analyze the plasma samples without washing steps, thereby reducing the operating procedures for non-professionals and promoting detection efficiency. The whole detection process can be completed in less than 15 min. This novel immunoassay offers a reliable and favorable analytical result by detecting the real samples, indicating that it holds great potential as a new alternative for biomolecule detection in complex samples, for the early detection of cardiac specific biomarkers.
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Colorantes Fluorescentes/química , Inmunoensayo , Microesferas , Nanoestructuras/química , Oxazinas/química , Troponina I/sangre , Acrilatos/química , Anticuerpos/química , Cromatografía de Afinidad , Emulsiones , Humanos , Límite de Detección , Nanoestructuras/ultraestructura , Sistemas de Atención de Punto , Reproducibilidad de los Resultados , Reología , Estireno/químicaRESUMEN
BACKGROUND: Malaria presents a considerable threat to public health. Histidine-rich protein 2 (HRP 2) is the major protein released into human blood upon infection by Plasmodium falciparum. In this study, we aimed to evaluate the immunogenicity of HRP 2 exon II and the efficacy of novel monoclonal antibodies (mAbs) against HRP 2 for Point-of-Care Test (POCT). METHODS: The recombinant protein was expressed in soluble form in E. coli and used to immunize mice for mAb production. Two IgG1 mAbs (1A5 and 1C10) with high affinity, specificity and sensitivity for both native and recombinant HRP 2 were selected after fusion of mouse spleen with myeloma cells. The affinity constant of 1A5 and 1C10 were 7.15 and 4.91 × 10-7 L/mol, respectively. Subsequently, an immunochromatograhic assay was used for screening of clinical samples in endemic regions of China and Myanmar. RESULTS: The immunochromatographic test retrospectively showed an overall sensitivity of 99.07%, and specificity of 100%. Sensitivity at parasite densities < 200, 200-2000, and > 2000 parasites/µL was 87.5, 98.7, and 100%, respectively. CONCLUSIONS: These results suggest that HRP 2 exon II contains immunogenic sites similar to those of the native antigen and can be used for the development of mAbs suitable for malaria diagnosis in endemic communities.
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Anticuerpos Monoclonales/inmunología , Antígenos de Protozoos/análisis , Cromatografía de Afinidad/métodos , Malaria Falciparum/diagnóstico , Plasmodium falciparum/aislamiento & purificación , Sistemas de Atención de Punto , Proteínas Protozoarias/análisis , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Antiprotozoarios/inmunología , Anticuerpos Antiprotozoarios/aislamiento & purificación , Antígenos de Protozoos/inmunología , China , Pruebas Diagnósticas de Rutina/métodos , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina G/aislamiento & purificación , Ratones , Mianmar , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
BACKGROUND: The low sensitivity and specificity of Plasmodium falciparum diagnostic tests pose a serious health threat to people living in endemic areas. The objective of the study was to develop a rapid assay for the detection of histidine-rich protein 2 (HRP2) of P. falciparum in whole blood by immunofluorescence chromatographic technology. METHODS: A total of 1163 positive and negative blood samples were screened. The double-antibody sandwich assay was used to establish the kit and its performance was evaluated for sensitivity, specificity, accuracy, precision, stability, and clinical effectiveness. RESULTS: The cut-off level of detection of the kit was 25 parasites/µl. Common interfering substances in human blood specimens, such as bilirubin, triglyceride and cholesterol had no significant effect on HRP2 antigen detection. The precision of the kit was run with different concentration of standard calibrators and the values were less than 10 %. The performance of this diagnostic kit in the detection of the calibrators has shown that a shelf life of about 12 months gives a more reliable result. Among clinical samples tested, the HRP2 test kit and the reference products had good coincidence rate in a parallel experiment and this test kit had a more sensitive detecting level to the target protein than the reference kits used in this study. The specificity and sensitivity for this test were 99.6 % (800/803) and 99.7 % (1160/1163), respectively. CONCLUSIONS: A novel HRP2 immunofluorescence detection method was developed in this study. Overall performance evaluation indicated that the kit has a rapid, high sensitivity and on-spot method for detecting P. falciparum.
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Antígenos de Protozoos/aislamiento & purificación , Cromatografía/métodos , Pruebas Diagnósticas de Rutina/métodos , Malaria Falciparum/diagnóstico , Plasmodium falciparum/fisiología , Proteínas Protozoarias/aislamiento & purificación , Técnica del Anticuerpo Fluorescente , Sensibilidad y EspecificidadRESUMEN
OBJECTIVES: To develop a rapid, sensitive and specific assay for quantification of serum heart-type fatty acid binding protein (H-FABP) based on immunofluorescence of specific monoclonal antibodies. DESIGN AND METHODS: We generated novel H-FABP-directed monoclonal antibodies by cloning of spleen cells of mice immunized with H-FABP. Epitopes were mapped and antigen affinity was assessed by surface plasmon resonance (SPR). The H-FABP specific monoclonal antibodies were coupled to fluorescent beads and sprayed onto a nitrocellulose membrane facilitating quantification of H-FABP by immunofluorescence. Reagent cross-reactivity, interference resistance, accuracy and sensitivity were examined. A total of 103 clinical samples were used to compare the sensitivity and specificity of the new assay to a commercially available Randox kit. RESULTS: This new assay could be finished within 15 min, with sensitivity reaching 1 ng/ml. In a trial of 103 clinical serum samples, the new testing kit results were highly correlated with those from the Randox kit (R(2) = 0.9707). Using the Randox kit as the reference kit, the sensitivity of the new assay was 98.25%, and specificity was 100%. CONCLUSIONS: An immunofluorescence-based H-FABP assay employing novel monoclonal antibodies could rapidly, specifically and sensitively detect H-FABP in serum samples, providing an effective method for rapid clinical assessment of H-FABP index in the clinic.
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Anticuerpos Monoclonales de Origen Murino/química , Proteínas de Unión a Ácidos Grasos/sangre , Animales , Unión Competitiva , Biomarcadores/sangre , Cromatografía , Proteína 3 de Unión a Ácidos Grasos , Proteínas de Unión a Ácidos Grasos/inmunología , Técnica del Anticuerpo Fluorescente Directa , Humanos , Límite de Detección , Ratones Endogámicos BALB C , Isquemia Miocárdica/sangre , Unión ProteicaRESUMEN
BACKGROUND: Misdiagnosis of malaria by commercial rapid diagnostic tests (RDTs) is a major cause of concern in the diagnosis of malaria. This retrospective study was aimed at assessing the relative performance of four RDTs with emphasis on the detection of two Plasmodium vivax antigens: aldolase and lactate dehydrogenase (LDH). METHODS: Three commercially available Plasmodium LDH or aldolase antigen detection kits (One Step Malaria P.f/P.v, ParaHit Total ver. 1.0, SD Bioline Malaria) and an anti-P. vivax aldolase-specific monoclonal antibody (mAb) pair 1C3-12 F10 were evaluated with P. vivax positive as well as non-P. vivax samples and healthy samples using blood smear examination as standard. Each test was read according to the manufacturer's instructions. RESULTS: MAb 1C3-12 F10 pair targeting P. vivax-specific aldolase exhibited very good specificity and sensitivity of 100 and 97.4%, respectively. Positive predictive value (PPV) and negative predictive value (NPV) of 100 and 99.5%, respectively, were also observed. The anti-P. vivax LDH in the One-Step Malaria P.f/P.v test showed sensitivity, specificity, PPV and NPV of 93.5, 98.0, 88.9 and 98.8%, respectively. ParaHit Total ver. 1.0 targeting the pan-aldolase antigen showed sensitivity, specificity of 97.4 and 99.6%, respectively. PPV and NPV were both 99.5%. SD Bioline had sensitivity, specificity, PPV and NPV of 93.5, 100, 100 and 98.8%, respectively. The overall sensitivity and specificity of all four RDTs were acceptable, especially for the aldolase detection tests. Five (6.5%) of the P. vivax-positive samples (n = 77) that were confirmed by microscopic examination as well as the two aldolase detection RDTs (mAb 1C3-12 F10 and ParaHit Total ver.1.0) were undetected by the two LDH detection RDTs (One Step Malaria P.f/P.v and SD Bioline). Similarly, two positive samples (2.6%) that were positively confirmed by the LDH detection RDTs were also undetected by the aldolase detection test kits. CONCLUSION: Aldolase and LDH antigens perform differently in different P. vivax samples; hence there is a high risk of misdiagnosis when monoclonal antibodies are used against only one particular antigen in the test. A combination of both aldolase and LDH in RDTs for the rapid diagnosis of P. vivax will enhance the sensitivity of the assay and reduce misdiagnosis.
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Antígenos de Protozoos/sangre , Pruebas Diagnósticas de Rutina/métodos , Fructosa-Bifosfato Aldolasa/sangre , L-Lactato Deshidrogenasa/sangre , Malaria Vivax/diagnóstico , Sistemas de Atención de Punto , Anticuerpos Monoclonales , Anticuerpos Antiprotozoarios , Humanos , Inmunoensayo/métodos , Estudios Retrospectivos , Sensibilidad y EspecificidadRESUMEN
The development of an advanced analytical platform with regard to SARS-CoV-2 is crucial for public health. Herein, we present a machine learning platform based on paper-assisted ratiometric fluorescent sensors for highly sensitive detection of the SARS-CoV-2 RdRp gene. The assay involves target-induced rolling circle amplification to generate magnetic DNAzyme, which is then detectable using the paper-assisted ratiometric fluorescent sensor. This sensor detects the SARS-CoV-2 RdRp gene with a visible-fluorescence color response. Moreover, leveraging different fluorescence responses, the ResNet algorithm of machine learning assists in accurately identifying fluorescence images and differentiating the concentration of the SARS-CoV-2 RdRp gene with over 99% recognition accuracy. The machine learning platform exhibits exceptional sensitivity and color responsiveness, achieving a limit of detection of 30 fM for the SARS-CoV-2 RdRp gene. The integration of intelligent artificial vision with the paper-assisted ratiometric fluorescent sensor presents a novel approach for the on-site detection of COVID-19 and holds potential for broader use in disease diagnostics in the future.
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Técnicas Biosensibles , COVID-19 , ADN Catalítico , Humanos , SARS-CoV-2 , COVID-19/diagnóstico , Técnicas Biosensibles/métodos , Colorantes Fluorescentes , Fenómenos Magnéticos , ARN Polimerasa Dependiente del ARNRESUMEN
BACKGROUND: Most rapid diagnostic tests (RDTs) currently used for malaria diagnosis cannot distinguish the various Plasmodium infections. The development of a Plasmodium vivax specific RDTs with high sensitivity to sufficiently differentiate the two most common Plasmodium infections would be very crucial for disease treatment and control. METHOD: Plasmodium vivax aldolase gene (PvALDO) was amplified from the extracted genomic DNA and constructed into pET30a vector. Plasmodium vivax aldolase protein was successfully expressed in Escherichia coli in soluble form and the overall purity was over 95% after one-step affinity chromatography purification. The purified products were used for the immunization of mice and rabbits. Rabbit polyclonal antibodies generated were deployed to develop a novel antibody-capture ELISA for hybridoma screening. RESULTS: Three PvALDO specific mAbs (14C7, 15F1 and 5H7) with high affinities were selected and used in immunochromatographic test strips. Clinical blood samples (n=190) collected from Yunnan (China) were used for evaluation and the RDT's sensitivity for P. vivax was 98.33% (95% Confidence Interval (CI): 91.03% to 99.72%) compared with microscopic examination. There was specificity of 99.23% (95% CI: 95.77% to 99.87%) for P. vivax. Only one Plasmodium falciparum sample was detected among the P. falciparum samples (n=20). All Plasmodium malariae samples (n=2) as well as healthy uninfected samples (n=108) were negative. Overall performance of this RDT was excellent with positive predictive value (PPV) and negative predictive value (NPV) of 98.33% and 99.23%, respectively, at 95% CI and a very good correlation with microscopic observations (kappa value, K=0.9757). Test strips show high sensitivity even at 6.25 ng/ml of recombinant P. vivax aldolase (rPvALDO). CONCLUSION: This study further elucidates the possibility of developing aldolase-specific RDTs which can differentiate the different Plasmodium infections and improve accurate diagnosis of malaria. This RDT could adequately differentiate between P. vivax and P. falciparum infections. The novel mAb screening method developed here could find application in the screening of highly specific antibodies against other antigens.
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Anticuerpos Monoclonales , Anticuerpos Antiprotozoarios , Antígenos de Protozoos/sangre , Pruebas Diagnósticas de Rutina/métodos , Malaria Vivax/diagnóstico , Parasitología/métodos , Plasmodium vivax/aislamiento & purificación , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Antiprotozoarios/aislamiento & purificación , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , China , Técnicas de Laboratorio Clínico/métodos , Femenino , Fructosa-Bifosfato Aldolasa/sangre , Fructosa-Bifosfato Aldolasa/genética , Fructosa-Bifosfato Aldolasa/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Plasmodium vivax/inmunología , Valor Predictivo de las Pruebas , Conejos , Sensibilidad y EspecificidadRESUMEN
OBJECTIVE: To investigate the effects of natural drug curcumin (Cur) on apoptosis of lens epithelial cell (LEC) in vitro and its mechanism. METHODS: The bovine LEC were cultured with Cur, the ultrastructure changes were observed under transmission electron microscope (TEM), the DNA content and mitochondrial transmembrane potential (DeltaPsim) changes were studied by flow cytometry (FCM). RESULTS: The typical morphological changes of LEC apoptosis in Cur group detected by TEM included chromatin condensation and aggregation at the periphery of the nucleons and nuclear fragmentation. The DNA content of LEC in Cur group decreased time-dependently. The DNA content was significantly lower than that of the control group (P < 0.01). The DeltaPsim of LEC in Cur group was decreased, appeared in early stage (8 hours) and reached the maximum after 72 hours. The difference of DeltaPsim of LEC between Cur group and the control group was significant (P < 0.01). CONCLUSIONS: Cur can remarkably induce apoptosis of LEC in vitro. Cur induced LEC apoptosis is caused by decrease of DNA content in LEC nucleus. Collapse of DeltaPsim in cytoplasm induced by Cur results in the irreversible apoptosis process of LEC. This is the early event of LEC apoptosis. LEC apoptosis induced by Cur may pass through two pathways: nuclear pathway and cytoplasmic pathway. The apoptosis of LEC induced by Cur may be the cellular and molecular mechanisms of reducing lens posterior capsular opacification by Cur. Cur may become an effective and low toxic medication for the prevention and treatment of after-cataract.
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Apoptosis/efectos de los fármacos , Curcumina/farmacología , Células Epiteliales/ultraestructura , Cristalino/citología , Animales , Catarata/prevención & control , Bovinos , Células Cultivadas , Fragmentación del ADN , Citometría de Flujo , Cápsula del Cristalino/patología , Potenciales de la Membrana , Mitocondrias/efectos de los fármacosRESUMEN
Human immunodeficiency virus type 1 (HIV-1) p24 protein is the most abundant viral protein of HIV-1. This protein is secreted in blood serum at high levels during the early stages of HIV-1 infection, making it a biomarker for early diagnosis. In this study, a colloidal gold immunochromatographic assay (GICA) was established for detecting p24 protein using mouse monoclonal antibodies (mAbs). The HIV-1 p24 protein was expressed in E. coli strain BL21 and the purified protein was used to immunize mice. Stable hybridoma cell lines secreting anti-p24 monoclonal antibodies were obtained after ELISA screening and subcloning by limiting dilution. 34 different capture and labeling mAb pairs were selected by a novel antibody-capture indirect sandwich ELISA and then applied in GICA to detect p24 protein. The GICA method has a limit of detection (LOD) of 25 pg/mL and could detect p24 protein in all 10 positive samples obtained from the National Reference of HIV-1 p24 antigen. Out of 153 negative samples tested, 3 false positives results were obtained. The overall specificity of this test was 98.03%. The good sensitivity and specificity of this method make it a suitable alternative to provide a more convenient and efficient tool for early diagnosis of HIV infection.
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Anticuerpos Monoclonales/inmunología , Cromatografía de Afinidad/métodos , Oro Coloide/química , Proteína p24 del Núcleo del VIH/inmunología , VIH-1/aislamiento & purificación , Proteínas Recombinantes/inmunología , Animales , Productos Biológicos , Escherichia coli , Proteína p24 del Núcleo del VIH/genética , Proteína p24 del Núcleo del VIH/metabolismo , Infecciones por VIH/diagnóstico , Infecciones por VIH/virología , VIH-1/inmunología , Humanos , Límite de Detección , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
In this study, an indirect competitive enzyme-linked immunoassay (ic-ELISA) based on monoclonal antibody for 4(5)-Methylimidazole (4-MI) detection was described. The artificial antigens were prepared by conjugating bovine serum albumin (BSA) or ovalbumin (OVA) with the hapten of 4-MI. And monoclonal antibody, evaluated by ic-ELISA, was obtained by immunizing BABL/c mice. After optimizing, a standard curve for ic-ELISA detection on 4-MI was obtained with the linear detection range of 0.64-20.48 mg/L. The cross-reactivity (CR) of all the structural analogues of 4-MI was less than 5.62%. The recoveries of 4-MI in caramels detection were ranged from 88.69% to 114.09%, with relative standard deviation (n=3) below 8.07%. The results suggested that the established ic-ELISA is promising for 4-MI commercial detection in caramels.
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Anticuerpos Monoclonales/inmunología , Dulces/análisis , Animales , Bioensayo , Carbohidratos , Inmunoadsorbentes , Ratones , Ratones Endogámicos BALB CRESUMEN
BACKGROUND: Procalcitonin (PCT) has been recognized as a biomarker in severe inflammation, infection and sepsis. PCT detection in serum requires sensitive and specific antibodies. In this study, we generated monoclonal antibodies (mAbs) and developed fluorescent immunochromatographic assay for PCT detection. METHODS: Human recombinant PCT was used as immunogen. mAbs against PCT were developed and applied to fluorescent immunochromatographic assay for PCT detection in clinical samples. RESULTS: Out of 35 hybridoma cell lines secreting antibodies against the recombinant PCT, five sensitive and specific cell lines were selected and designated as F6, G2, C2, D2 and E5. All these antibodies have no cross reaction with calcitonin or calcitonin gene-related peptides (CGRP). After screening for pairing, mAb F6 was labeled with fluorescent microspheres and C2 was coated on a nitrocellulose membrane for immunochromatographic test. All 35 clinical samples were detected by the mAb F6-C2 test strips and the bioMérieux PCT assay. The test strips showed high specificity and sensitivity for PCT. Good correlation was observed between our immunochromatographic test strips and the bioMérieux PCT assay (R(2):0.986). CONCLUSIONS: These newly developed anti-PCT mAbs and fluorescent immunochromatographic assay can serve as important diagnostic tools for a fast, reliable and point-of-care testing for easy determination of PCT in serum and diagnosis of bacterial infection, inflammation or sepsis.
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Calcitonina/sangre , Cromatografía de Afinidad/métodos , Fluorescencia , Precursores de Proteínas/sangre , Animales , Anticuerpos Monoclonales/inmunología , Reacciones Antígeno-Anticuerpo , Calcitonina/genética , Calcitonina/inmunología , Péptido Relacionado con Gen de Calcitonina , China , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Precursores de Proteínas/genética , Precursores de Proteínas/inmunología , Proteínas Recombinantes/sangre , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
The possibility of using variable domain heavy-chain antibodies (VHH antibodies) as diagnostic tools for dengue virus (DENV) type 2 NS1 protein was investigated and compared with the use of conventional monoclonal antibodies. After successful expression of DENV type 2 NS1 protein, the genes of VHH antibodies against NS1 protein were biopanned from a non-immune llama library by phage display. VHH antibodies were then expressed and purified from Escherichia coli. Simultaneously, monoclonal antibodies were obtained by the conventional route. Sequence analysis of the VHH antibodies revealed novel and long complementarity determining regions 3 (CDR3). Epitope mapping was performed via a phage display peptide library using purified VHH and monoclonal antibodies as targets. Interestingly, the same region of NS1, which comprises amino acids 224HWPKPHTLW232, was conserved for both kinds of antibodies displaying the consensus motif histidine-tryptophan-tryptophan or tryptophan-proline-tryptophan. The two types of antibodies were used to prepare rapid diagnostic kits based on immunochromatographic assay. The VHH antibody immobilized rapid diagnostic kit showed better sensitivity and specificity than the monoclonal antibody immobilized rapid diagnostic kit, which might be due to the long CDR3 regions of the VHH antibodies and their ability to bind to the pocket and cleft of the targeted antigen. This demonstrates that VHH antibodies are likely to be an option for developing point-of-care tests against DENV infection.
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Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Virus del Dengue/inmunología , Dengue/inmunología , Cadenas Pesadas de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/inmunología , Pruebas Inmunológicas/métodos , Secuencia de Aminoácidos , Animales , Cromatografía de Afinidad , Dengue/diagnóstico , Dengue/virología , Ensayo de Inmunoadsorción Enzimática , Mapeo Epitopo , Femenino , Proteínas Inmovilizadas/inmunología , Cadenas Pesadas de Inmunoglobulina/química , Región Variable de Inmunoglobulina/química , Cinética , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Péptidos/química , Péptidos/inmunología , Pliegue de Proteína , Juego de Reactivos para Diagnóstico , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Sensibilidad y Especificidad , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Resonancia por Plasmón de Superficie , Proteínas no Estructurales Virales/aislamiento & purificaciónRESUMEN
We assessed a colloidal gold immunochromatographic assay (GICA) for rapid detection of influenza A (H7N9) and compared it with reverse-transcription-polymerase chain reaction (RT-PCR) and viral culture. Samples from 35 H7N9 infected patients were collected, including 45 throat swab samples, 56 sputum samples, and 39 feces samples. All samples were tested by GICA, viral culture, and RT-PCR. GICA specifically reacted with recombinant HA proteins, virus lysates, and clinical samples from H7 subtype viruses. Compared with RT-PCR, GICA demonstrated low sensitivity (33.33%) but high specificity (97.56%). The positive rate of GICA tests for samples collected in the period from 8 to 21 days after contact with poultry was much higher than those for samples collected before or after this period. Compared with viral culture, GICA showed sensitivity of 91.67% and specificity of 82.03%. Sputum specimens were more likely to test positive for H7N9 virus than samples from throat swabs and feces. The GICA-based H7 test is a reliable, rapid, and convenient method for the screening and diagnosis of influenza A (H7N9) disease, especially for the sputum specimens with high viral load. It may be helpful in managing H7N9 epidemics and preliminary diagnosis in early stages in resource-limited settings.
Asunto(s)
Cromatografía de Afinidad/métodos , Subtipo H7N9 del Virus de la Influenza A/aislamiento & purificación , Gripe Humana/diagnóstico , Gripe Humana/virología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Virología/métodos , Animales , Perros , Heces/virología , Oro , Humanos , Subtipo H7N9 del Virus de la Influenza A/genética , Subtipo H7N9 del Virus de la Influenza A/inmunología , Células de Riñón Canino Madin Darby , Faringe/virología , Sensibilidad y Especificidad , Esputo/virología , Carga Viral , Cultivo de VirusRESUMEN
BACKGROUND: Since human infection with the novel H7N9 avian influenza virus was identified in China in March 2013, the relatively high mortality rate and possibility of human-to-human transmission have highlighted the urgent need for sensitive and specific assays for diagnosis of H7N9 infection. METHODOLOGY/PRINCIPAL FINDINGS: We developed a rapid diagnostic test for the novel avian influenza A (H7N9) virus using anti-hemagglutinin (HA) monoclonal antibodies specifically targeting H7 in an immunochromatographic assay system. The assay limit of detection was 103.5 pfu/ml or 103TCID50 of H7N9 virus. The assay specifically detected H7N9 viral isolates and recombinant HA proteins of H7 subtypes including H7N7 and H7N9, but did not react with non-H7 subtypes including H1N1, H3N2, H5N1, H5N9, and H9N2. The detection sensitivity was 59.4% (19/32) for H7N9 patients confirmed by RT-PCR. Moreover, the highest sensitivity of 61.5% (16/26) was obtained when testing H7N9 positive sputum samples while 35.7% (5/14) of nasopharyngeal swabs and 20% (2/10) of fecal samples tested positive. No false positive detection was found when testing 180 H7N9 negative samples. CONCLUSIONS/SIGNIFICANCE: Our novel rapid assay can specifically detect H7 HA antigen, facilitating rapid diagnosis for prevention and control of the on-going H7N9 epidemic.