Fluorescent neutralization and adherence inhibition assays
Inventors
Kachurin, Anatoly • Kachurina, Olga • Wittman, Vaughan • TAPIA, Tenekua
Assignees
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Publication Number
US-8778347-B2
Publication Date
2014-07-15
Expiration Date
Abstract
The present invention comprises rugged, inexpensive, reliable, and sensitive laboratory assays of antibody-based viral neutralization activity and antibody-based viral adherence inhibition activity. The assays use inactivated, fluorescently-labeled virus, allowing the tests to be performed without extensive safety precautions. The interaction of the labeled virus with target cells is monitored using flow cytometric methods. A preferred embodiment uses simple and inexpensive flow cytometry methodologies and equipment, such as bead array readers used as simplified flow cytometers. The assays are rapid, taking no longer than a few hours and are readily conducted by a trained technician. The assays are sensitive because they use labeled viruses at low concentrations and determine neutralizing and blocking capacity of sera and antibody at low concentrations. The methods are appropriate for high-throughput screening of large panels of samples.
Core Innovation
The invention provides a method for determining a viral neutralizing activity of a test antibody by using a fluorescently-labeled virus and measuring fluorescence after cellular endocytosis. A population of target cells is incubated with the mixture of the test antibody and the fluorescently-labeled virus under conditions permitting endocytosis of the labeled virus, and fluorescence associated with the surface-bound labeled virus is quenched before measuring fluorescence of labeled virus endocytozed by the target cells.
The core readout relies on endocytosis-based neutralization measured through fluorescence quenching of fluorescence of the labeled virus bound to the surface of the cells. The method includes incubating target cells with a quencher of fluorescence bound to the surface, staining the population of target cells with a dye, and comparing the measured fluorescence with fluorescence measured in a control where the labeled virus was not incubated with a test antibody.
In addition to the basic endocytosis/neutralization workflow, refinements specify using an inactivated fluorescently-labeled virus and selecting fluorescence quenchers that act at the cell surface, including antibody-based quenchers. Detection formats are refined to include flow cytometer or bead array reader approaches, and the neutralizing activity is characterized as blocking labeled virus entry into target cells.
Claims Coverage
The independent claim is clm-00001. The claim set covers a fluorescence-quenching, endocytosis-based neutralization assay workflow and refines it with virus inactivation options, specific fluorescence quencher configurations, specific quenching dyes, and defined detection instrumentation, plus an explicit characterization of neutralizing activity as blocking entry.
Endocytosis-based fluorescence neutralization readout with surface quenching
Incubating a test antibody with a fluorescently-labeled virus, incubating target cells with the mixture under conditions permitting endocytosis of the labeled virus, incubating the cells with a quencher of fluorescence of the labeled virus bound to the surface of the cells, staining the cells with a dye, measuring fluorescence of labeled virus endocytozed by the target cells, and comparing with a control lacking incubation with the test antibody to determine neutralizing activity.
Inactivated fluorescently-labeled virus for neutralization testing
Using an inactivated virus in the assay, with inactivation performed by betapropiolactone (BPL) or by UV or gamma irradiation.
Antibody-conjugate fluorescence quencher bound to cell surface label
Specifying that the fluorescence quencher is an antibody that specifically binds the fluorescent label of the labeled virus and is conjugated to at least one quenching compound.
Quenching dye selection as QSY-9 or QSY-21
Specifying that the quenching dye used is either quenching dye QSY-9 or quenching dye QSY-21.
Fluorescence detection using flow cytometer or bead array reader
Detecting fluorescence with a flow cytometer or a bead array reader.
Neutralizing activity defined as blocking labeled virus entry
Characterizing the antibody’s neutralizing activity as blocking labeled virus entry into target cells.
Overall, the claims center on an endocytosis-based neutralization determination using fluorescently-labeled virus, surface fluorescence quenching, cell staining, and comparison against a no-antibody control. Dependent claim refinements add specific inactivation options, antibody-conjugate quenchers, named quenching dyes, defined detection instrumentation, and an explicit entry-blocking characterization of neutralizing activity.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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