Sep 07, 2020
HolographicDiagnostics: Automated Virus Binding Assay
- Rushna Quddus1,
- Kaitlynn Snyder1,
- Lauren Altman1,
- Laura A Philips2,
- David G. Grier1,
- Andrew D. Hollingsworth1,
- Kent Kirshenbaum1
- 1New York University;
- 2Spheryx Inc
- Coronavirus Method Development Community
- Grier Group
Protocol Citation: Rushna Quddus, Kaitlynn Snyder, Lauren Altman, Laura A Philips, David G. Grier, Andrew D. Hollingsworth, Kent Kirshenbaum 2020. HolographicDiagnostics: Automated Virus Binding Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.bkpgkvjw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 02, 2020
Last Modified: September 07, 2020
Protocol Integer ID: 41416
Keywords: Immunoassay, Holographic Particle Characterization, SARS-CoV-2,
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Abstract
This protocol describes the steps required to perform a holographic immunoassay for SARS-CoV-2 whole virus particles. The assay uses an xSight holographic particle characterization instrument (Spheryx, Inc.) to monitor the diameter of specifically functionalized probe beads. The diameter of the beads increases by a few nanometers as targets bind to the surface. xSight detects and reports this change, yielding an estimate for the concentration of analytes in the sample. The method for holographic immunoassays (applied to antibody binding assays) is described in
Y. Zagzag, M. F. Soddu, A. D. Hollingsworth and D. G. Grier, Holographic molecular binding assays, Scientific Reports 10, 1932 (2020) and K. Snyder, R. Quddus, A. D. Hollingsworth and K. Kirshenbaum, Holographic Immunoassays: Direct Detection of Antibodies Binding to Colloidal Spheres, submitted for publication (2020).
Image Attribution
David G. Grier
Materials
MATERIALS
HD Test KitCatalog #HD SARS-CoV-2 TK-1
Safety warnings
Patient samples must be handled and disposed of appropriately.
Before start
Sealed patient samples must be heat-treated to inactivate viruses in patient samples before samples are processed in a BSL2 facility.
Sample Collection
Sample Collection
10m
10m
2 mL saliva $2
Subject passively collects saliva for testing.
Equipment
SDNA-1000
NAME
Whole Saliva Collection Kit
TYPE
Spectrum Solutions
BRAND
NA
SKU
10m
Sample Preparation
Sample Preparation
1h 45m
1h 45m
65 °C
Heat treat patient sample to inactivate virus.
Safety information
Patient samples must remain sealed until they are heat treated. BSL3 facilities are required to work with untreated samples. Heat-treated samples may be opened for processing in BSL2 facilities.
Note
Heat treatment may disrupt virus particles, liberating the antigens that are targets for the assay. This is not a problem because holographic immunoassays can detect dissolved proteins as well as dispersed virus particles.
30m
20 µL HD SARS-CoV-2 test kit $1
Add HolographicDiagnostics test kit to inactivated saliva sample.
31m
120 rpm, Room temperature
Incubate sample with test kit.
1h 31m
1000 rpm, Room temperature
Concentrate test beads by centrifugation.
1h 45m
Transfer sample to 96-well plate
Note
Sample barcode must be associated with the well in analytical software.
Currently, this transfer is performed manually using a barcode scanner.
Adoption of an automated sample preparation system also will automate this step
Sample Analysis
Sample Analysis
21m
21m
Transfer 96-well plate to xStream sample robot
Equipment
xStream
NAME
Sample handling robot
TYPE
Spheryx
BRAND
xStream
SKU
For each sample in 96-well plate, xStream transfers sample to xCell microfluidic chip for measurement in xSight.
Equipment
xCell
NAME
Microfluidic sample holder
TYPE
Spheryx
BRAND
xCell-8
SKU
30 µL $40/8 tests
Transfer concentrated beads to first available reservoir in xCell.
Note
xCell has 8 reservoirs, permitting up to 8 independent tests to be performed with a single chip. The cost per chip will decrease with volume purchases.
xStream automatically loads and disposes of xCells as needed.
1m
Perform holographic characterization measurement on loaded sample
Equipment
xSight
NAME
Holographic Particle Characterizer
TYPE
Spheryx
BRAND
xSight
SKU
xStream inserts xCell in xSight and initiates sample analysis
Note
xSight settings
wavelength: 445 nm
analytical volume: 1 uL
presaved ROI for each type of test bead in test kit
18m
Results of test are reported out.
Expected result
Report population mean diameter for each type of bead in test kit
HD SARS-CoV-2 TK-1 includes beads for three biomarkers (SARS-CoV-2 S1, S2 and M) and one negative control (H1N1).
20m
Sample Disposal
Sample Disposal
Dispose of used sample containers, pipette tips and xCells
Safety information
Used sample containers, pipette tips and xCells must be disposed in compliant biohazard containers. xStream deposits used xCells in suitable containers. Manual disposal is required for all other consumable items.