Benjamin D. Grant

Intellectual Ventures (United States)

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David D. Thomas

University of Minnesota

Tory Schaaf

Minneapolis VA Medical Center

Bernhard H. Weigl

Intellectual Ventures (United States)

Luis F. Alonzo

Intellectual Ventures (United States)

Kevin P. Nichols

Intellectual Ventures (United States)

Caitlin E. Anderson

Intellectual Ventures (United States)

Veronika Glukhova

Bellevue Hospital Center

David S. Boyle

Academia Sinica

Kurt C. Peterson

Fluorescence Innovations (United States)

Gregory D. Gillispie

Fluorescence Innovations (United States)

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University of Washington

Intellectual Ventures (United States)

Bellevue Hospital Center

University of Minnesota

Seattle University

University of Minnesota System

Program for Appropriate Technology in Health

Rice University

Hospital de Câncer de Barretos

Infectious Diseases Research Collaboration

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A low-cost platform for cervical cancer detection

Benjamin D. Grant

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307. Diagnostic Test Kinetics, Infectivity, and Immunological Responses Among Unvaccinated Adults During Acute SARS-CoV-2 Infection

Open Forum Infectious Diseases (2022)

Paul K. Drain Ronit R. Dalmat Meagan J. Bemer Elvira Budiawan Jennifer F. Morton

Abstract Background Appropriate diagnostic testing can be used to inform infection control measures and reduce SARS-CoV-2 transmission, yet the test kinetics, infectivity, and immunological responses during acute, non-severe SARS-CoV-2 infection need clarity. Methods We conducted a prospective cohort study between Nov 2020-July 2021 in Seattle, Washington of 95 unvaccinated, immunocompetent adults with no prior SARS-CoV-2 infection. Nasal swabs (nasopharyngeal and anterior) and blood serum samples were serially collected at six visits over two months. Viral RNA, N and S antigen concentrations, and viral growth/infectivity were measured from nasal samples. Anti-S total antibody and IgG assays were performed on serum. We fit loess curves to quantitative data corresponding to each testing modality by days since symptom onset (DSSO) and compared qualitative test results across time points to demonstrate time-dependent agreement of PCR, N antigen, and culture results. Generalized estimating equations were used to approximate relative risk of culture positivity (a proxy for infectiousness) for positive vs. negative test results (antigen and PCR), stratified by presence/absence of symptoms and DSSO. Sampling Schema Nasal swabs and venous blood were collected at visits 1-4; venous blood only at visits 5-6. All participants were enrolled within 14 days of symptom onset (median: 6) and 7 days of a positive test (median: 4). Results Infections in this cohort (median age: 29y) were mild (no hospitalization). Median (IQR) time to negative result was 11 (4), 13 (6), and 20 (7) DSSO for culture growth, N antigen, and PCR tests, respectively. Viral RNA quantities declined more slowly than antigen and culturable virus; antibody titers rose rapidly 5-15 DSSO and plateaued 20-30 DSSO. All culture-positive samples collected 0-5 DSSO were positive by PCR, but relative risk of culture positivity (infectiousness) for positive vs. negative PCR results declined 6-10 DSSO. Relative risk of culture positivity for positive vs. negative antigen results was consistently high 0-10 DSSO, with similar results when stratified by presence of symptoms. Diagnostic test kinetics and immunological responses Diagnostic test kinetics and immunological responses measured in adults with non-severe, symptomatic SARS-CoV-2 infection: loess trendlines and 95% confidence intervals are given for SARS-CoV-2 viral load (calculated from PCR Ct value using a calibration curve), TCID50 from viral culture, mean concentrations of nucleocapsid and spike antigen proteins, and anti-S total and IgG antibody concentrations. Conclusion The results reinforce the importance of molecular PCR testing as a highly sensitive diagnostic tool but with limited utility as an indicator of viral culturability and likely infectiousness. N antigen testing may be a preferable diagnostic test within two weeks of symptom onset, especially 6-10 DSSO, because it more closely correlates with culture growth over the course of infection. Disclosures Daphne Hamilton, BA, Roche (spouse is employed by Roche): Employee Alexander L. Greninger, MD, PhD, Abbott: Contract Testing|Cepheid: Contract Testing|Gilead: Grant/Research Support|Gilead: Contract Testing|Hologic: Contract Testing|Merck: Grant/Research Support|Novavax: Contract Testing|Pfizer: Contract Testing Geoffrey S. Gottlieb, MD, PhD, Abbott Molecular Diagnostics: Grant/Research Support|Alere Technologies: Grant/Research Support|BMGF: Grant/Research Support|BMS: Grant/Research Support|Cerus Corp.: Grant/Research Support|Gilead Sciences: Grant/Research Support|Janssen Pharmaceutica: Grant/Research Support|Merck & Co: Grant/Research Support|Roche Molecular Systems: Grant/Research Support|THERA Technologies/TaiMed Biologics: Grant/Research Support|ViiV Healthcare: Grant/Research Support.

Infectivity

10.1093/ofid/ofac492.385

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Screening Antibodies Raised Against the Spike Glycoprotein of SARS-CoV-2 to Support the Development of Rapid Antigen Assays

Jason L. Cantera David M. Cate Allison Golden Roger Peck Lorraine Lillis

The spike glycoprotein of SARS-CoV-2 is a highly conserved surface protein and as such may represent a good target for immunoassay detection. We screened a variety of antibodies that were reactive to the S glycoprotein in a highly sensitive liquid immunoassay format and also on paper-based or lateral flow assay (LFA) to assess their analytical performance. Our findings included significant variation in performance when using different sources of S antigen. We identified several antibody pairs that had an LOD of below 10 pg/mL in the liquid immunoassay format with the lowest being 3 pg/mL. The antibodies were highly specific to SARS-Cov-2 based on cross reactivity screening with other human CoVs. The LFA screening found some different optimal antibody pairs from the pool of candidate antibodies used but a several antibodies were observed to have high performance with either immunoassay format.

10.26434/chemrxiv.12899672.v1

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SARS-CoV-2 Coronavirus Nucleocapsid Antigen-Detecting Half-Strip Lateral Flow Assay Toward the Development of Point of Care Tests Using Commercially Available Reagents

Analytical Chemistry (2020)

Benjamin D. Grant Caitlin E. Anderson John R. Williford Luis F. Alonzo Veronika Glukhova

The SARS-CoV-2 pandemic has created an unprecedented need for rapid diagnostic testing to enable the efficient treatment and mitigation of COVID-19. The primary diagnostic tool currently employed is reverse transcription polymerase chain reaction (RT-PCR), which can have good sensitivity and excellent specificity. Unfortunately, implementation costs and logistical problems with reagents during the global SARS-CoV-2 pandemic have hindered its universal on demand adoption. Lateral flow assays (LFAs) represent a class of diagnostic that, if sufficiently clinically sensitive, may fill many of the gaps in the current RT-PCR testing regime, especially in low- and middle-income countries (LMICs). To date, many serology LFAs have been developed, though none meet the performance requirements necessary for diagnostic use cases, primarily due to the relatively long delay between infection and seroconversion. However, on the basis of previously reported results from SARS-CoV-1, antigen-based SARS-CoV-2 assays may have significantly better clinical sensitivity than serology assays. To date, only a very small number of antigen-detecting LFAs have been developed. Development of a half-strip LFA is a useful first step in the development of any LFA format. In this work, we present a half-strip LFA using commercially available antibodies for the detection of SARS-CoV-2. We have tested this LFA in buffer and measured an LOD of 0.65 ng/mL (95% CI of 0.53 to 0.77 ng/mL) ng/mL with recombinant antigen using an optical reader with sensitivity equivalent to a visual read. Further development, including evaluating the appropriate sample matrix, will be required for this assay approach to be made useful in a point of care setting, though this half-strip LFA may serve as a useful starting point for others developing similar tests.

Seroconversion

Point of care

Pandemic

10.1021/acs.analchem.0c01975

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SUPR: Spectral Unmixinig Plate Reader for Live-Cell FRET Biosensor Drug Screening

Biophysical Journal (2016)

Tory Schaaf Ji Li Samantha L. Yuen Prachi Bawaskar Benjamin D. Grant

Autofluorescence

Acceptor

Plate reader

10.1016/j.bpj.2015.11.2011

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310. Longitudinal Assessment of Point-of-Care LumiraDx Antigen and Antibody Test Performance During Acute SARS-CoV-2 Infection and Recovery

Open Forum Infectious Diseases (2022)

Ronit R. Dalmat Roshni Prabhu Marie Bauer Matthew H Ikuma Daphne Hamilton

Abstract Background Rapid COVID-19 tests can offer significant advantages and reduce health disparities. The LumiraDx SARS-CoV-2 platform can perform microfluidic fluorescence assays for the rapid detection of SARS-CoV-2 antigen (Ag) and antibodies (Ab). We evaluated both tests in a longitudinal cohort to evaluate performance during acute SARS-CoV-2 infection and recovery. Methods We collected nasal samples from 71 unique participants at four clinic visits spanning 0-21 days since symptom onset (DSSO); blood samples were collected from the same participants over six visits spanning 0-87 DSSO. For Ag testing, 232 anterior nasal swabs were assayed by: 1) the LumiraDx Ag test, 2) a laboratory-based electrochemiluminescence immunoassay for N Ag, 3) RT-PCR (Hologic Panther Fusion), and 4) culture (growth in VeroE6AT cells). For Ab testing, 308 serum samples were assayed by: 1) the LumiraDx Ab test and 2) Roche Elecsys Anti-S SARS-CoV-2 total Ab test. Measures of concordance [positive predictive agreement (PPA), negative predictive agreement (NPA), and Cohen’s Kappa (Κ)] were estimated for qualitative results of the LumiraDx tests versus corresponding lab reference tests. Confidence intervals were estimated via bootstrapping. Results LumiraDx Ag results had strong agreement with lab N-Ag results (Κ > 0.80) across all samples. Between 0-5 days, agreement was perfect, except for one sample resulting positive by LumiraDx Ag and negative by lab Ag. Agreement with PCR results was moderate overall (Κ=0.60), though substantial (Κ > 0.6) for both 0-5 DSSO (PPA=0.96/NPA=0.80) and 6-10 DSSO (PPA=0.96/NPA=0.59). Agreement with culture results was moderate overall (Κ=0.46): substantial (Κ=0.6) between 0-5 DSSO (PPA=0.96/NPA=0.60) and fair (Κ=0.29) between 6-10 DSSO (PPA=1.0/NPA=0.32). LumiraDx Ab results showed almost perfect agreement with lab Ab results across all samples (Κ=0.88), with substantial agreement (Κ > 0.7) for samples collected 0-10 DSSO (PPA=0.93/NPA=0.89) and 11-28 DSSO (PPA=0.99/NPA=0.69). Longitudinal agreement of LumiraDx antigen test result and culture positivity, by PCR Ct value. Nasal samples grouped by participant (lines) and agreement of results between LumiraDx antigen test result and culture positivity (proxy for infectiousness). Conclusion LumiraDx rapid tests perform well compared to more costly and time-consuming lab methods of Ag and Ab detection. The rapid Ag test may be helpful in identifying patients infectious between 0-5 DSSO, given the substantial concordance of the rapid Ag test and culture positivity. Disclosures Daphne Hamilton, BA, Roche (spouse is employed by Roche): Employee.

Concordance

Point-of-Care Testing

Point of care

Kappa

Gold standard (test)

10.1093/ofid/ofac492.388

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Red-Shifted Fluorescent Proteins Improve FRET Biosensors for High-throughput Fluorescence Lifetime Screening

Biophysical Journal (2018)

Tory Schaaf Ang Li Benjamin D. Grant Prachi Bawaskar Evan Kleinboehl

Alexa Fluor

Plate reader

10.1016/j.bpj.2017.11.955

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Inhibition of Tumor Necrosis Factor Receptor 1 Signaling by Small Molecules

The FASEB Journal (2017)

Chih Hung Lo Nagamani Vunnam Andrew K. Lewis Ting‐Lan Chiu Benjamin E. Brummel

Tumor necrosis factor receptor 1 (TNFR1) is a member of the TNFR superfamily which can be activated by binding to one of two cognate ligands, tumor necrosis factor (TNF‐α) or lymphotoxin‐α (LT‐α), via its extracellular domain. Ligand binding leads to network formation and signaling cascade of IκBα degradation and NF‐κB activation, resulting in inflammatory and autoimmune diseases. Current treatments, such as etanercept and infliximab, reduce symptoms through sequestration of free ligands. However, these treatments are expensive and induce dangerous side‐effects due to off‐target inhibition of other related TNF receptors that are not involved in the disease condition. As a consequence, there is a desperate need for receptor‐specific treatments, which hold the promise of overcoming the limitations of conventional therapeutics. It has been proposed that TNFR1 pre‐ligand assembly domain (PLAD), a portion of the extracellular region of TNFR1 that mediates receptor‐chain association essential for signaling, is an important therapeutic target in inflammatory arthritis. This motivates the discovery of small‐molecule inhibitors that could bind to TNFR1 PLAD to disrupt receptor‐receptor interactions and inhibit downstream signaling. In this study, we have engineered a biosensor exhibiting fluorescence resonance energy transfer (FRET) by attaching fluorophores to the intracellular domain of TNFR1. By using a fluorescence lifetime screening platform, two hit compounds were identified to inhibit TNFR1 function. These small‐molecule inhibitors reduce FRET and disrupt PLAD‐PLAD interaction without ablating ligand binding. The biosensor and the fluorescence lifetime screening approach reported here provide a very promising high‐throughput drug discovery platform for TNF receptors, as well as oligomeric receptors in general. Support or Funding Information This work is supported by National Institutes of Health Grant R01 GM107175 and R42 DA037622

10.1096/fasebj.31.1_supplement.609.11

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Spectral Unmixing Plate Reader: High-Throughput, High-Precision FRET Assays in Living Cells

SLAS DISCOVERY (2016)

Tory Schaaf Kurt C. Peterson Benjamin D. Grant David D. Thomas Gregory D. Gillispie

We have developed a microplate reader that records a complete high-quality fluorescence emission spectrum on a well-by-well basis under true high-throughput screening (HTS) conditions. The read time for an entire 384-well plate is less than 3 min. This instrument is particularly well suited for assays based on fluorescence resonance energy transfer (FRET). Intramolecular protein biosensors with genetically encoded green fluorescent protein (GFP) donor and red fluorescent protein (RFP) acceptor tags at positions sensitive to structural changes were stably expressed and studied in living HEK cells. Accurate quantitation of FRET was achieved by decomposing each observed spectrum into a linear combination of four component (basis) spectra (GFP emission, RFP emission, water Raman, and cell autofluorescence). Excitation and detection are both conducted from the top, allowing for thermoelectric control of the sample temperature from below. This spectral unmixing plate reader (SUPR) delivers an unprecedented combination of speed, precision, and accuracy for studying ensemble-averaged FRET in living cells. It complements our previously reported fluorescence lifetime plate reader, which offers the feature of resolving multiple FRET populations within the ensemble. The combination of these two direct waveform-recording technologies greatly enhances the precision and information content for HTS in drug discovery.

Autofluorescence

Plate reader

10.1177/1087057116679637

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High-resolution microendoscopy (HRME): A low-cost, point-of-care alternative to colposcopy and biopsies?

Gynecologic Oncology (2015)

Kathleen M. Schmeler J.T.G. Fregnani Benjamin D. Grant José Resende C. Scapulatempo‐Neto

Atypia

10.1016/j.ygyno.2015.01.040

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