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Threshold-based quantification in a multi-line lateral flow assay via computationally designed capture efficiency David J Gasperino, Daniel Leon, Barry Lutz, David M Cate, Kevin P. Nichols, David Bell, and Bernhard Weigl Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00440 • Publication Date (Web): 23 Apr 2018 Downloaded from http://pubs.acs.org on April 23, 2018
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Analytical Chemistry
Threshold-based quantification in a multi-line lateral flow assay via computationally designed capture efficiency David J. Gasperino1+, Daniel Leon2+, Barry Lutz2, David M. Cate1, Kevin P. Nichols1, David Bell1, and Bernhard Weigl1, 2 1 Intellectual Ventures, Bellevue, WA, USA 2 University of Washington, Seattle, WA, USA + these authors contributed equally to this work
ABSTRACT: Lateral flow assays (LFAs) are widely-used for yes/no detection of analytes, but they are not well-suited for quantification. We show that the sensitivity of the test line in a lateral flow assay can be tuned to appear at a specific sample concentration by varying the density of capture molecules at the test line, and that when test lines tuned for different responses are combined into a single test strip, lines appear at specific thresholds of sample concentration. We also developed a model based on mass-action kinetics that accurately described test line signal and shape over a wide matrix of capture molecule and sample concentrations in single-line strips. The model was used to design a three line test strip with lines designed to appear at logarithmically-spaced sample concentrations, and experiments showed a remarkable match to predictions. The response of this "graded ladder bar" format is due to the effect of test line concentration on tuned capture efficiency at each tests line, not on sample depletion effects, and the effect is maintained whether a system is under kinetic or equilibrium control. These features enable design of non-linear responses (logarithmic here) and suggest robustness for different systems. Thus, the graded ladder bar format could be a useful tool for applications requiring quantification of sample concentrations over a wide dynamic range.
on-the-spot rather than waiting for a laboratory test4. LFAs have generally been limited to applications where a binary (e.g. “yes” or “no”) readout is sufficient and interpretation of hue or test line intensity is not necessary. However, many laboratory tests, including the ubiquitous enzyme immunoassays (EIA), allow quantification of analyte concentration.
Figure 1. Schematic describing the antibody detection mechanism within a threshold-based multi-line LFA. In this test format, test lines have increasing concentration of immobilized capture antigen in the flow direction, and each test line captures an increasing fraction of the total analyte based on the influence of capture antigen concentration on kinetics and equilibrium binding. This approach to visual thresholding extends the linear dynamic range of the assay beyond what is achievable in a single test line format. INTRODUCTION Lateral flow assays (LFAs) have attracted considerable attention for research and diagnostic point-of-need applications1–3. Rapid diagnostic tests (RDTs) including LFAs represent a multi-billion dollar industry due to mobility, ease-of-use, low cost, and rapid time-to-result that allows widespread use for analyzing samples
Quantitation in immunoassays is important for measuring disease indicators (e.g., pathogens, antibody responses, inflammatory indicators, blood biochemistry) or for monitoring the efficacy of therapeutic treatment, among other things 5–7. Detecting normal/abnormal disease biomarker levels in the sample can direct medical intervention and also serve as a surveillance tool8. For instance, blood bank screening in countries with malaria transmission could be performed with serology LFAs to quantify antibody levels in sera9,10. Recent infection is typically signified by elevated anti-malaria antibodies compared with an individual infected less recently, or never11. Potential donors are triaged if high antibody concentrations are detected because they may be a vector for transmission. In practice, however, anti-malaria antibody concentrations vary over four orders of magnitude from less than 0.1 to over 100 µg/mL12. Children are less resistant to malaria than adults and tend to generate weaker immune responses when infected, implying that the relevant concentration range in children is different from adults. With semi-quantitative ranges of ~1 log, typical single-threshold LFAs are not adequate to discern infected/not-infected in both children and adults. Another example is quantitation of C-reactive protein (CRP) levels, for which the clinically-relevant range spans several orders of magnitude (
