Academic literature on the topic 'Cell-based-ELISA'
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Journal articles on the topic "Cell-based-ELISA"
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Shankar, Gopi, and Donald A. Cohen. "Enhanced Cytokine Detection by a Novel Cell Culture-Based Elisa." Journal of Immunoassay 18, no. 4 (November 1997): 371–88. http://dx.doi.org/10.1080/01971529708005828.
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Ahmadzadeh, Maryam, Farzaneh Farshdari, Leila Nematollahi, Shayan Maleknia, and Elham Mohit. "Optimization of HER2-based and cell-based ELISA for detection of trastuzumab biosimilar." Koomesh journal 22, no. 2 (April 1, 2020): 341–50. http://dx.doi.org/10.29252/koomesh.22.2.341.
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FRIIS, TINA, BIRGITTE KJAER SORENSEN, ANNE-MARIE ENGEL, JORGEN RYGAARD, and GUNNAR HOUEN. "A quantitative ELISA-based co-culture angiogenesis and cell proliferation assay." APMIS 111, no. 6 (June 2003): 658–68. http://dx.doi.org/10.1034/j.1600-0463.2003.1110609.x.
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Bengleil, Mudafara, and Jeffrey R. Fry. "In situ cell-based ELISA for determination of multiple heat shock proteins." Toxicology 226, no. 1 (September 2006): 71. http://dx.doi.org/10.1016/j.tox.2006.05.095.
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Tizard, E. J., E. Baguley, G. R. Hughes, and M. J. Dillon. "Antiendothelial cell antibodies detected by a cellular based ELISA in Kawasaki disease." Archives of Disease in Childhood 66, no. 2 (February 1, 1991): 189–92. http://dx.doi.org/10.1136/adc.66.2.189.
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Franciotta, Diego, Gianvito Martino, Elena Brambilla, Elisabetta Zardini, Vera Locatelli, Alessandra Bergami, Carmine Tinelli, Gaetano Desina, and Vittorio Cosi. "TE671 Cell-based ELISA for Anti-Acetylcholine Receptor Antibody Determination in Myasthenia Gravis." Clinical Chemistry 45, no. 3 (March 1, 1999): 400–405. http://dx.doi.org/10.1093/clinchem/45.3.400.
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Abstract Background: Acetylcholine receptor (AChR) from human muscles is the antigen used currently in radioimmunoprecipitation assays (RIPAs) for the determination of anti-AChR antibodies in the diagnosis of myasthenia gravis (MG). Our aim was to develop and validate an ELISA using TE671 cells as the source of AChR. Methods: After TE671 cell homogenization, the crude AChR extract was used for plate coating. Anti-AChR antibodies were determined in 207 MG patients and in 77 controls. Results: The mean intra- and interassay CVs (for two samples with different anti-AChR antibody concentrations) were 9.7% and 15.7%, respectively. Test sensitivity and specificity, for generalized MG, were 79.5% (95% confidence interval, 72.8–85.0%) and 96.1% (89.0–99.1%). The detection limit was 2 nmol/L. Anti-AChR antibody concentrations from 53 MG patients, as tested with our ELISA, showed good agreement with an RIPA with a mean difference (SD) of 1.0 (5.6) nmol/L. Conclusion: Our ELISA is a simple screening test for the diagnosis of MG and enables rapid and inexpensive patient follow-up.
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Zarletti, Gianpaolo, Massimo Tiberi, Veronica De Molfetta, Maurizio Bossù, Elisa Toppi, Paola Bossù, and Giuseppe Scapigliati. "A Cell-Based ELISA to Improve the Serological Analysis of Anti-SARS-CoV-2 IgG." Viruses 12, no. 11 (November 8, 2020): 1274. http://dx.doi.org/10.3390/v12111274.
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Knowledge of the antibody-mediated immune response to SARS-CoV-2 is crucial to understand virus immunogenicity, establish seroprevalence, and determine whether subjects or recovered patients are at risk for infection/reinfection and would therefore benefit from vaccination. Here, we describe a novel and simple cell-ELISA specifically designed to measure viral spike S1-specific IgG produced in vitro by B cells in peripheral blood mononuclear cell (PBMC) cultures from a cohort of 45 asymptomatic (n = 24) and symptomatic (n = 21) individuals, with age ranging from 8 to 99 years. All subjects underwent ELISA serological screening twice, at the same time as the cell-ELISA (T2) as well as 35–60 days earlier (T1). Cryopreserved PBMCs of healthy donors obtained years before the COVID-19 pandemic were also included in the analysis. The preliminary results presented here show that out of 45 tested subjects, 16 individuals (35.5%) were positive to the cell-ELISA, 11 (24.5%) were concomitantly positive in the serological screening (T1 and/or T2), and only one person was exclusively positive in ELISA (T1) and negative in cell-ELISA, though values were close to the cutoff. Of note, five individuals (11.2%) tested negative in ELISA but positive in cell-ELISA and thus, they appear to have circulating B cells that produce antibodies against SARS-CoV-2, likely at levels that are undetectable in the serum, which challenges the negative results of the serological screening. The relative level of in vitro secreted IgG was measurable in positive subjects, ranging from 7 to 50 ng/well. Accordingly, all anti-SARS-CoV-2 antibody-positive subjects previously reported moderate to severe symptoms attributable to COVID-19, even though the RT-PCR data were rarely available to confirm viral infection. Overall, the described cell-ELISA might be an effective method for detecting subjects who encountered the virus in the past, and thus helpful to improve serological ELISA tests in the case of undetectable/equivocal circulating IgG levels, and a suitable and improved tool to better evaluate SARS-CoV-2-specific humoral immunity in the COVID-19 pandemic.
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Chuang, Kuo-Hsiang, Shey-Cherng Tzou, Ta-Chun Cheng, Chien-Han Kao, Wei-Lung Tseng, Jentaie Shiea, Kuang-Wen Liao, et al. "Measurement of Poly(ethylene glycol) by Cell-Based Anti-poly(ethylene glycol) ELISA." Analytical Chemistry 82, no. 6 (March 15, 2010): 2355–62. http://dx.doi.org/10.1021/ac902548m.
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Khodashenas, Shabanali, Saeed Khalili, and Mehdi Forouzandeh Moghadam. "A cell ELISA based method for exosome detection in diagnostic and therapeutic applications." Biotechnology Letters 41, no. 4-5 (April 8, 2019): 523–31. http://dx.doi.org/10.1007/s10529-019-02667-5.
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Stults, Cheryl L. M., and Bruce A. Macher. "Measurement of β-galactosyltransferase activity in cell extracts with an ELISA-based assay." Archives of Biochemistry and Biophysics 280, no. 1 (July 1990): 20–26. http://dx.doi.org/10.1016/0003-9861(90)90512-w.
Full textDissertations / Theses on the topic "Cell-based-ELISA"
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Ng, Hoi-yee Iris, and 吳凱怡. "Serological diagnosis of influenza B virus infection in pigs : a comparison of the hemagglutination inhibition assay and the cell-based ELISA assay." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hdl.handle.net/10722/193797.
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Background
Swine influenza virus (SIV) was first isolated in the United States in 1930 and was thereafter widely reported in many countries. Most SIVs that have been identified are influenza A viruses. There was no report of influenza B viruses isolated in swine. Seroepidemiological study in UK has shown a low seroprevalence of influenza B antibody in pigs. The primary serological test used to detect influenza antibody is the hemagglutination inhibition (HI)test. Enzyme-linked immunosorbent assay (ELISA) are also available commercially for detection of antibodies against influenza A viruses but not for the detection of influenza B antibodies.
Objectives
1) To examine the prevalence of influenza B antibodies in pig sera sampled at the abattoir in Hong Kong.
2) To develop the cell-based ELISA assay for the detection of antibodies against influenza A and B viruses.
3) To compare the cell-based ELISA assays with three commercial ELISA kits, namely the IDVet ID Screen influenza A antibody competition ELISA, the IDEXX Influenza A Ab test and the IDEXX AI MultiS-Screen Ab test using swine sera.
4) To test swine sera using the influenza B cell-based ELISA assay to complement data on swine seroprevalence obtained with HI tests.
Methods
The first part of this study involved HI screening of 4643 pig sera from 2009 to 2012. These sera were tested for the presence of antibodies against B/Brisbane/60/2008 and B/Wisconsin/1/2010whichrepresent the B/Victoria and B/Yamagata lineages respectively.
The second part of this study involved the development and performance evaluation of the cell-based ELISA assays. The cell-based ELISA assays were developed using influenza virus infected cells as the capture antigens and fluorescence-labelled anti-IgG antibody as the detection antibody. The viruses that were used to prepare the assays were A/California/04/2009, B/Brisbane/60/2008 and B/Wisconsin/1/2010. All three cell-based ELISA assays were tested with WHO reference sera and swine sera and the results were analyzed using paired t-test and receiver operating characteristic analysis. In addition, the results of the influenza A cell-based ELISA assay were compared with the commercial ELISA assay using Fisher’s exact two-tailed test, Pearson’s correlation analysis and Bland-Altman plot.
Results
A low prevalence (0.28%; 95%CI: 0.16%-0.47%) of influenza B antibody was observed inthe swine sera samples. The seroprevalence for B/Victoria was higher than that of B/Yamagatain 2010to2012. Co-existence of B/Victoria and B/Yamagata antibodies were found in the swine population during 2010 and 2011.
The influenza A cell-based ELISA was found to have low sensitivity (64.1%;95%CI: 52.4%-74.4%) and high specificity (94.7%; 95%CI:80.9%-99.1%) when compared with the commercial ELISA assays. In contrast, using HI as the reference test influenza B cell-based ELISA prepared using B/Wisconsin/1/2010 infected cells were shown to have high sensitivity (92.31%; 95%CI:64.0%-99.8%) but low specificity (63.16%;95%CI:38.4%-83.7%) in detection of influenza B antibodies in swine sera.
Conclusion
Sporadic transmission of influenza B virus may occur in swine but there is no evidence for efficient and sustained transmission of the virus between them. Cell-based ELISA assay prepared with B/Wisconsin/1/2010 may be considered as an alternative screening testprior to HI subtyping.published_or_final_version
Public Health
Master
Master of Public Health
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Hata, Misako. "Comparison of a novel cell-based reporter assay and a competitive binding ELISA for the detection of thyrotropin-receptor (TSHR) autoantibodies (TRAb) in Graves' disease patients." Ohio : Ohio University, 2010. http://www.ohiolink.edu/etd/view.cgi?ohiou1262099140.
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Nording, Malin. "Rapid sample preparation and bioanalytical techniques for efficient screening of organic pollutants in the environment." Doctoral thesis, Umeå universitet, Kemi, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-842.
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Large numbers of samples often need to be prepared and analysed in surveys of organic pollutants in the environment, but while the methods commonly used in such surveys can provide abundant detail they are generally costly, time-consuming and require large amounts of resources, so there is a need for simpler techniques. The work underlying this thesis assessed the potential utility of more convenient sample preparation and bioanalytical techniques for rapidly screening various environmental matrices that could be useful complements to higher resolution methods. Initially, the utility of a simplified extraction technique followed by an enzyme-linked immunosorbent assay (ELISA) for detecting polycyclic aromatic hydrocarbons (PAHs) in authentic (i.e. unspiked) contaminated soils was explored. The results showed that there are relationships between the structure and cross-reactivity among compounds that often co-occur with target PAHs. However, their potential contribution to deviations between estimates of total PAH contents of soils obtained using ELISA and gas chromatography-mass spectrometry (GC-MS) based reference methods were limited. Instead, the cross-reactivity of target PAHs and the failure to extract all of the PAHs prior to the ELISA determinations were the main reasons for these deviations. Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) were detected in food and feed matrices, as well as in authentic contaminated soils using different bioanalytical techniques – ELISA and two cell-based bioassays: CAFLUX and CALUX (chemically activated fluorescent/luciferase gene expression) assays. In addition, enhanced sample preparation techniques based on accelerated solvent extraction (ASE) were developed. ASE with integrated carbon fractionation (ASE-C) in combination with CAFLUX produced estimates of PCDD and PCDF contents in fish oil and fish meal that agreed well with results obtained using reference methods. Furthermore, results from ELISA and GC-high resolution MS analyses of extracts of PCDD- and PCDF-contaminated soil samples obtained using an adjusted ASE-C technique were strongly correlated. Finally, the thesis reports the first experiments in which the results of CAFLUX, CALUX, and ELISA determinations of PCDDs and PCDFs in extracts of authentic contaminated soil samples were evaluated and compared to those obtained using a reference method. All of the bioanalytical techniques were found to be sufficiently sensitive, selective, and accurate for use in screening in compliance with soil quality assessment criteria. Overall, the improved sample preparation and bioanalytical techniques examined proved to be useful potential complements to conventional methods, enhancing the analytical framework for PAHs, PCDDs, and PCDFs. However, further validation has to be undertaken before they are applied on a large-scale.
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bhardwaj, vinay. "Label-free surface-enhanced Raman spectroscopy-linked immunosensor assay (SLISA) for environmental surveillance." FIU Digital Commons, 2015. http://digitalcommons.fiu.edu/etd/2321.
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The contamination of the environment, accidental or intentional, in particular with chemical toxins such as industrial chemicals and chemical warfare agents has increased public fear. There is a critical requirement for the continuous detection of toxins present at very low levels in the environment. Indeed, some ultra-sensitive analytical techniques already exist, for example chromatography and mass spectroscopy, which are approved by the US Environmental Protection Agency for the detection of toxins. However, these techniques are limited to the detection of known toxins. Cellular expression of genomic and proteomic biomarkers in response to toxins allows monitoring of known as well as unknown toxins using Polymerase Chain Reaction and Enzyme Linked Immunosensor Assays. However, these molecular assays allow only the endpoint (extracellular) detection and use labels such as fluorometric, colorimetric and radioactive, which increase chances of uncertainty in detection. Additionally, they are time, labor and cost intensive. These technical limitations are unfavorable towards the development of a biosensor technology for continuous detection of toxins. Federal agencies including the Departments of Homeland Security, Agriculture, Defense and others have urged the development of a detect-to-protect class of advanced biosensors, which enable environmental surveillance of toxins in resource-limited settings.
In this study a Surface-Enhanced Raman Spectroscopy (SERS) immunosensor, aka a SERS-linked immunosensor assay (SLISA), has been developed. Colloidal silver nanoparticles (Ag NPs) were used to design a flexible SERS immunosensor. The SLISA proof-of-concept biosensor was validated by the measurement of a dose dependent expression of RAD54 and HSP70 proteins in response to H2O2 and UV. A prototype microchip, best suited for SERS acquisition, was fabricated using an on-chip SLISA to detect RAD54 expression in response to H2O2. A dose-response relationship between H2O2 and RAD54 is established and correlated with EPA databases, which are established for human health risk assessment in the events of chemical exposure. SLISA outperformed ELISA by allowing RISE (rapid, inexpensive, simple and effective) detection of proteins within 2 hours and 3 steps. It did not require any label and provided qualitative information on antigen-antibody binding. SLISA can easily be translated to a portable assay using a handheld Raman spectrometer and it can be used in resource-limited settings. Additionally, this is the first report to deliver Ag NPs using TATHA2, a fusogenic peptide with cell permeability and endosomal rupture release properties, for rapid and high levels of Ag NPs uptake into yeast without significant toxicity, prerequisites for the development of the first intracellular SERS immunosensor.
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Chen, Yi-Jou, and 陳易柔. "Development of a novel poly-protein G expressing cell to enhance detection sensitivity of antibody based enzyme-linked immunosorbent assay(ELISA)." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/9gtsfh.
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碩士臺北醫學大學
生藥學研究所
102
Enzyme-linked immunosorbent assay (ELISA) has been widely used as an analytical tool in medicine and in various biotechnological applications. However, low sensitivity is a weakness of this method. We have developed a novel poly-protein G cell-based ELISA plate based on fixing membrane poly-protein G expressing cell on ELISA plate; thereby increasing the coating amount and detective sensitivity of antibody. Functional protein G or poly-protein G was stably expressed on cell membrane. The anti-PEG antibodies which were coated on poly-protein G cell-based ELISA plate was found to preserve PEG-binding activity. The antibody-coating capacity of the poly-protein G cell-based ELISA plate was 2−18 times higher than traditional ELISA plate. More importantly, the sensitivity for detecting PEG and pegylation molecular (PEG10K、PEG2K-Lipo-dox and PEGASYS) using anti-PEG antibodies coated on poly-protein G cell-based ELISA plate was significantly greater than traditional ELISA plate. In addition, the poly-protein G cell-based ELISA plate can be used to prepare a sandwich ELISA assay resulting in an increase in the coating amount of antibodies on ELISA plate; preserving the specificity and orientation of antibodies. Therefore, the poly-protein G cell-based ELISA plate ai a rald replacement for the traditional ELISA plate and is applicable in various industries.
Book chapters on the topic "Cell-based-ELISA"
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Molnár, Elek. "Cell-Based Enzyme-Linked Immunosorbent Assay (Cell-ELISA) Analysis of Native and Recombinant Glutamate Receptors." In Methods in Molecular Biology, 47–54. New York, NY: Springer New York, 2019. http://dx.doi.org/10.1007/978-1-4939-9077-1_4.
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Oubihi, Mohamed, Ken Kitajima, Kazukiyo Kobayashi, Takahiro Adachi, Naohito Aoki, and Tsukasa Matsuda. "Development of an Elisa Based Assay for Measuring Glycosyltransferase Activity Using Synthetic Glycopolymers." In Animal Cell Technology: Basic & Applied Aspects, 171–76. Dordrecht: Springer Netherlands, 1998. http://dx.doi.org/10.1007/978-94-011-5161-0_30.
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Logozzi, Mariantonia, Rossella Di Raimo, Davide Mizzoni, and Stefano Fais. "Immunocapture-based ELISA to characterize and quantify exosomes in both cell culture supernatants and body fluids." In Methods in Enzymology, 155–80. Elsevier, 2020. http://dx.doi.org/10.1016/bs.mie.2020.06.011.
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Elfiani, Elfiani, and Huntari Harahap. "Influence Engulfment Cell Motility-1 (ELMO-1) Protein and Matrix Metalloproteases-9 (MMP-9) in Diabetic Nephropathy Patients." In Urinary Tract Infection and Nephropathy - Insights into Potential Relationship. IntechOpen, 2022. http://dx.doi.org/10.5772/intechopen.98192.
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Engulfment and Cell Motility-1 (ELMO-1) are well-known genes in Asia that can cause diabetic nephropathy in people with Diabetes Mellitus type-2. The increase in ELMO-1 protein affects Matrix Metalloproteases-9 (MMP-9) levels, both of which can cause chronic glomerular injury through dysregulation of Extra Cellular Matrix metabolism and decreased adhesive properties of endothelial cells to kidney structures. This study aims to prove differences in ELMO-1 and MMP-9 protein levels in type-2 Diabetes Mellitus (DM) patients with Diabetic Nephropathy compared to those without Diabetic Nephropathy. This study is a comparative observational study with venous blood samples taken from 60 patients with type-2 DM patients without Diabetic Nephropathy as a control and type-2 DM group with Diabetic Nephropathy cases diagnosed based on the criteria of Glomerular Filtration Rate and Albumin-to-Creatinine Ratio. In this study, the levels of ELMO-1 and MMP-9 proteins were checked by ELISA (Enzyme-linked Immunosorbent Assay). The results showed that the mean plasma ELMO-1 value was higher in the Diabetes Mellitus type-2 group with Diabetic Nephropathy than without the Diabetic Nephropathy group (t-test, p = 0.025). The mean plasma MMP-9 value was higher in the DM with Diabetic Nephropathy group rather than in the DM without Diabetic Nephropathy group (t-test, p = 0.032). Conclusion ELMO-1 and MMP-9 levels were higher in Diabetes Mellitus type-2 with diabetic nephropathy.
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De Martino, Mara, Camille Daviaud, and Claire Vanpouille-Box. "ELISA-based quantification of type I IFN secretion by irradiated cancer cells." In Methods in Cell Biology. Elsevier, 2022. http://dx.doi.org/10.1016/bs.mcb.2022.01.004.
Full textConference papers on the topic "Cell-based-ELISA"
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Mahmoud, M., F. Hammerschmidt, H. Scheuerlein, and P. J. Gaffney. "IMMUNOASSAYS FOR SINGLE CHAIN URINARY-TYPE PLASMINOGEN ACTIVATOR (SCUPA) IN PLASMA AND IN CELL CULTURE SUPERNATANTS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643604.
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Monoclonal antibodies (mabs) to SCUPA have been generated in balb/C mice by conventional means and have been demonstrated to have no crossreactivity with two chain urinary-type plasminogen activator (TCUPA). These mabs have been used to develop two types of specific assay for SCUPA. Mabs coated on polyvinyl plates in conjunction with polyclonal antibodies (pabs) to TCUPA have allowed the development of a catcher-tag ELISA using alkaline phosphatase-label led goat anti-rabbit IgG as a final step. The sensitivity range of the assay was 0.5 - 10.0 iu/ml. A second bioimmunoassay (BIA) using SCUPA mabs as catcher and inplate development with glu-plasminogen and S-2251 has yielded an assay with a sensitivity range of 0.5 - 10.0 iu/ml. The international unitage ascribed in these assays was derived by comparing the hydrolysis of S-2444 by the I.S. for TCUPA with the purified SCUPA following full activation with plasmin.Using these assays it was found that normal pooled plasmas contained about 1.0 iu of SCUPA antigen which was fully inhibited such that no activity was evident by the BIA assay for SCUPA. This suggests that urokinase in plasma is present in two forms: SCTJPA bound to inhibitor and TCUPA which is biologically active when assayed using a BIA based on immobilised pabs to TCUPA. Cell supernatants from cultured human lung fibroblasts yield a SCTJPA/TCUPA ratio of 70/30 using S-2444 chromogenic assay following a plasmin-mediated SCTJPA-TCUPA conversion step. It was also shown that, in these cell supernatants, SCTJPA was secreted with no inhibitor bound to it, since the BIA and ELISA data were quite similar. A curious feature of these assays, which is as yet unexplained, is the observation that urokinase (both plasmin activated SCTJPA and TCUPA), when immunologically adsorbed on to the PVC-immobilised specific mabs or pabs used in this study, readily activated plasminogen but showed no hydrolytic activity on the chromogenic substrate, S-2444.
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Wang, Wei, Hamada A. Aboubakr, James Vang, Victor Brenk, Sagar M. Goyal, and James Collins. "Nanomagnetic Biosensor for the Detection of Porcine Interferon Gamma." In 2017 Design of Medical Devices Conference. American Society of Mechanical Engineers, 2017. http://dx.doi.org/10.1115/dmd2017-3375.
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Due to the anatomical and physiological similarities to humans that include similar heart size, flow rate, skin, liver enzymes and bone healing, porcine models as a powerful investigational platform have been widely used in research areas such as diabetes, obesity and islet transplantation [1]. The advantages of relative low cost, ease in handling and comparatively short period of breeding time may make swine provide a promising solution to the shortage of human donors and difficulty in isolating purified islets from adult human in future. Porcine cytokines play a significant role in innate immunity, apoptosis, angiogenesis, cell growth and differentiation. They are involved in cellular responses, maintenance of homeostasis, and disease states such as inflammatory disease, cardiovascular disease, and cancer. Thus, the technologies to analyze the expression of cytokines are developed rapidly and are still hot topics. The traditional approach for cytokine detection and quantification is the use of an enzyme-linked immunosorbent assay (ELISA). However, its inability to do multiplex test calls for more robust detection system. Biochip-based assay for the detection of biological agents using giant magnetoresistive (GMR) sensors and magnetic nanoparticles have emerged recently [2, 3]. It is proved that the nanomagnetic biosensor technology has advantages of low cost, high sensitivity, multiplexity, and real-time signal readout. The integration of GMR biosensor and use of weak magnetic fields allow to eventually realize point-of-care and portability. In addition, interferon gamma (IFNγ) is one of the most important porcine cytokines, and is associated with a number of autoinflammatory and autoimmune diseases. In this work, IFNγ is selected as a model target for the detection of porcine cytokine using nanomagnetic GMR biosensor.
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Camacho, M., A. Fabra, F. Carretero, M. Borrell, I. Millet, and M. L. Rutllant. "A MONOCLONAL BASED ELISA FOR HUMAN u-PA QUANTIFICATION." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644844.
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An ELISA has been developped for quantifiing the anti gen levels of u-PA present in human plasma, tissue and cell extracts, conditioned medium and others biological fluids.The assay was set up using PVC plates coated with rabbit anti u-PA IgG and a monoclonal antibody against human u-PA as second antibody (UKM23 obtained in our laboratory as previously described).Detection was performed with a rabbit anti-mouse IgG conjugated with horseradish peroxidase.By immunoblotting technique the monoclonal antibody used UKM23 , recognizes all human molecular weight spe cies and an additional band of 81 KD in human plasma. Also recognizes the u-PA present in conditioned medium from HT-1080 cell line.The detection limit of ELISA assay is 0,1 ng of total u-PA.The first assay in human plasma from healty volun ters, shows u-PA levels of 4,39 ± 0,94 ng/ml.This study was supported by grants from the CAICYTn9 1258/81 and 3628/86.
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Al-Ansari, Dana E., Nura A. Mohamed, Isra Marei, Huseyin Yalcin, and Haissam Abou-Saleh. "Assessment of Metal Organic Framework as Potential Drug Carriers in Cardiovascular Diseases." In Qatar University Annual Research Forum & Exhibition. Qatar University Press, 2020. http://dx.doi.org/10.29117/quarfe.2020.0127.
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Background: Cardiovascular diseases (CVDs) are considered the major cause of death worldwide. Therapeutic delivery to the cardiovascular system may play an important role in the successful treatment of a variety of CVDs, including atherosclerosis, ischemic-reperfusion injury, and microvascular diseases. Despite their clinical benefits, current therapeutic drugs are hindered by their short half-life and systemic side effects. This limitation could be overcome using controlled drug release with the potential for targeted drug delivery using a nanomedicine approach. In the current study, we have assessed the use of a highly porous nano-sized preparation of iron-based Metal-organic Framework (MOF) commonly referred to as MIL-89 as potential drug carriers in the cardiovascular system. Aims: To assess the effect of MOFs on the viability and cytotoxicity of human vascular cells and the cellular uptake in vitro, and the organ-system toxicity of MOF in vivo using the Zebrafish model. Methods: Human pulmonary endothelial cells (HPAECs) and pulmonary smooth muscle cells (HPASMCs) were treated with variable concentrations of MOFs. The viability, cytotoxicity and anti-inflammatory effects were measured using AlamarBlue, LDH assay and ELISA. The cellular uptake of MOFs were assessed using light, confocal, and transmission electron microscopes and EDS analysis. Moreover, Zebrafish embryos were cultured and treated with MOFs-nanoparticles at 0 hours post fertilization (hpf) followed by different organ-specific assays at 24, 48, and 72 hpf. Results: Although MOFs affect the viability at high concentrations, it does not cause any significant cytotoxicity on HPAECs and HPASMCs. Interestingly, MOFs were shown to have an anti-inflammatory effect. Microscopic images showed an increased (concentration-dependent) cellular uptake of MOFs and transfer to daughter cells in both cell types. Moreover, the in vivo study showed that high concentrations of MOFs delay zebrafish embryos hatching and cause heart deformation, which is currently investigated using cardiotoxicity markers. Conclusion: MOFs is a promising nanoparticle prototypes for drug delivery in the cardiovascular system with high cellular uptake and anti-inflammatory effects. Further investigations of MOFs, including diseased models and drug- loaded formulation is required.
Reports on the topic "Cell-based-ELISA"
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Gafny, Ron, A. L. N. Rao, and Edna Tanne. Etiology of the Rugose Wood Disease of Grapevine and Molecular Study of the Associated Trichoviruses. United States Department of Agriculture, September 2000. http://dx.doi.org/10.32747/2000.7575269.bard.
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Rugose wood is a complex disease of grapevines, characterized by modification of the woody cylinder of affected vines. The control of rugose wood is based on the production of healthy propagation material. Detection of rugose wood in grapevines is difficult and expensive: budwood from tested plants is grafted onto sensitive Vitis indicators and the appearance of symptoms is monitored for 3 years. The etiology of rugose wood is complex and has not yet been elucidated. Several elongated clostero-like viruses are consistently found in affected vines; one of them, grapevine virus A (GVA), is closely associated with Kober stem grooving, a component of the rugose wood complex. GVA has a single-stranded RNA genome of 7349 nucleotides, excluding a polyA tail at the 3' terminus. The GVA genome includes five open reading frames (ORFs 1-5). ORF 4, which encodes for the coat protein of GVA, is the only ORF for which the function was determined experimentally. The original objectives of this research were: 1- To produce antisera to the structural and non-structural proteins of GVA and GVB and to use these antibodies to establish an effective detection method. 2- Develop full length infectious cDNA clones of GVA and GVB. 3- Study the roll of GVA and GVB in the etiology of the grapevine rugose wood disease. 4- Determine the function of Trichovirus (now called Vitivirus) encoded genes in the virus life cycle. Each of the ORFs 2, 3, 4 and 5 genes of GVA were cloned and expressed in E. coli and used to produce antisera. Both the CP (ORF 4) and the putative MP (ORF 3) were detected with their corresponding antisera in-GVA infected N. benthamiana and grapevine. The MP was first detected at an early stage of the infection, 6-12 h after inoculation, and the CP 2-3 days after inoculation. The MP could be detected in GVA-infected grapevines that tested negative for CP, both with CP antiserum and with a commercially available ELISA kit. Antisera to ORF 2 and 5 encoded proteins could react with the recombinant proteins but failed to detect both proteins in GVA infected plants. A full-length cDNA clone of grapevine virus A (GVA) was constructed downstream from the bacteriophage T7 RNA polymerase promoter. Capped in vitro transcribed RNA was infectious in N. benthamiana and N. clevelandii plants. Symptoms induced by the RNA transcripts or by the parental virus were indistinguishable. The infectivity of the in vitro-transcribed RNA was confirmed by serological detection of the virus coat and movement proteins and by observation of virions by electron microscopy. The full-length clone was modified to include a gus reporter gene and gus activity was detected in inoculated and systemic leaves of infected plants. Studies of GVA mutants suggests that the coat protein (ORF 4) is essential for cell to cell movement, the putative movement protein (ORF 3) indeed functions as a movement protein and that ORF 2 is not required for virus replication, cell to cell or systemic movement. Attempts to infect grapevines by in-vitro transcripts, by inoculation of cDNA construct in which the virus is derived by the CaMV 35S promoter or by approach grafting with infected N. benthamiana, have so far failed. Studies of the subcellular distribution of GFP fusion with each of ORF 2, 3 and 4 encoded protein showed that the CP fusion protein accumulated as a soluble cytoplasmatic protein. The ORF 2 fusion protein accumulated in cytoplasmatic aggregates. The MP-GFP fusion protein accumulated in a large number of small aggregates in the cytoplasm and could not move from cell to cell. However, in conditions that allowed movement of the fusion protein from cell to cell (expression by a PVX vector or in young immature leaves) the protein did not form cytoplasmatic aggregates but accumulated in the plasmodesmata.
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Jordan, Ramon L., Abed Gera, Hei-Ti Hsu, Andre Franck, and Gad Loebenstein. Detection and Diagnosis of Virus Diseases of Pelargonium. United States Department of Agriculture, July 1994. http://dx.doi.org/10.32747/1994.7568793.bard.
Full textAbstract:
Pelargonium (Geranium) is the number one pot plant in many areas of the United States and Europe. Israel and the U.S. send to Europe rooted cuttings, foundation stocks and finished plants to supply a certain share of the market. Geraniums are propagated mainly vegetatively from cuttings. Consequently, viral diseases have been and remain a major threat to the production and quality of the crop. Among the viruses isolated from naturally infected geraniums, 11 are not specific to Pelargonium and occur in other crops while 6 other viruses seem to be limited to geranium. However, several of these viruses are not sufficiently characterized to conclude that they are distinct agents and their nomenclature and taxonomy are confusing. The ability to separate, distinguish and detect the different viruses in geranium will overcome obstacles te developing effective detection and certification schemes. Our focus was to further characterize some of these viruses and develop better methods for their detection and control. These viruses include: isolates of pelargonium line pattern virus (PLPV), pelargonium ringspot virus (PelRSV), pelargonium flower break virus (PFBV), pelargonium leaf curl (PLCV), and tomato ringspot virus (TomRSV). Twelve hybridoma cell lines secreting monoclonal antibodies specific to a geranium isolate of TomRSV were produced. These antibodies are currently being characterized and will be tested for the ability to detect TomRSV in infected geraniums. The biological, biochemical and serological properties of four isometric viruses - PLPV, PelRSV, and PFBV (and a PelRSV-like isolate from Italy called GR57) isolated from geraniums exhibiting line and ring pattern or flower break symptoms - and an isolate ol elderbeny latent virus (ELV; which the literature indicates is the same as PelRSV) have been determined Cloned cDNA copies of the genomic RNAs of these viruses were sequenced and the sizes and locations of predicted viral proteins deduced. A portion of the putative replicase genes was also sequenced from cloned RT-PCR fragments. We have shown that, when compared to the published biochemical and serological properties, and sequences and genome organizations of other small isometric plant viruses, all of these viruses should each be considered new, distinct members of the Carmovirus group of the family Tombusviridae. Hybridization assays using recombinant DNA probes also demonstrated that PLPV, PelRSV, and ELV produce only one subgenomic RNA in infected plants. This unusual property of the gene expression of these three viruses suggests that they are unique among the Carmoviruses. The development of new technologies for the detection of these viruses in geranium was also demonstrated. Hybridization probes developed to PFBV (radioactively-labeled cRNA riboprobes) and to PLPV (non-radioactive digoxigenin-labeled cDNAs) were generally shown to be no more sensitive for the detection of virus in infected plants than the standard ELISA serology-based assays. However, a reverse transcriptase-polymerase chain reaction assay was shown to be over 1000 times more sensitive in detecting PFBV in leaf extracts of infected geranium than was ELISA. This research has lead to a better understanding of the identity of the viruses infecting pelargonium and to the development of new tools that can be used in an improved scheme of providing virus-indexed pelargonium plants. The sequence information, and the serological and cloned DNA probes generated from this work, will allow the application of these new tools for virus detection, which will be useful in domestic and international indexing programs which are essential for the production of virus-free germplasm both for domestic markets and the international exchange of plant material.
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Altstein, Miriam, and Ronald J. Nachman. Rational Design of Insect Control Agent Prototypes Based on Pyrokinin/PBAN Neuropeptide Antagonists. United States Department of Agriculture, August 2013. http://dx.doi.org/10.32747/2013.7593398.bard.
Full textAbstract:
The general objective of this study was to develop rationally designed mimetic antagonists (and agonists) of the PK/PBAN Np class with enhanced bio-stability and bioavailability as prototypes for effective and environmentally friendly pest insect management agents. The PK/PBAN family is a multifunctional group of Nps that mediates key functions in insects (sex pheromone biosynthesis, cuticular melanization, myotropic activity, diapause and pupal development) and is, therefore, of high scientific and applied interest. The objectives of the current study were: (i) to identify an antagonist biophores (ii) to develop an arsenal of amphiphilic topically active PK/PBAN antagonists with an array of different time-release profiles based on the previously developed prototype analog; (iii) to develop rationally designed non-peptide SMLs based on the antagonist biophore determined in (i) and evaluate them in cloned receptor microplate binding assays and by pheromonotropic, melanotropic and pupariation in vivo assays. (iv) to clone PK/PBAN receptors (PK/PBAN-Rs) for further understanding of receptor-ligand interactions; (v) to develop microplate binding assays for screening the above SMLs. In the course of the granting period A series of amphiphilic PK/PBAN analogs based on a linear lead antagonist from the previous BARD grant was synthesized that incorporated a diverse array of hydrophobic groups (HR-Suc-A[dF]PRLa). Others were synthesized via the attachment of polyethylene glycol (PEG) polymers. A hydrophobic, biostablePK/PBAN/DH analog DH-2Abf-K prevented the onset of the protective state of diapause in H. zea pupae [EC50=7 pmol/larva] following injection into the preceding larval stage. It effectively induces the crop pest to commit a form of ‘ecological suicide’. Evaluation of a set of amphiphilic PK analogs with a diverse array of hydrophobic groups of the formula HR-Suc-FTPRLa led to the identification of analog T-63 (HR=Decyl) that increased the extent of diapause termination by a factor of 70% when applied topically to newly emerged pupae. Another biostablePK analog PK-Oic-1 featured anti-feedant and aphicidal properties that matched the potency of some commercial aphicides. Native PK showed no significant activity. The aphicidal effects were blocked by a new PEGylated PK antagonist analog PK-dF-PEG4, suggesting that the activity is mediated by a PK/PBAN receptor and therefore indicative of a novel and selective mode-of-action. Using a novel transPro mimetic motif (dihydroimidazole; ‘Jones’) developed in previous BARD-sponsored work, the first antagonist for the diapause hormone (DH), DH-Jo, was developed and shown to block over 50% of H. zea pupal diapause termination activity of native DH. This novel antagonist development strategy may be applicable to other invertebrate and vertebrate hormones that feature a transPro in the active core. The research identifies a critical component of the antagonist biophore for this PK/PBAN receptor subtype, i.e. a trans-oriented Pro. Additional work led to the molecular cloning and functional characterization of the DH receptor from H. zea, allowing for the discovery of three other DH antagonist analogs: Drosophila ETH, a β-AA analog, and a dF analog. The receptor experiments identified an agonist (DH-2Abf-dA) with a maximal response greater than native DH. ‘Deconvolution’ of a rationally-designed nonpeptide heterocyclic combinatorial library with a cyclic bis-guanidino (BG) scaffold led to discovery of several members that elicited activity in a pupariation acceleration assay, and one that also showed activity in an H. zea diapause termination assay, eliciting a maximal response of 90%. Molecular cloning and functional characterization of a CAP2b antidiuretic receptor from the kissing bug (R. prolixus) as well as the first CAP2b and PK receptors from a tick was also achieved. Notably, the PK/PBAN-like receptor from the cattle fever tick is unique among known PK/PBAN and CAP2b receptors in that it can interact with both ligand types, providing further evidence for an evolutionary relationship between these two NP families. In the course of the granting period we also managed to clone the PK/PBAN-R of H. peltigera, to express it and the S. littoralis-R Sf-9 cells and to evaluate their interaction with a variety of PK/PBAN ligands. In addition, three functional microplate assays in a HTS format have been developed: a cell-membrane competitive ligand binding assay; a Ca flux assay and a whole cell cAMP ELISA. The Ca flux assay has been used for receptor characterization due to its extremely high sensitivity. Computer homology studies were carried out to predict both receptor’s SAR and based on this analysis 8 mutants have been generated. The bioavailability of small linear antagonistic peptides has been evaluated and was found to be highly effective as sex pheromone biosynthesis inhibitors. The activity of 11 new amphiphilic analogs has also been evaluated. Unfortunately, due to a problem with the Heliothis moth colony we were unable to select those with pheromonotropic antagonistic activity and further check their bioavailability. Six peptides exhibited some melanotropic antagonistic activity but due to the low inhibitory effect the peptides were not further tested for bioavailability in S. littoralis larvae. Despite the fact that no new antagonistic peptides were discovered in the course of this granting period the results contribute to a better understanding of the interaction of the PK/PBAN family of Nps with their receptors, provided several HT assays for screening of libraries of various origin for presence of PK/PBAN-Ragonists and antagonists and provided important practical information for the further design of new, peptide-based insecticide prototypes aimed at the disruption of key neuroendocrine physiological functions in pest insects.