Capillary flow-driven immunoassay platform for COVID-19 antigen diagnostics

Link, Jeremy S.; Carrell, Cody S.; Jang, Ilhoon; Barstis, Elijah J.O.; Call, Zachary D.; Bellows, Rae A.; O'Donnell-Sloan, John J.; Terry, James S.; Anderson, Loran B.R.; Panraksa, Yosita; Geiss, Brian J.; Dandy, David S.; Henry, Charles S.

doi:10.1016/j.aca.2023.341634

Cited by 3 publications

(10 citation statements)

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“…Conditions for the N-protein assay had been optimized in previous work. 42 To optimize the HA assay, the single-channel CaDI device described previously was used, and the locations of conjugate release pads and NC membrane are shown in Figure 2A. Manufacturing the single-channel devices in bulk is slightly easier, giving us a quick way to determine the optimal 2°Ab-HRP concentrations and TMB volumes to use.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…The multiplex device used in this work consists of two separate immunoassays. Conditions for the N-protein assay had been optimized in previous work . To optimize the HA assay, the single-channel CaDI device described previously was used, and the locations of conjugate release pads and NC membrane are shown in Figure A.…”

Section: Resultsmentioning

confidence: 99%

“…39,42 Therefore, the concentrations of 2°Ab-HRP and volume of the TMB substrate used in the N-protein assay were the same as from previous work. 42 The running buffer in which the sample was diluted was made using 1.5× stable peroxide buffer (Thermo Fisher Scientific, 34062) at pH 6.5 with 150 mM NaCl, 0.1% Igepal CA630, and 0.1% Tween-20. The influenza A H1N1 assay has never been performed on this type of capillary-driven immunoassay device (CaDI).…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…To optimize the conditions for the HA side of the multiplex device, the HA assay was performed on the single-channel CaDI device outlined in previous work. 42 First, TMB volume was held constant at 30 μL while the 2°Ab-HRP concentration was adjusted between 5 and 60 μg/mL. Devices were run in triplicate with running buffer containing either 0 or 50 ng/mL HA (11085-V08B, SinoBiological).…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…Initial optimization for the H1N1 assay was performed on a single-channel device that was developed in previous work. 42 Once conditions were chosen, dose−response curves were generated for H1N1 hemagglutinin and SARS-CoV-2 nucleocapsid protein (N-protein) on the multiplex device with detection limits of 840 and 133 pg/mL, respectively. The amount of SARS-CoV-2 N-protein found in blood and saliva from positive patients ranges from 1 to 1 × 10 5 pg/mL for the first 2 weeks after infection; our device is well within that range.…”

Section: ■ Introductionmentioning

confidence: 99%

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Multiplexed Capillary-Flow Driven Immunoassay for Respiratory Illnesses

Link,

O’Donnell-Sloan,

Curdts

et al. 2024

Anal. Chem.

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Multiplexed analysis in medical diagnostics is widely accepted as a more thorough and complete method compared to single-analyte detection. While analytical methods like polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) exist for multiplexed detection of biomarkers, they remain time-consuming and expensive. Lateral flow assays (LFAs) are an attractive option for point-of-care testing, and examples of multiplexed LFAs exist. However, these devices are limited by spatial resolution of test lines, large sample volume requirements, cross-reactivity, and poor sensitivity. Recent work has developed capillary-flow microfluidic ELISA platforms as a more sensitive alternative to LFAs; however, multiplexed detection on these types of devices has yet to be demonstrated. In the aftermath of the initial SARS-CoV-2 pandemic, the need for rapid, sensitive point-of-care devices has become ever clearer. Moving forward, devices that can distinguish between diseases with similar presenting symptoms would be the ideal home diagnostic. Here, the first example of a multiplexed capillary-flow immunoassay device for the simultaneous detection of multiple biomarkers is reported. From a single sample addition step, the reagents and washing steps required for two simultaneous ELISAs are delivered to spatially separated test strips. Visual results can be obtained in <15 min, and images captured with a smartphone can be analyzed for quantitative data. This device was used to distinguish between and quantify H1N1 hemagglutinin (HA) and SARS-CoV-2 nucleocapsid protein (N-protein). Using this device, analytical detection limits of 840 and 133 pg/mL were obtained for hemagglutinin and nucleocapsid protein, respectively. The presence of one target in the device did not increase the signal on the other test line, indicating no cross-reactivity between the assays. Additionally, simultaneous detection of both N-protein and HA was performed as well as simultaneous detection of Nprotein and human C-reactive protein (CRP). Elevated levels of CRP in a patient infected with SARS-CoV-2 have been shown to correlate with more severe outcomes and a greater risk of death as well. To further expand on the simultaneous detection of two biomarkers, CRP and N-protein were detected simultaneously, and the presence of SARS-CoV-2 N-protein did not interfere with the detection of CRP when both targets were present in the sample.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%