Recipient Organization
APOLLODX , LLC
825 NORTH 300 WEST
SALT LAKE CITY,UT 841031459
Performing Department
R&D
Non Technical Summary
In Harvard's publication,Roadmap to Pandemic Resilience, Danielle Allen and co-authors clearly stated that for "a safe social reopening" of the US, there needs to be 5 million coronavirus tests performper dayin early June and 20 million per day in, ideally, late July.Nobel Laureate economist Paul Romer stated that the number is 35 million tests per day.As of 8/29/20, the CDC reported 82,779,999 tests have been performed in the US since the beginning.This total is far short of these prominent experts' projections.Bill Gates further accentuates this gap in his statement 'obtaining test results more than 48 hours after sample acquisition "are a complete waste".Results should be provided within 24 hours.'Currently, test results often are not provided for 3, 7, and in some cases more than 14 days.This is unacceptable.In short, the US needs much more testing capabilities.Many segments of medical diagnostics are heterogeneous: no single testing technology or vendor satisifying the market need.For coronavirus testing, commercial labs, the main provider of test results during this pandemic, are not sufficiently capable as the above statistics indicate.The concept of the 'drive-through testing center' has largely been a misnomer: these centers are not for testing; they are sample collection centers that ship samples to commercial labs for testing.Most sampling for the 83 million tests has occurred in these drive-through sampling centers.Because the sophisticated, equipment-intensive, high throughput commercial labs are not the solution as is evident, other resources must be testing resources including hospitals, clinics, physician offices within the medical complex but also non-medical facilities as well including schools, businesses, places of worship, and, ultimately, homes. This is especially true in sparsely populated rural areas.Control of this rampant pandemic willonlytake place with a multiplicity of places where testing can occur are implemented.Understanding this profound need for a geometric expansion of testing drives the requirement for extensive decentralized testing.This mandates testing technologies that are more widely accessible from an equipment, procedural and training points of view.Accessibility can be implemented through equipment-free or low-cost equipment-based diagnostic technologies.Reducing the complexity and number of steps from sample acquisition to test result drives procedural simplicity. If training people with no educational background in laboratory sciences can be successfully performed by experts with the decentralized, simply, rapid technology, this is a recipe for spreading the burden of dramatically increasing the sites where coronavirus testing is performed.This is possible with the ApolloDx system.This proposal addresses some of these challenges with a simple, inexpensive, rapid testing technology.Will ApolloDx's solution serve all market segments?No. ApolloDx's technology is not a high throughput technology; it will not be used in commercial labs to process thousands of samples in one location within a day.In ApolloDx's instrument's current form, It is unlikely that many homes will purchase ApolloDx's current potentiostat - the instrument that removes subjectivity from evaluating test results.ApolloDx's potentiostat is much more capable in than current glucose electrochemical sensors because ApolloDx's instrument can detect concentrations of the substance in a patient's sample a billion-fold lower in concentration than glucose meters can.As such, ApolloDx's meters are more expensive.This will limit sales of the ApolloDx system from marketing success in many homes.But, there are many other market segments as listed above in which the ApolloDx system will serve well.There are extremely important features of the ApolloDx system that are not and will not included in other decentralized, simple coronavirus diagnostic systems including:Objective assessment of test results andAn integrated gateway to contact tracingObjectively determined test results.Objective assessment is extraordinarily important in infectious disease testing especially for any diagnostic system that is used by laypeople.Why? Because the substances appearing in samples from patients potentially tainted with an infectious organismdo notalways appear in abundant concentrations.Consequently, some samples will present as 'borderline positive' requiring the judgment of an un- or under-trained individual in judging whether the sample should be declared positive or not.With the experience leading a R&D group at DuPont developing HIV/AIDS test in the 1980's when AIDS was nearly 100% fatal, this project's PI has first-hand knowledge that interpreting subjectively read diagnostic tests as declared positive or negative depends on multiple factors including:scrutiny of the individual reading the test device,the clarity of the lighting (artificial versus natural) when viewing the test and, most importantly,the concentration of the substance to be detected (in this project, coronavirus antigen) on the test device.SUBJECTIVITY CAN BE COMPLETELY ELIMINATED by using a diagnostic system that uses a meter...an instrument with a precisely defined, invariant cutoff between positive and negative samples.This is inherent in the ApolloDx diagnostic system for all of its assays including this project's coronavirus antigen assay.Integrated contact tracing gateway.ApolloDx system's operation is super simple: the potentiostat is a slave to the operational instructions from the smart device.Once the test strip containing test sample is inserted into the potentiostat and the electrochemical interrogation of that test strip is completed, the results are automatically relayed to the smart device.Once resident on the smart device, these test results and all demographic information identifying the test sample are relayed to a remote server within local and/or national public health authorities to communicate with the individual who provided the sample to implement contact tracingin real time.Because all infectious diseases are combatted with the three-pronged approach of 'test-quarantine-contact trace', the ApolloDx system is instrumentally important in enabling two out of the three of these to combat this pervasive pandemic.In this Phase I project, ApolloDx will demonstrate the feasibility of using its diagnostic system to detect coronavirus antigen. ApolloDx will integrate a technique that one of its consultants used to detect another virus with the published claim that the technique was at least equal in sensitivity to PCR. This is very significant because PCR is widely accepted as the presumed state-of-the-art in terms of sensitivity and is the technology used in commercial labs to diagnose coronavirus infection since the beginning of the pandemic.The key difference in combining ApolloDx's test system with this special technique is fundamentally important for controlling the pandemic for the following reasons:Results are available in approximately 10 minutes,at the point of sample acquisition; no transportation of samples requiredApolloDx's diagnostic system is inexpensive and simple enough (like a glucose test system that tests for coronavirus) that it can be used by individuals in many settings with minimal trainingThe system provides a gateway to contact tracingApolloDx believes that for these reasons and the preliminary data predicting success of this approach that the Company's diagnostic system can and will meaningfully contribute to controlling the pandemic starting in facilities in great need for rapid answers: this country's meat packing facilities and beyond, in many rural settings.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Goals / Objectives
The goals of this project are both broad and technical in orientation. The board-minded objectives include:Dailyscreening until the pandemic is deemed under control nationally but particularly locally in rural communities...To couple monitoring temperatures of all employees before entering facility withFollow-up screening of those employees with elevated temperatures with a sensitive diagnostic test designed to detect a coronavirus analyte at early stage after exposure, NOT an antibody test.Additional statistical sampling of employees to capture asymptomatic infections.Link test results to contact tracing to immediately screen all other individuals potentially exposed to the individual screened positive for coronavirus.And with this above process,To most effectively minimize or eliminate the spread of the infection.To identify and monitor asymptomatics a mechanism through which they can be identified and isolated as necessary.To close the gap in meat packing facilities between operational shutdowns and business as normal.To recapture more of the economic health in rural communities experienced before the coronavirus pandemic spreads further.From a technical perspective, the goals of this project are to:Establish ApolloDx's platform's capability to detect the SARS-CoV-2 virus' antigenThis will require purchasing matched pairs of antibodies that have already been utilized for producing working ELISA (central laboratory) tests.Immobilize the primary (capture) antibody to graphene oxide deposited on a carbon electrochemical electrode - just as ApolloDx has initiated work on already with a model system for an unrelated virus.Either directly conjugate the secondary (reporter) antibody to an enzyme (horseradish peroxidase or lactate dehydrogenase) or use a generic anti-specie-enzyme conjugate. (In other words, if a mouse monoclonal demonstrates the best results (good limit of detection sensitivity) in this diagnostic mechanism, an anti-mouse antibody-enzyme conjugate will be used. Perform optimizations across a wide range for the critical variables (concentration of primary and second antibodies used, incubation times, pH, concentration of electrochemically (redox) species (e.g. methylene blue), peroxidase substrate concentration (e.g. hydrogen peroxide) etc.) to obtain an electrochemical signal sufficient to demonstrate assay detection feasibility. In their latest advisory, the FDA does not set a quantitative limit of detection (LOD) for coronavirus tests but that the quantitative performance of the test be documented as described in this recommendation. ApolloDx will follow this recommendation.Demonstrate feasibility of coronavirus detection by testing a serial dilution series of the recombinant spike protein target sample in a) first, buffer and b) second, resuspension in nasopharyngeal eluate from non-infected people.Establish specificity of developed test versus common recombinant respiratory viruses.Secure three non-infectious recombinant or biologically inactivated respiratory virus samples from among influenza viruses, respiratory syncytial virus (RSV), parainfluenza viruses, and respiratory adenoviruses.Test these samples at equimolar concentrations with the SARS-CoV-2 antigen in ApolloDx's assay and examine results for interference from these samples. As described above for sensitivity analysis, this specificity will be documented. Evaluate a range of nasopharyngeal swab eluates equivalent to 1x, 3x, 5x and 7x the normal concentration of eluate in 500µL of elution buffer. Look for interference in the electrochemical signal with increasing eluate concentration.?Endeavor to reduce test performance for <10' TAT (turnaround time). This is a 'nice to have' not a 'must have'.Write a downloadable smart device app to collect test subject information that will a) merge with sample and consumable test device barcode information, b) the coronavirus antigen test results and c) connect this de-identified information with iOS and Android operating systems to enable contact tracing.
Project Methods
The Gates Foundation has supported work to identify matched pairs of antibodies that do not interfere with one another in binding four sources of coronavirus antigen including clinical samples.Although this excellent study focuses on selecting the ideal antibodies for lateral flow immunoassays (LFA), some elements of the study serve this electrochemical approach precisely well.The quirk about LFAs is that the solvent front migrating through the nitrocellulose strip results in extreme pressure for the capture antibodies to bind target (antigen) extremely quickly.In technical parlance, the Kon rate must be very high whereas the Koff (desorption) rate is low (slow). While these factors are of paramount importance in LFA, in a 10-minute electrochemical assay such as is described in this proposal, idealized Kon and Koff rates are contributory to a fast-acting diagnostic mechanism, but not of the same paramount importance.Therefore, this recently published Gates report examining 673 antibody pairs for optimized immunoassay performance are a starting point in assembling the ApolloDx electrochemical assay for coronavirus antigen.The report provides clear and exhaustively accumulated evidence for the best four antibody pairs.ApolloDx will order these four paired antibodies to prepare for assembling the electrochemical prototype devices.With these antibody pairs acquired, ApolloDx's scientists will prepare coronavirus biosensors using the methods described in the paper published by the scientist chosen to prove feasibility for this electrochemical coronavirus assay.Method to prepare the coronavirus diagnostic consumable component1.To a clean glassy carbon electrode (GCE), a 5 μL of graphene oxide (GO) from a GO-ethanol stock solution (5 mL) was casted on a clean GCE (GCE/GO) and dried in air for 5 ± 1 mins at room temperature.GO surface functional group (-COOH and NH2) was activated by treating with 1:1 ratio of EDC-NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride-N-hydroxysuccinimide)(10 mg/mL) solution for 5 mins.2.Methylene blue (MB) dye was immobilized on GCE/GO by immersing the working electrode in 5mg MB dissolved 500 μL ethanol solution for 5 mins.Using a standard potentiostat, this electrode was pre-treated by performing continuous cyclic voltammograms (CV) in pH 7 PBS at scan rate (v) = 50 mV/sec for twenty cycles in pH 7 PBS.3.Primary (capture) antibody (Ab1) modified the GCE/GO@MB working electrode by drop-casting 1 μL of the capture antibody (1 mg/mL) and dried for 5 mins at room temperature.4.The electrode was treated with 1% (w/v) (1 mg/mL) bovine serum albumin (BSA) in 0.1 M PBS, pH 7 and dried at room temperature for 5 mins to block the non-specific binding sites and then rinsed with milli-Q water to remove unbound BSA.At this point, the electrochemical electrode derivatized to capture coronavirus antigen is prepared.This is the consumable in ApolloDx's test system.Method to test samples with the coronavirus diagnostic consumable component1.Incubation of the test sample (inactivated or recombinant coronavirus antigen) will be performed by incubating the above modified electrode with 1 μL of antigen and dried for 5 minutes in room temperature.2.The electrode exposed to sample was overlaid with 1 μL of enzyme (either HRP or lactate dehydrogenase) labelled secondary (detector) antibody (Ab2-ENZ) (1 mg/mL), dried for 5 mins in room temperature and washed with water. Then, GCE/GO@MB-Ab1-Ag-BSA-Ab2-ENZ was subjected to CV study with 500 μM H2O2dissolved pH7 PBS at v=10mV/sec.By this method, this immunosensor will be able sense the presence or absence of coronavirus antigen in a inactivated or recombinant coronavirus sample (Phase I) or lysed swab sample (Phase II) within 10 ± 1 min time.The above protocol is what ApolloDx choses as a starting point in proving feasibility of this electrochemical system to detect coronavirus antigen.There may be some cursory optimizations needed to tweak assay performance to illustrate the feasibility of this diagnostic system.These are described in the proposal in "Phase I Technical Objectives".The other 'methods' used in this Phase I project include employing an enzyme (either HRP or lactate dehydrogenase) antibody (Ab2) conjugate.This/these enzyme-Ab conjugate(s) will either be directly ordered as a product or prepared in ApolloDx's laboratories.If the latter, the biotinylated enzyme will be ordered from widely available commercial stocks along with the avidinylated antibodies (of the four capture antibodies, Ab2 noted as the best detector antibodies in the Gates coronavirus report) and conjugated relying upon the extraordinary avidin-biotin binding affinity (Kd=10-15M) using standard lab procedures.