scholarly journals Epitope Binning of Novel Monoclonal Anti F1 and Anti LcrV Antibodies and Their Application in a Simple, Short, HTRF Test for Clinical Plague Detection

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 285
Author(s):  
Adva Mechaly ◽  
Einat B. Vitner ◽  
Yinon Levy ◽  
David Gur ◽  
Moria Barlev-Gross ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Blood Culture ◽  
Virulence Factor ◽  
Protein Fraction ◽  
Time Resolved Fluorescence ◽  
Calcium Response ◽  
Time Resolved ◽  
Disease Biomarkers ◽  
Antibody Purification

Mouse monoclonal antibodies were raised against plague disease biomarkers: the bacterial capsular protein fraction 1 (F1) and the low-calcium response—LcrV virulence factor (Vag). A novel tandem assay, employing BioLayer Interferometry (BLI), enabled the isolation of antibodies against four different epitopes on Vag. The tandem assay was carried out with hybridoma supernatants, circumventing the need for antibody purification. The BioLayer assay was further adopted for characterization of epitope-repetitive antigens, enabling the discovery of two unique epitopes on F1. The selected antibodies were purified and applied as “oligo-clonal” reagents for the immuno-detection of both biomarkers. The developed Homogenous Time Resolved Fluorescence (HTRF) tests were short (10 min) and simple (no washing steps), allowing for detection of 10 ng/mL F1 and 2.5 ng/mL Vag. The tests were successfully applied for detection of disease biomarkers produced by various Y. pestis strains during growth in blood culture vials.

scholarly journals Synthesis and Characterization of Time-resolved Fluorescence Probes for Evaluation of Competitive Binding to Melanocortin Receptors

2013 ◽  
Vol 21 (17) ◽  
pp. 5029-5038 ◽  
Author(s):  
Ramesh Alleti ◽  
Josef Vagner ◽  
Dilani Chathurika Dehigaspitiya ◽  
Valerie E. Moberg ◽  
N.G.R.D. Elshan ◽  
...  
Keyword(s):  
Competitive Binding ◽  
Fluorescence Probes ◽  
Synthesis And Characterization ◽  
Melanocortin Receptors ◽  
Time Resolved Fluorescence ◽  
Time Resolved

Measurement of estrone-3-glucuronide in urine by rapid, homogeneous time-resolved fluoroimmunoassay.

1989 ◽  
Vol 35 (4) ◽  
pp. 555-559 ◽  
Author(s):  
G Barnard ◽  
F Kohen ◽  
H Mikola ◽  
T Lövgren
Keyword(s):  
Monoclonal Antibodies ◽  
Liquid Phase ◽  
Clinical Utility ◽  
Ovarian Function ◽  
Fluorescence Immunoassay ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Europium Chelate ◽  
Covalently Linked ◽  
Sensitivity Specificity

Abstract We describe a liquid-phase nonseparation time-resolved fluorescence immunoassay for measuring estrone-3-glucuronide in undiluted urine. The sensitivity, specificity, and accuracy are similar to those for a conventional separation fluoroimmunoassay or radioimmunoassay, but the speed, convenience, precision, reliability, and clinical utility of the new method are more advantageous. The labeled antigen, a fluorescent europium chelate covalently linked to estrone-3-glucuronide, is incubated for 10 min with a limited concentration of polyclonal or monoclonal antibodies to estrone-3-glucuronyl-6-bovine serum albumin and 10 microL of standard or sample (undiluted urine) in microtiter wells. The fluorescence emanating from the antibody-free label, which is proportional to the concentration of estrone-3-glucuronide in the standard or sample, is then measured in a time-resolved fluorometer. The method is useful for monitoring ovarian function in women.

Characterization of Plasma-Modified Fluoropolymer Surfaces Using Steady-State and Time-Resolved Fluorescence Spectroscopy

1994 ◽  
Vol 48 (5) ◽  
pp. 630-637 ◽  
Author(s):  
Ming Li ◽  
Michaeleen L. Pacholski ◽  
Frank V. Bright
Keyword(s):  
Steady State ◽  
Fluorescence Spectroscopy ◽  
Sulfonyl Chloride ◽  
Sodium Dodecylsulfate ◽  
Covalent Immobilization ◽  
Ambient Conditions ◽  
Time Resolved Fluorescence ◽  
Decay Kinetics ◽  
Time Resolved

Poly(hexafluoropropylene-co-tetrafluoroethylene) (FEP) has been widely used in biotechnology because of its unique surface properties and biocompatibility. Recent work from our group has shown that plasma discharge-modified FEP can be used as the substratum for development of a very stable immunosensor. This result has prompted us to study further this new surface under ambient conditions. In this paper, we report on the covalent immobilization of a pyrene residue (-Py) onto FEP-APS (FEP-aminopropyl silane) surfaces and the characterization of FEP-APS-Py using steady-state and time-resolved fluorescence spectroscopy. Among the immobilization schemes tested, we found that the covalent coupling of pyrene-sulfonyl chloride to FEP-APS is the easiest and yields the most photostable FEP-APS-Py derivative. Steady-state emission spectra of FEP-APS-Py in contact with H2O, β-cyclodextrin (β-CD), and sodium dodecylsulfate (SDS) aqueous solutions differ considerably from those of Py-SO3 in solution. Time-resolved fluorescence spectroscopy of FEP-APS-Py demonstrates that the decay kinetics are strongly affected by the presence of ionic quenchers and molecular oxygen, as well as β-CD and SDS. The results are consistent with the suggestion that the APS-Py moiety undergoes a slow time-dependent reconfiguration at the FEP/APS interface.

scholarly journals Characterization of F2N12S in Cell Membranes using Time-Resolved Fluorescence Techniques

2018 ◽  
Vol 114 (3) ◽  
pp. 98a-99a
Author(s):  
Donald S. Anderson ◽  
Matthew J. Sydor ◽  
Harmen B. Steele ◽  
J.B.A. Ross ◽  
Holian Andrij
Keyword(s):  
Cell Membranes ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Fluorescence Techniques
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