TITLE: HolographicDiagnostics: Automated Virus Binding Assay
AUTHORS: Rushna Quddus, Kaitlynn Snyder, Lauren Altman, Laura Philips, David G. Grier, Andrew D. Hollingsworth, Kent Kirshenbaum

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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</div><div class = "text-block"><span>Y. Zagzag, M. F. Soddu, A. D. Hollingsworth and D. G. Grier, </span><a href="http://physics.nyu.edu/grierlab/binding06c" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Holographic molecular binding assays</span></a><span>, </span><span style = "font-style:italic;">Scientific Reports </span><span style = "font-weight:bold;">10</span><span>, 1932 (2020) and K. Snyder, R. Quddus, A. D. Hollingsworth and K. Kirshenbaum, </span><a href="http://physics.nyu.edu/grierlab/immunoassay02c" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Holographic Immunoassays: Direct Detection of Antibodies Binding to Colloidal Spheres</span></a><span>, submitted for publication (2020).</span></div></div>

[STEPS]
?. [Sample Collection]
Subject passively collects saliva for testing.
[saliva]
?. [Sample Preparation]
Heat treat patient sample to inactivate virus.
65 °C
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.
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.
?. [Sample Preparation]
Add HolographicDiagnostics test kit to inactivated saliva sample.
[HD SARS-CoV-2 test kit]
?. [Sample Preparation]
Incubate sample with test kit.
33
?. [Sample Preparation]
Concentrate test beads by centrifugation.
Centrifuge: 1000 33
?. [Sample Analysis]
For each sample in 96-well plate, xStream transfers sample to xCell microfluidic chip for measurement in xSight.Transfer concentrated beads to first available reservoir in xCell.
30 µl
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.
?. [Sample Analysis]
Perform holographic characterization measurement on loaded samplexStream inserts xCell in xSight and initiates sample analysis
xSight settingswavelength: 445 nmanalytical volume: 1 uLpresaved ROI for each type of test bead in test kit
?. [Sample Analysis]
Results of test are reported out.
Report population mean diameter for each type of bead in test kitHD SARS-CoV-2 TK-1 includes beads for three biomarkers (SARS-CoV-2 S1, S2 and M) and one negative control (H1N1).
?. [Sample Disposal]
Dispose of used sample containers, pipette tips and xCells
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.
?. [Sample Preparation]
Transfer sample to 96-well plate
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]
Transfer 96-well plate to xStream sample robot