Accelerated Development of a COVID-19 Lateral Flow Test in an Academic Setting: Lessons Learned

ConspectusThe COVID-19 pandemic further demonstrated the need for usable, reliable, and cost-effective point-of-care diagnostics that can be broadly deployed, ideally for self-testing at home. Antigen tests using more-detectable reporter labels (usually at the cost of reader complexity) achieve better diagnostic sensitivity, supporting the value of higher-analytical-sensitivity reporter technologies in lateral flow.We developed a new approach to simple, inexpensive lateral flow assays (LFAs) of great sensitivity, based on the glow stick peroxyoxalate chemistry widely used in emergency settings and in children’s toys. At the peak of the COVID-19 pandemic, we had the opportunity to participate in the pandemic-driven NIH Rapid Acceleration of Diagnostics (RADx) initiative aiming to develop a deployable lateral flow diagnostic for SARS-CoV-2 nucleoprotein based on our novel glow stick-inspired light-emitting reporter technology. During this project, we screened more than 250 antibody pairs for analytical sensitivity and specificity directly in LFA format, using recombinant nucleoprotein and then gamma-irradiated virions spiked into negative nasal swab extracts. Membranes and other LFA materials and swabs and extraction reagent components also were screened and selected. Optimization of conjugate preparation and spraying as well as pretreatment/conditioning of the sample pad led to the final optimized LFA strip. Technology development also included optimization of excitation liquid enclosed in disposable droppers, design of a custom cartridge and smartphone-based reader, and app development, even a prototype reader usable with any mobile phone. Excellent preclinical performance was first demonstrated with contrived samples and then with leftover clinical samples. Moving beyond traditional academic focus areas, we were able to establish a quality management system (QMS), produce large numbers of customized LFA cassettes by contract injection molding, build in-house facilities to assemble and store thousands of complete tests for verification and validation and usability studies, and source kitting/packaging services and quality standard reagents and build partnerships for clinical translation, regulatory guidance, scale up, and market deployment. We were not able to bring this early stage technology to the point of commercialization within the limited time and resources available, but we did achieve strong proof-of-concept and advance translational aspects of the platform including initial high-performance LFAs, reading by the iPhone app using only a $2 plastic dark box with no lens, and convenient, usable excitation liquid packaging in droppers manufacturable in very large numbers.In this Account, we aim to provide a concise overview of our 18-month sprint toward the practical development of a deployable antigen lateral flow assay under pandemic conditions and the challenges and successes experienced by our team. We highlight what it takes to coach a technically savvy but commercially inexperienced academic team through the accelerated translation of an early stage technology into a useful product. Finally, we provide a guided tutorial and workflow to empower others interested in the rapid development of translatable LFAs

Increasing Binding Efficiency via Reporter Shape and Flux in a Viral Nanoparticle Lateral-Flow Assay

To identify factors controlling the performance of reporter particles in a sensitive lateral-flow assay (LFA), we investigated the effect of the flux and shape of filamentous bacteriophage (phage) on the performance of phage LFAs. Phage of three different lengths and diameters were modified with biotin and AlexaFluor 555 as binding and read-out elements, respectively. The binding efficiencies of the functionalized phage were tested in a fibrous glass LFA membrane modified with avidin. The total binding rate, quantified using real-time particle counting and particle image velocimetry, decreased monotonically with the average bulk flux of phage through the membrane. At the pore scale, more phage bound in regions with faster local flow, confirming that both average and local flux increased binding. The number of bound phage increased with the aspect ratio of the phage and scaled with the phage surface area, consistent with a binding interaction controlled by the number of recognition elements on the surface. Together, these results indicate that increasing the likelihood that recognition elements on the surface of phage encounter the fibers enhances the assay binding efficiency and suggests one origin for the improved performance of nonspherical phage reporters

Fig 3 -

(A) Visual detection of the dilution series of purified RPA products run on commercial gold nanoparticle-based LFA strips. (B) Smartphone images of the dilution series of purified RPA products run on in-house-made LFA strips with SBMSO nanophosphor reporters. (C) Normalized TL/CL intensity ratio of SBMSO reporters against the concentration of purified DNA amplicons. Three trials were run for each concentration, then the average was calculated. The red line signifies the detection limit cutoff, taken as the mean plus three times the standard deviation (μ+3σ) of the no-analyte control LFAs.</p

Additional file 1: of Sero-epidemiological status and risk factors of toxoplasmosis in pregnant women in Northern Vietnam

Questionnaire-English. (PDF 136 kb

dsDNA standard curve obtained from the QuantiFluor dsDNA system.

The inset shows the fluorescence obtained with 4 μL of 5X diluted purified and unpurified RPA products (in red) and their respective dsDNA concentrations. According to the standard curve, the dsDNA amount of purified and unpurified samples is 16.23 and 9.37 ng/well, respectively. Therefore, the dsDNA concentration of the undiluted purified and unpurified amplicons is 20.3 and 11.7 ng/μL, respectively. (TIF)</p

A 3-D printed phone accessory with minimal optical hardware, containing a lens and a bundle of inexpensive plastic optical fibers but no electronic components, was used as a dark imaging compartment which was designed to hold a universal LFA cartridge (MICA-125; DCN Diagnostics) such that the result window of the cartridge is aligned with the rear camera of the iPhone 5S and occupies most of the field of view when the cartridge is fully inserted into the attachment.

A proprietary software application, “Luminostics”, controls the flash and the rear camera of the iPhone. The flash excites the nanophosphors for ~3 s, and, after switching off the flash, the camera captures the images after a ~100 ms time delay. The camera captures four images and generates the average result. We have described the iPhone reader in more detail in our previous publications [17,24]. (TIF)</p

Fig 4 -

(A) Visual detection of the dilution series of unpurified RPA products run on commercial gold nanoparticle-based LFA strips. (B) Smartphone images of the dilution series of unpurified RPA products run on in-house-made LFA strips with SBMSO nanophosphor reporters. (C) Normalized TL/CL intensity ratio of SBMSO reporters against the concentration of unpurified DNA amplicons. Three trials were run for each concentration, then the average was calculated. The red line signifies the detection limit cutoff, taken as the mean plus three times the standard deviation (μ+3σ) of the no-analyte control LFAs.</p

Additional file 2: of Sero-epidemiological status and risk factors of toxoplasmosis in pregnant women in Northern Vietnam

Questionnaire-Vietnamese. (PDF 587 kb

Fig 5 -

(A) Visual detection of RPA-amplified Leishmania parasite DNA dilution series (unpurified RPA products), run on commercial gold nanoparticle-based LFA strips. (B) Smartphone images of the RPA-amplified Leishmania parasite DNA dilution series (unpurified RPA products), run on in-house-made LFA strips with SBMSO nanophosphor reporters. (C) Normalized TL/CL intensity ratio of SBMSO reporters against the number of parasites added per RPA reaction. Three trials were run for each concentration, and the average was calculated. The red line signifies the detection limit cutoff, taken as the mean plus three times the standard deviation (μ+3σ) of the no-analyte control LFAs.</p