Lateral flow assays (LFAs) are rapid, inexpensive, easy-to-manufacture and -use tests widely employed in medical and environmental applications, particularly in low resource settings. Historically, ...LFAs have been stigmatized as having limited sensitivity. However, as their global usage expands, extensive research has demonstrated that it is possible to substantially improve LFA sensitivity without sacrificing their advantages. In this critical review, we have compiled state-of-the-art approaches to LFA sensitivity enhancement. Moreover, we have organized and evaluated these approaches from a system-level perspective, as we have observed that the advantages and disadvantages of each approach have arisen from the integrated and tightly interconnected chemical, physical, and optical properties of LFAs.
The performance, field utility, and low cost of lateral flow assays (LFAs) have driven a tremendous shift in global health care practices by enabling diagnostic testing in previously unserved ...settings. This success has motivated the continued improvement of LFAs through increasingly sophisticated materials and reagents. However, our mechanistic understanding of the underlying processes that drive the informed design of these systems has not received commensurate attention. Here, we review the principles underpinning LFAs and the historical evolution of theory to predict their performance. As this theory is integrated into computational models and becomes testable, the criteria for quantifying performance and validating predictive power are critical. The integration of computational design with LFA development offers a promising and coherent framework to choose from an increasing number of novel materials, techniques, and reagents to deliver the low-cost, high-fidelity assays of the future.
Inexpensive, simple, rapid diagnostics are necessary for efficient detection, treatment, and mitigation of COVID-19. Assays for SARS-CoV2 using reverse transcription polymerase chain reaction ...(RT-PCR) offer good sensitivity and excellent specificity, but are expensive, slowed by transport to centralized testing laboratories, and often unavailable. Antigen-based assays are inexpensive and can be rapidly mass-produced and deployed at point-of-care, with lateral flow assays (LFAs) being the most common format. While various manufacturers have produced commercially available SARS-Cov2 antigen LFAs, access to validated tests remains difficult or cost prohibitive in low-and middle-income countries. Herein, we present a visually read open-access LFA (OA-LFA) using commercially-available antibodies and materials for the detection of SARS-CoV-2. The LFA yielded a Limit of Detection (LOD) of 4 TCID.sub.50 /swab of gamma irradiated SARS-CoV-2 virus, meeting the acceptable analytical sensitivity outlined by in World Health Organization target product profile. The open-source architecture presented in this manuscript provides a template for manufacturers around the globe to rapidly design a SARS-CoV2 antigen test.
The developing world does not have access to many of the best medical diagnostic technologies; they were designed for air-conditioned laboratories, refrigerated storage of chemicals, a constant ...supply of calibrators and reagents, stable electrical power, highly trained personnel and rapid transportation of samples. Microfluidic systems allow miniaturization and integration of complex functions, which could move sophisticated diagnostic tools out of the developed-world laboratory. These systems must be inexpensive, but also accurate, reliable, rugged and well suited to the medical and social contexts of the developing world.
Immunoassays are important for the detection of proteins to enable disease identification and monitor treatment, but many immunoassays suffer from sensitivity limitations. The development of digital ...assays has enabled highly sensitive biomarker detection and quantification, but the necessary devices typically require precisely controlled volumes to reduce biases in concentration estimates from compartment size variation. These constraints have led to systems that are often expensive, cumbersome, and challenging to operate, confining many digital assays to centralized laboratories. To overcome these limitations, we have developed a simplified digital immunoassay performed in polydisperse droplets that are prepared without any specialized equipment. This polydisperse digital droplet immunoassay (ddIA) uses proximity ligation to remove the need for wash steps and simplifies the system to a single reagent addition step. Using interleukin-8 (IL-8) as an example analyte, we demonstrated the concept with samples in buffer and diluted whole blood with limits of detection of 0.793 pM and 1.54 pM, respectively. The development of a one-pot, washless assay greatly improves usability compared to traditional immunoassays or digital-based systems that rely heavily on wash steps and can be run with common and readily available laboratory equipment such as a heater and simple fluorescent microscope. We also developed a stochastic model with physically meaningful parameters that can be utilized to optimize the assay and enable quantification without standard curves, after initial characterization of the parameters. Our polydisperse ddIA assay serves as an example of sensitive, lower-cost, and simpler immunoassays suitable for both laboratory and point-of-care applications.
Immunochromatographic or lateral flow assays (LFAs) are inexpensive, easy to use, point-of-care medical diagnostic tests that are found in arenas ranging from a doctor's office in Manhattan to a ...rural medical clinic in low resource settings. The simplicity in the LFA itself belies the complex task of optimization required to make the test sensitive, rapid and easy to use. Currently, the manufacturers develop LFAs by empirical optimization of material components (e.g., analytical membranes, conjugate pads and sample pads), biological reagents (e.g., antibodies, blocking reagents and buffers) and the design of delivery geometry. In this paper, we will review conventional optimization and then focus on the latter and outline analytical tools, such as dynamic light scattering and optical biosensors, as well as methods, such as microfluidic flow design and mechanistic models. We are applying these tools to find non-obvious optima of lateral flow assays for improved sensitivity, specificity and manufacturing robustness.
The rapid onset of the global COVID-19 pandemic has led to challenges for accurately diagnosing the disease, including supply shortages for sample collection, preservation, and purification. ...Currently, most diagnostic tests require RNA extraction and detection by RT-PCR; however, extraction is expensive and time-consuming and requires technical expertise. With these challenges in mind, we report extraction-free, multiplexed amplification of SARS-CoV-2 RNA from 246 clinical samples, resulting in 86% sensitivity and 100% specificity. The multiplex RT-PCR uses the CDC singleplex targets and has an LoD of 2 c/μL. We also report on amplification using a range of master mixes in different transport media. This work can help guide which combinations of reagents will enable accurate results when availability of supplies changes throughout the pandemic. Implementing these methods can reduce complexity and cost, minimize reagent usage, expedite time to results, and increase testing capacity.
Introduction: Biomarkers are objective indications of a medical state that can be measured accurately and reproducibly. Traditional biomarkers enable diagnosis of disease through detection of ...disease-specific molecules, disease-mediated molecular changes, or distinct physiological or anatomical signatures.
Areas covered: This work provides a framework for selecting biomarkers that are most likely to provide useful information about a patient's disease state. Though the authors emphasize markers related to disease, this work is also applicable to biomarkers for monitoring physiological changes such as ovulation or pregnancy. Additionally, the scope was restricted to biomarkers that are amenable to analytical detection across a range of health care levels, including low resource settings. The authors describe trade-offs between biomarkers' sensitivity/specificity for a disease-causing agent, the complexity of detection, and how this knowledge can be applied to the development of diagnostic tests. This report also details additional assessment criteria for successful tests.
Expert commentary: Biomarker selection should primarily be driven by an attempt to answer an explicit clinical question (preferably causative relationship of the biomarker to disease-state), and only then by test development expediency (ease of detection). This framework is useful for stakeholders from test developers to clinicians to identify the trade-offs for diagnostic biomarkers for any use case.
Detection of tuberculosis at the point-of-care (POC) is limited by the low sensitivity of current commercially available tests. We describe a diagnostic accuracy field evaluation of a prototype urine ...Tuberculosis Lipoarabinomannan Lateral Flow Assay (TB-LAM LFA) in both HIV-positive and HIV-negative patients using fresh samples with sensitivity and specificity as the measures of accuracy. This prototype combines a proprietary concentration system with a sensitive LFA. In a prospective study of 292 patients with suspected pulmonary tuberculosis in Uganda, the clinical sensitivity and specificity was compared against a microbiological reference standard including sputum Xpert MTB/RIF Ultra and solid and liquid culture. TB-LAM LFA had an overall sensitivity of 60% (95%CI 51–69%) and specificity of 80% (95%CI 73–85%). When comparing HIV-positive (N = 86) and HIV-negative (N = 206) patients, there was no significant difference in sensitivity (sensitivity difference 8%, 95%CI -11% to +24%, p = 0.4351) or specificity (specificity difference -9%, 95%CI -24% to +4%, p = 0.2051). Compared to the commercially available Alere Determine TB-LAM Ag test, the TB-LAM LFA prototype had improved sensitivity in both HIV-negative (difference 49%, 95%CI 37% to 59%, p<0.0001) and HIV-positive patients with CD4+ T-cell counts >200cells/μL (difference 59%, 95%CI 32% to 75%, p = 0.0009). This report is the first to show improved performance of a urine TB LAM test for HIV-negative patients in a high TB burden setting. We also offer potential assay refinement solutions that may further improve sensitivity and specificity.
Rapid tests for SARS-COV-2 infection are important tools for pandemic control, but current rapid tests are based on proprietary designs and reagents. We report clinical validation results of an ...open-access lateral flow assay (OA-LFA) design using commercially available materials and reagents, along with RT-qPCR and commercially available comparators (BinaxNOW® and Sofia®). Adult patients with suspected COVID-19 based on clinical signs and symptoms, and with symptoms ≤7 days duration, underwent anterior nares (AN) sampling for the OA-LFA, Sofia®, BinaxNOW ™, and RT-qPCR, along with nasopharyngeal (NP) RT-qPCR. Results indicate a positive predictive agreement with NP sampling as 69% (60% -78%) OA-LFA, 74% (64% - 82%) Sofia®, and 82% (73% - 88%) BinaxNOW™. The implication for these results is that we provide an open-access LFA design that meets the minimum WHO target product profile for a rapid test, that virtually any diagnostic manufacturer could produce.
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