Fluorescent neutralization and adherence inhibition assays
Inventors
Kachurin, Anatoly • Kachurina, Olga • Wittman, Vaughan • TAPIA, Tenekua
Assignees
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Publication Number
US-9541545-B2
Publication Date
2017-01-10
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 relates to fluorescent antibody-based viral neutralization assays, including fluorescent neutralization (fmNt) and fluorescent adherence inhibition (fADI) assays. In these assays, a test antibody is incubated with a fluorescently-labeled virus to form a mixture, and target cells are exposed under conditions subduing endocytosis and permitting cell surface adherence so that labeled virus remains adhered to the cell surface rather than internalized. Fluorescence is then measured to determine inhibitory activity based on reduced cell-surface signal compared with a control without test antibody.
The fluorescently-labeled virus is used to report different functional outcomes depending on assay conditions. For fADI, inhibitory activity is determined by measuring fluorescence of labeled virus adhered to the surface of target cells and comparing it to a no-antibody control. For fmNt, neutralizing activity is determined through measurement of fluorescent signal associated with endocytosed virus, providing a readout associated with virus entry.
The approach is implemented using inactivated, fluorescently labeled virus and flow cytometric readout, including a BioPlex bead array reader as a simplified flow cytometer option. The described labeling and fluorescence management include fluorescent labels on virus, including biotinylated virus with streptavidin-phycoerythrin, and the use of surface-fluorescence quenchers and quenching strategies to reduce background signal. Multiple virus families and label types are described, and proof-of-concept comparisons are reported showing correlations with CDC/WHO microneutralization and improved sensitivity versus classical assay formats.
Claims Coverage
The partial content provides one independent claim and indicates dependent claims that refine assay components. Overall, the inventive coverage is structured around five core inventive elements: antibody incubation with a fluorescently-labeled virus, target-cell exposure under conditions subduing endocytosis while allowing cell-surface adherence, fluorescence measurement of adhered labeled virus, comparison to a no-antibody control, and determination of inhibitory activity from the comparison.
Incubating test antibody with fluorescently-labeled virus
Incubating a test antibody in vitro with a fluorescently-labeled virus to form a mixture.
Subduing endocytosis while permitting cell surface adherence
Incubating a population of target cells with the mixture under conditions subduing endocytosis and permitting cell surface adherence of the labeled virus to the target cells.
Measuring cell-surface fluorescence of adhered labeled virus
Measuring fluorescence of labeled virus adhered to the surface of the target cells.
Comparing with no-antibody control to determine inhibitory activity
Comparing the fluorescence measured with fluorescence of fluorescently-labeled virus in a control experiment where the labeled virus was not incubated with a test antibody, thereby determining an inhibitory activity of a test antibody.
Flow cytometer or bead array reader fluorescence detection
Detecting the labeled virus fluorescence using a flow cytometer or a bead array reader.
Optional target-cell staining prior to measurement
Staining a population of target cells with a dye before measuring them.
Red and infrared dye for bead array cell classification
Using a dye with red and infrared fluorescence to enable classification of the target cells in a bead array reader.
Inhibition represents blocking labeled virus entry
Specifying that the inhibitory activity blocks a labeled virus from entering target cells.
Fluorescently-labeled virus selected from specified virus families
Using a virus selected from specified virus families, where the selected labeled virus is conjugated to a fluorescent label.
The independent method claim covers an inhibitory activity assay based on incubating a test antibody with a fluorescently-labeled virus, exposing target cells under conditions that subdue endocytosis while allowing cell-surface adherence, measuring fluorescence of adhered labeled virus, and comparing to a no-antibody control to determine inhibitory activity. Dependent claim refinements further specify detection hardware, optional target-cell staining and bead-array-compatible red/infrared dye classification, mechanistic narrowing toward blocking entry, and use of labeled viruses selected from specified virus families.
Stated Advantages
Reduced safety concerns by using inactivated virus.
Rapid assay performance (about 1.5–4 hours).
Improved sensitivity by allowing measurement with low virus concentrations.
Compatibility with automation/high-throughput.
Lower cost versus conventional flow cytometers.
Improved sensitivity and correlation with classical HAI (hemagglutination inhibition).
fmNt correlation with CDC/WHO microneutralization using live virus.
Documented Applications
Determining inhibitory activity of a test antibody by measuring fluorescence of fluorescently-labeled virus adhered to target-cell surfaces under conditions subduing endocytosis.
Measuring neutralizing activity associated with virus entry using fluorescent neutralization (fmNt) as described through endocytosed fluorescence readout.
Measuring inhibitory activity associated with adherence blockade using fluorescent adherence inhibition (fADI) as described through surface-adhered fluorescence readout.
Evaluating antibodies/sera against viruses and reporting correlations with CDC/WHO microneutralization and with classical HAI.
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