scholarly journals Analyte and label binding assay read by flow cytometry

1996 ◽  
Vol 42 (10) ◽  
pp. 1702-1708 ◽  
Author(s):  
J O Utgaard ◽  
J Frengen ◽  
T Stigbrand ◽  
A Ullen ◽  
R Schmid ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Binding Assay ◽  
Placental Alkaline Phosphatase ◽  
Sandwich Assay ◽  
Binding Partner ◽  
Hook Effect ◽  
Binding Partners ◽  
Unambiguous Determination ◽  
Label Antibody

Abstract A new immunometric two-site sandwich assay is introduced, in which a label-scavenging binding partner is added to the sample in addition to the analyte-binding partner. The scavenger binding partner binds excess label antibody, giving a signal proportional to the amount of excess label antibody in the sample solution. A set of two calibration curves is obtained from the two binding partners simultaneously, and a combination of the two signals gives an unambiguous determination of the analyte concentration, even for high analyte concentrations where the hook effect may occur. Two-particle immunofluorometric assays developed for placental alkaline phosphatase and human chorionic gonadotropin on the basis of this principle and yielding signals measured by flow cytometry gave rapid results (2 h) and had working ranges in excess of 5 and 6 orders of magnitude for the respective analytes.

scholarly journals Unambiguous determination of Plasmodium vivax reticulocyte invasion by flow cytometry

2016 ◽  
Vol 46 (1) ◽  
pp. 31-39 ◽  
Author(s):  
Jee-Sun Cho ◽  
Bruce Russell ◽  
Varakorn Kosasaivee ◽  
Rou Zhang ◽  
Yves Colin ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Plasmodium Vivax ◽  
Unambiguous Determination

scholarly journals Fluorescence Immunoassays Using CdSe/ZnS Core/Shell Quantum Dots for the Determination of Progesterone in Human Serum

2020 ◽  
Author(s):  
Hong Dinh Duong ◽  
Sung-Duk Oh ◽  
Jong Il Rhee
Keyword(s):  
Detection Limit ◽  
Human Serum ◽  
Fluorescence Intensity ◽  
Binding Assay ◽  
Sol Gel ◽  
Microtiter Plate ◽  
Linear Range ◽  
Sandwich Assay ◽  
Direct Binding

In this study, two heterogeneous fluorescence immunoassays using CdSe/ZnS quantum dot (QD) to label anti-progesterone antibody (P4Ab) for the determination of progesterone (P4) were performed in the wells of a 96-well microtiter plate. First, P4Ab was conjugated to hydrophilic CdSe/ZnS QDs via ethyl-3-(dimethylaminopropyl) carbodiimide(EDC)-N-hydroxysuccinimide) chemistry(NHS) (QDs-P4Ab conjugates). The QDs-P4Ab conjugate was employed as a second antibody in a sandwich assay, where the P4Ab was immobilized onto the 3-aminopropyltrimethoxysilane (APTMS) sol-gel membrane of the wells of a 96-well microtiter plate, and P4 was bound between the immobilized P4Ab and the QDs-P4Ab conjugate. In this assay, the fluorescence intensity of the QDs increased with increasing P4 concentrations. This assay had a detection limit of 553.9 pg/ml and a sensitivity of 18,251.96 pg/ml with a linear range of 2,184.6 – 117,082 pg/ml. In the direct binding assay, P4 was directly bound to the QDs-P4Ab conjugates immobilized onto the APTMS sol-gel membrane of the wells of a 96-well microtiter plate. In this direct binding assay the fluorescence intensity of the QDs decreased with increasing P4 concentrations, and this assay had a linear range of 28.95 – 26,607.7 pg/ml with a detection limit of 3.32 pg/ml and a sensitivity of 987.24 pg/ml. These fluorescence immunoassays have been successfully applied for the determination of P4 in real human serum, and the results were well correlated with those of a certified radioimmunoassay (RIA) method.

Corrigendum to “Unambiguous determination of Plasmodium vivax reticulocyte invasion by flow cytometry” [Int. J. Parasitol. 46 (2016) 31–39]

2017 ◽  
Vol 47 (4) ◽  
pp. 237
Author(s):  
Jee-Sun Cho ◽  
Bruce Russell ◽  
Varakorn Kosaisavee ◽  
Rou Zhang ◽  
Yves Colin ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Plasmodium Vivax ◽  
Unambiguous Determination

scholarly journals Optineurin links myosin VI to the Golgi complex and is involved in Golgi organization and exocytosis

2005 ◽  
Vol 169 (2) ◽  
pp. 285-295 ◽  
Author(s):  
Daniela A. Sahlender ◽  
Rhys C. Roberts ◽  
Susan D. Arden ◽  
Giulietta Spudich ◽  
Marcus J. Taylor ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Rna Interference ◽  
Vesicular Stomatitis Virus ◽  
Protein Interaction ◽  
Golgi Complex ◽  
Myosin Vi ◽  
Binding Partner ◽  
Binding Partners ◽  
Vesicular Stomatitis ◽  
Golgi Organization

Myosin VI plays a role in the maintenance of Golgi morphology and in exocytosis. In a yeast 2-hybrid screen we identified optineurin as a binding partner for myosin VI at the Golgi complex and confirmed this interaction in a range of protein interaction studies. Both proteins colocalize at the Golgi complex and in vesicles at the plasma membrane. When optineurin is depleted from cells using RNA interference, myosin VI is lost from the Golgi complex, the Golgi is fragmented and exocytosis of vesicular stomatitis virus G-protein to the plasma membrane is dramatically reduced. Two further binding partners for optineurin have been identified: huntingtin and Rab8. We show that myosin VI and Rab8 colocalize around the Golgi complex and in vesicles at the plasma membrane and overexpression of constitutively active Rab8-Q67L recruits myosin VI onto Rab8-positive structures. These results show that optineurin links myosin VI to the Golgi complex and plays a central role in Golgi ribbon formation and exocytosis.

scholarly journals Shared Components of the FRQ-Less Oscillator and TOR Pathway Maintain Rhythmicity in Neurospora

2021 ◽  
pp. 074873042199994
Author(s):  
Rosa Eskandari ◽  
Lalanthi Ratnayake ◽  
Patricia L. Lakin-Thomas
Keyword(s):  
Circadian Rhythms ◽  
Clock Genes ◽  
Nutrient Sensing ◽  
Mass Spectrometry Analysis ◽  
Growth Responses ◽  
Tor Pathway ◽  
Spectrometry Analysis ◽  
Binding Partner ◽  
Binding Partners ◽  
Free Running

Molecular models for the endogenous oscillators that drive circadian rhythms in eukaryotes center on rhythmic transcription/translation of a small number of “clock genes.” Although substantial evidence supports the concept that negative and positive transcription/translation feedback loops (TTFLs) are responsible for regulating the expression of these clock genes, certain rhythms in the filamentous fungus Neurospora crassa continue even when clock genes ( frq, wc-1, and wc-2) are not rhythmically expressed. Identification of the rhythmic processes operating outside of the TTFL has been a major unresolved area in circadian biology. Our lab previously identified a mutation ( vta) that abolishes FRQ-less rhythmicity of the conidiation rhythm and also affects rhythmicity when FRQ is functional. Further studies identified the vta gene product as a component of the TOR (Target of Rapamycin) nutrient-sensing pathway that is conserved in eukaryotes. We now report the discovery of TOR pathway components including GTR2 (homologous to the yeast protein Gtr2, and RAG C/D in mammals) as binding partners of VTA through co-immunoprecipitation (IP) and mass spectrometry analysis using a VTA-FLAG strain. Reciprocal IP with GTR2-FLAG found VTA as a binding partner. A Δ gtr2 strain was deficient in growth responses to amino acids. Free-running conidiation rhythms in a FRQ-less strain were abolished in Δ gtr2. Entrainment of a FRQ-less strain to cycles of heat pulses demonstrated that Δ gtr2 is defective in entrainment. In all of these assays, Δ gtr2 is similar to Δ vta. In addition, expression of GTR2 protein was found to be rhythmic across two circadian cycles, and functional VTA was required for GTR2 rhythmicity. FRQ protein exhibited the expected rhythm in the presence of GTR2 but the rhythmic level of FRQ dampened in the absence of GTR2. These results establish association of VTA with GTR2, and their role in maintaining functional circadian rhythms through the TOR pathway.

scholarly journals The Past, Present and Future of Flow Cytometry in Central Nervous System Malignancies

2021 ◽  
Vol 4 (1) ◽  
pp. 11
Author(s):  
Evrysthenis Vartholomatos ◽  
George Vartholomatos ◽  
George A. Alexiou ◽  
Georgios S. Markopoulos
Keyword(s):  
Central Nervous System ◽  
Flow Cytometry ◽  
Nervous System ◽  
Therapeutic Agents ◽  
Phenotypic Characterization ◽  
Cell Phenotype ◽  
Analytical Technique ◽  
The Past ◽  
Current Review

Central nervous system malignancies (CNSMs) are categorized among the most aggressive and deadly types of cancer. The low median survival in patients with CNSMs is partly explained by the objective difficulties of brain surgeries as well as by the acquired chemoresistance of CNSM cells. Flow Cytometry is an analytical technique with the ability to quantify cell phenotype and to categorize cell populations on the basis of their characteristics. In the current review, we summarize the Flow Cytometry methodologies that have been used to study different phenotypic aspects of CNSMs. These include DNA content analysis for the determination of malignancy status and phenotypic characterization, as well as the methodologies used during the development of novel therapeutic agents. We conclude with the historical and current utility of Flow Cytometry in the field, and we propose how we can exploit current and possible future methodologies in the battle against this dreadful type of malignancy.

scholarly journals KD determination from time-resolved experiments on live cells with LigandTracer and reconciliation with end-point flow cytometry measurements

2021 ◽  
Author(s):  
Diana Spiegelberg ◽  
Jonas Stenberg ◽  
Pascale Richalet ◽  
Marc Vanhove
Keyword(s):  
Flow Cytometry ◽  
Live Cells ◽  
Time Resolved ◽  
Surface Receptors ◽  
Full Characterization ◽  
End Point ◽  
Titration Curves ◽  
Kinetics Of

AbstractDesign of next-generation therapeutics comes with new challenges and emulates technology and methods to meet them. Characterizing the binding of either natural ligands or therapeutic proteins to cell-surface receptors, for which relevant recombinant versions may not exist, represents one of these challenges. Here we report the characterization of the interaction of five different antibody therapeutics (Trastuzumab, Rituximab, Panitumumab, Pertuzumab, and Cetuximab) with their cognate target receptors using LigandTracer. The method offers the advantage of being performed on live cells, alleviating the need for a recombinant source of the receptor. Furthermore, time-resolved measurements, in addition to allowing the determination of the affinity of the studied drug to its target, give access to the binding kinetics thereby providing a full characterization of the system. In this study, we also compared time-resolved LigandTracer data with end-point KD determination from flow cytometry experiments and hypothesize that discrepancies between these two approaches, when they exist, generally come from flow cytometry titration curves being acquired prior to full equilibration of the system. Our data, however, show that knowledge of the kinetics of the interaction allows to reconcile the data obtained by flow cytometry and LigandTracer and demonstrate the complementarity of these two methods.

scholarly journals Development of multiparametric analysis of human PBMC by flow cytometry: Determination of inflammatory and calcic profile

2021 ◽  
Vol 13 (2) ◽  
pp. 199-200
Author(s):  
C. Brun ◽  
A. Paccalet ◽  
T. Bochaton ◽  
M. Paillard ◽  
C. Crola Da Silva
Keyword(s):  
Flow Cytometry ◽  
Human Pbmc ◽  
Multiparametric Analysis

scholarly journals Flow Cytometry-Based Determination of Ploidy from Dried Leaf Specimens in Genomically Complex Collections of the Tropical Forage Grass Urochloa s. l.

Genes ◽  
2021 ◽  
Vol 12 (7) ◽  
pp. 957
Author(s):  
Paulina Tomaszewska ◽  
Till K. Pellny ◽  
Luis M. Hernández ◽  
Rowan A. C. Mitchell ◽  
Valheria Castiblanco ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Germplasm Collection ◽  
Forage Grass ◽  
Breeding Programs ◽  
Robust Identification ◽  
Ploidy Levels ◽  
Sustainable Improvement ◽  
Tropical Forage ◽  
Relative Differences

Urochloa (including Brachiaria, Megathyrus and some Panicum) tropical grasses are native to Africa and are now, after selection and breeding, planted worldwide, particularly in South America, as important forages with huge potential for further sustainable improvement and conservation of grasslands. We aimed to develop an optimized approach to determine ploidy of germplasm collection of this tropical forage grass group using dried leaf material, including approaches to collect, dry and preserve plant samples for flow cytometry analysis. Our methods enable robust identification of ploidy levels (coefficient of variation of G0/G1 peaks, CV, typically <5%). Ploidy of some 348 forage grass accessions (ploidy range from 2x to 9x), from international genetic resource collections, showing variation in basic chromosome numbers and reproduction modes (apomixis and sexual), were determined using our defined standard protocol. Two major Urochloa agamic complexes are used in the current breeding programs at CIAT and EMBRAPA: the ’brizantha’ and ’humidicola’ agamic complexes are variable, with multiple ploidy levels. Some U. brizantha accessions have odd level of ploidy (5x), and the relative differences in fluorescence values of the peak positions between adjacent cytotypes is reduced, thus more precise examination of this species is required. Ploidy measurement of U. humidicola revealed aneuploidy.

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