Development Of Lateral Flow Assay Research Articles

Development of a lateral flow immunoassay with HRP enhancement for spiked SARS-CoV-2 protein N detection in human saliva

The Coronavirus (SARS-CoV-2) is an enveloped RNA virus that caused a dangerous COVID-19 pandemic following an outbreak in Wuhan, China in 2019. In response to the pandemic, the development of lateral flow assays (LFAs) has been crucial for the detection of viruses, commonly targeting the spike (S) or nucleocapsid (N) protein in nasopharyngeal swab (NS) specimens. COVID-19, caused by the SARS-CoV-2 virus, predominantly manifests as a respiratory tract infection-like illness, characterized by symptoms such as fever, dry cough, upper airway congestion, runny nose, sore throat, myalgia, headache, and exhaustion. This study presents the development of an LFA targeting the N protein to detect the coronavirus in saliva specimens, using a sandwich format. The use of 80 nm gold nanoparticles (AuNPs) has been investigated, and the application of horseradish peroxidase (HRP) and TMB substrate has been employed to enhance the limit of detection (LOD). The results demonstrate a significant 200-fold improvement in LOD, from 10 to 50 pg/ml, for N protein spiked in saliva samples after the application of HRP-TMB. This finding highlights an important advancement towards the utilization of saliva samples in diagnostic applications.

A preliminary study to develop a lateral flow assay using recombinant GRA1 protein for the diagnosis of toxoplasmosis in stray cats

Toxoplasma gondii is a protozoan parasite that may infect many mammals including humans. Cats are one of the main sources of infection for humans. Therefore, routine screening of cats with tests that are inexpensive, rapid, and do not require sophisticated laboratory equipment is important. In this study, a lateral flow assay (LFA) was designed to rapidly diagnose toxoplasmosis in cats. For this purpose, we selected GRA1 protein of T. gondii due to its high antigenicity in diagnostic and vaccine studies. We further analyzed the immunological properties of GRA1 protein using in silico tools. Then, we expressed and purified recombinant GRA1 (rGRA1) protein and used it during the development of LFA to detect toxoplasmosis in serum samples (n = 40) of cats. According to the results, rGRA1 protein has negative GRAVY value, high aliphatic index, alpha helix, random coil and 12 B cell epitopes. The in silico data supported the high antigenic properties of rGRA1 protein and showed that it can be a good antigen candidate for LFA. Among 30 cat positive serum samples, 27 were found positive by the LFA while seronegative sera (n = 10) were negative by the LFA. The preliminary data showed that the LFA has high sensitivity (90 %) and specificity (100 %). When we used high responsive cat sera (i.e. sera that have optical density > 0.5 with ELISA) the sensitivity value reached 100 %. These results showed that rGRA1 protein is a good candidate to develop a LFA for rapid diagnosis of toxoplasmosis in cats.

Development of lateral flow assays to detect host proteins in cattle for improved diagnosis of bovine tuberculosis.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis) infection in cattle, is an economically devastating chronic disease for livestock worldwide. Efficient disease control measures rely on early and accurate diagnosis using the tuberculin skin test (TST) and interferon-gamma release assays (IGRAs), followed by culling of positive animals. Compromised performance of TST and IGRA, due to BCG vaccination or co-infections with non-tuberculous mycobacteria (NTM), urges improved diagnostics. Lateral flow assays (LFAs) utilizing luminescent upconverting reporter particles (UCP) for quantitative measurement of host biomarkers present an accurate but less equipment- and labor-demanding diagnostic test platform. UCP-LFAs have proven applications for human infectious diseases. Here, we report the development of UCP-LFAs for the detection of six bovine proteins (IFN-γ, IL-2, IL-6, CCL4, CXCL9, and CXCL10), which have been described by ELISA as potential biomarkers to discriminate M. bovis infected from naïve and BCG-vaccinated cattle. We show that, in line with the ELISA data, the combined PPDb-induced levels of IFN-γ, IL-2, IL-6, CCL4, and CXCL9 determined by UCP-LFAs can discriminate M. bovis challenged animals from naïve (AUC range: 0.87-1.00) and BCG-vaccinated animals (AUC range: 0.97-1.00) in this cohort. These initial findings can be used to develop a robust and user-friendly multi-biomarker test (MBT) for bTB diagnosis.

The Current and Future States of Diagnostic Tests for Histoplasmosis with a Focus on People with HIV and Disseminated Histoplasmosis.

Histoplasmosis is caused by Histoplasma capsulatum and, although endemic in large parts of the world, is often underrecognized in many locations. In addition to underrecognition, inadequate availability of diagnostic tests is a major contributor to poor outcomes in disseminated disease in people with HIV. For those with advanced HIV and disseminated disease, antibody testing is less useful. Culture and histopathology can be useful in this situation, but each has limitations, including variable sensitivity by site and, in the case of culture, the need for a biosafety level three laboratory and a long period of growth. Antigen testing has proven useful for disseminated histoplasmosis due to the excellent sensitivity of urine. Yet, turnaround is slower than ideal due to use in a limited number of centers. The development of lateral flow assays has the potential to make for true rapid point-of-care assays for histoplasmosis, but in order to meet that promise, the tests must be widely available and affordable.

Aptamer-based colorimetric and lateral flow assay approaches for the detection of toxic metal ions, thallium(i) and lead(ii).

Thallium(i) and lead(ii) ions are heavy metals and extremely toxic. These metals are environmental pollutants, posing a severe risk to the environment and human health. In this study, two approaches were examined using aptamer and nanomaterial-based conjugates for thallium and lead detection. The first approach utilized an in-solution adsorption-desorption approach to develop colorimetric aptasensors for the detection of thallium(i) and lead(ii) using gold or silver nanoparticles. The second approach was the development of lateral flow assays, and their performance was tested with thallium (limit of detection is 7.4 μM) and lead ion (limit of detection is 6.6 nM) spiked into real samples. The approaches assessed are rapid, inexpensive, and time efficient with the potential to become the basis for future biosensor devices.

A Novel Ratiometric Fluorescent Approach for the Modulation of the Dynamic Range of Lateral Flow Immunoassays

AbstractThe majority of lateral flow assays (LFAs) use single‐color optical labels to provide a qualitative naked‐eye detection, however this detection method displays two important limitations. First, the use of a single‐color label makes the LFA prone to results misinterpretation. Second, it does not allow the precise modulation of the sensitivity and dynamic range of the test. To overcome these limitations, a ratiometric approach is developed. In particular, using anti‐HIgG functionalized red‐fluorescent quantum dots on the conjugate pad (as target dependent labels) and blue‐fluorescent nanoparticles fixed on the test line (as target independent reporters), it is possible to generate a wide color palette (blue, purple, pink, red) on the test line. It is believed that this strategy will facilitate the development of LFAs by easily adjusting their analytical properties to the needs required by the specific application.

Development of Lateral Flow Assay for the Detection of Tamm Horsfall Protein in Urine

Tamm Horsfall Protein (THP) is the most abundant urinary protein with concentrations of 20-200 mg/day and is known to be involved in a myriad of renal pathological processes such as renal stones and inflammation. THP is produced by renal epithelial cells and is the most abundant urinary protein, which tends to aggregate at higher concentrations. Therefore, our study was aimed to facilitate the detection of THP in urine by a rapid, efficient, and optimum detection using a lateral flow assay system. To develop the assay system, THP was isolated and purified from the urine of a healthy individual and used to generate antibodies in a white male New Zealand rabbit. These anti-THP antibodies were purified from the rabbit serum using the Protein-A column and further conjugated with gold nanoparticles. Gold nanoparticles (GNPs) of the size of 21 nm were synthesized by the chemical reduction method. Conjugation of gold nanoparticles and anti-THP antibodies was optimized by varying pH and concentrations. The developed lateral flow assay was tested with purified THP, and its sensitivity was calculated as 1 mg/ml of THP. This lateral flow immunoassay can detect THP in urine and can be beneficial for the detection of a various pathological or physiological phenomenon involving urinary Tamm Horsefall Protein secretions.

Screening Antibodies Raised against the Spike Glycoprotein of SARS-CoV-2 to Support the Development of Rapid Antigen Assays.

Severe acute respiratory coronavirus-2 (SARS-CoV-2) is a novel viral pathogen and therefore a challenge to accurately diagnose infection. Asymptomatic cases are common and so it is difficult to accurately identify infected cases to support surveillance and case detection. Diagnostic test developers are working to meet the global demand for accurate and rapid diagnostic tests to support disease management. However, the focus of many of these has been on molecular diagnostic tests, and more recently serologic tests, for use in primarily high-income countries. Low- and middle-income countries typically have very limited access to molecular diagnostic testing due to fewer resources. Serologic testing is an inappropriate surrogate as the early stages of infection are not detected and misdiagnosis will promote continued transmission. Detection of infection via direct antigen testing may allow for earlier diagnosis provided such a method is sensitive. Leading SARS-CoV-2 biomarkers include spike protein, nucleocapsid protein, envelope protein, and membrane protein. This research focuses on antibodies to SARS-CoV-2 spike protein due to the number of monoclonal antibodies that have been developed for therapeutic research but also have potential diagnostic value. In this study, we assessed the performance of antibodies to the spike glycoprotein, acquired from both commercial and private groups in multiplexed liquid immunoassays, with concurrent testing via a half-strip lateral flow assays (LFA) to indicate antibodies with potential in LFA development. These processes allow for the selection of pairs of high-affinity antispike antibodies that are suitable for liquid immunoassays and LFA, some of which with sensitivity into the low picogram range with the liquid immunoassay formats with no cross-reactivity to other coronavirus S antigens. Discrepancies in optimal ranking were observed with the top pairs used in the liquid and LFA formats. These findings can support the development of SARS-CoV-2 LFAs and diagnostic tools.

Production of highly sensitive monoclonal antibody and development of lateral flow assays for phallotoxin detection in urine.

Phallotoxins, toxic cyclopeptides found in wild poisonous mushrooms, are predominant causes of fatal food poisoning. For the early and rapid diagnosis mushroom toxin poisoning, a highly sensitive and robust monoclonal antibody (mAb) against phallotoxins was produced for the first time. The half-maximum inhibition concentration (IC50) values of the mAb-based indirect competitive ELISAs for phallacidin (PCD) and phalloidin (PHD) detection were 0.31ngmL-1 and 0.35ngmL-1, respectively. In response to the demand for rapid screening of the type of poisoning and accurate determination of the severity of poisoning, colloidal gold nanoparticle (GNP) and time-resolved fluorescent nanosphere (TRFN) based lateral flow assays (LFA) were developed. The GNP-LFA has a visual cut-off value of 3.0ngmL-1 for phallotoxins in human urine sample. The TRFN-LFA provides a quantitative readout signal with detection limit of 0.1ngmL-1 in human urine sample. In this study, urine samples without pretreatment were used directly for the LFA strip tests, and both two LFAs were able to accomplish analysis within 10min. The results demonstrated that LFAs based on the newly produced, highly sensitive, and robust mAb were able to be used for both rapid qualitative screening of the type of poisoning and accurate quantitative determination of the severity of poisoning after accidental ingestion by patients of toxic mushrooms.

Update on Non-Culture-Based Diagnostics for Invasive Fungal Disease.

Diagnostic tests for fungi provide the mycological evidence to strengthen diagnosis of invasive fungal disease. Conventional microbiology and histopathology have their limitations. Recognizing this, there have been attempts at developing new methods to improve yield of diagnosing invasive fungal disease (IFD). The recent focus has been on non-culture-based antigen detection and molecular methods. The use of antigen detection of IFD through 1,3-β-D-glucan and galactomannan assay have been expanded, followed by development of lateral flow assays, and in combination with other diagnostic modalities to further increase diagnostic yield. The molecular diagnostic front has seen initiatives to standardize polymerase chain reaction methodologies to detect fungi and anti-fungal resistance, new platforms such as the T2Candida Biosystems and foray into fungal metagenomics. As these newer assays undergo stringent validation before incorporation into the diagnostic algorithm, the clinician needs to be mindful of their bedside utility as well as their limitation.

Low-cost, open-source 3D printed antibody dispenser for development and small-scale production of lateral flow assay strips

We present a low cost, 3D printed open-source antibody dispenser that can be easily built and used for the development of lateral flow assay (LFA) strips. The fabrication of LFA strips need dispensing of antibodies or antigens in a linear fashion and commercially available dispensers typically cost from few thousands to few tens of thousands of US dollars. In this paper, an antibody dispenser was built by using 3D printed and commercially available parts, which cost no more than 30 USD. This paper presents a detailed instruction on how to assemble the printer and how to achieve a specific line width for the dispensed antibody. By using syringe needles of different gauges, line width ranging from 0.23 to 1.8 mm can be dispensed, and by varying the speed controller, the dispensing needle’s speed can be varied between 2.8 and 3.8 cm/s. We demonstrate uniform dispensing of anti-C-reactive protein (CRP) antibody and anti-rabbit antibody to draw a test line and a control line, which are used for the detection of CRP. The dispenser can also be equipped with two dispensing needles to allow simultaneous dispensing of multiple reagents, which can be useful for LFA strip development.

Development of lateral flow assay based on anti-IBDV IgY for the rapid detection of Gumboro disease in poultry

The poultry industry faces serious problems against infectious diseases, including Gumboro, which is caused by contagious bursal disease virus (IBDV). IBDV infects the bursa of Fabricius (BF), a lymphoid organ for controlling the B-cell maturation. Thus, it can trigger the secondary infection's vulnerability, leading to the high mortality and morbidity of the chicken. Moreover, managing the Gumboro post-outbreaks also requires considerable time and costs. Besides vaccination programs, the early detection of IBDV is vital as an outbreak control strategy. The most popular diagnostic tool is a lateral flow immunoassay or a rapid test that meets ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, and Deliverable to end-users) criteria. In this study, the lateral flow immunoassay was successfully developed based on anti-IBDV IgY as the bio receptor. Anti-IBDV IgY was successfully isolated from Isa Brown's egg yolk. The detection system showed an acceptable affinity against the inactivated IBDV sample (1.5 × 103 TCID50). In addition, it did not react with avian influenza and Newcastle disease viruses, demonstrating a good specificity of the test.

Tutorial: design and fabrication of nanoparticle-based lateral-flow immunoassays

Lateral-flow assays (LFAs) are quick, simple and cheap assays to analyze various samples at the point of care or in the field, making them one of the most widespread biosensors currently available. They have been successfully employed for the detection of a myriad of different targets (ranging from atoms up to whole cells) in all type of samples (including water, blood, foodstuff and environmental samples). Their operation relies on the capillary flow of the sample throughout a series of sequential pads, each with different functionalities aiming to generate a signal to indicate the absence/presence (and, in some cases, the concentration) of the analyte of interest. To have a user-friendly operation, their development requires the optimization of multiple, interconnected parameters that may overwhelm new developers. In this tutorial, we provide the readers with: (i) the basic knowledge to understand the principles governing an LFA and to take informed decisions during lateral flow strip design and fabrication, (ii) a roadmap for optimal LFA development independent of the specific application, (iii) a step-by-step example procedure for the assembly and operation of an LF strip for the detection of human IgG and (iv) an extensive troubleshooting section addressing the most frequent issues in designing, assembling and using LFAs. By changing only the receptors, the provided example procedure can easily be adapted for cost-efficient detection of a broad variety of targets.

Reliable performance of aggregation-induced emission nanoparticle-based lateral flow assay for norfloxacin detection in nine types of animal-derived food

The wide application of lateral flow assay (LFA) was limited by its low sensitivity and poor matric tolerance. Aggregation induced emission (AIE) materials show superior luminescence and good stability under close packing state, which may accelerate the development of LFA. However, the detection performance of the AIE-based LFA in different real samples was unclear. In this work, an AIE-LFA was established for norfloxacin (NOR) detection in nine types of animal-derived food. Results indicated that AIE-LFA had the average recovery range of 75.6%–95.1%, 78.6%–94.6%, 71.4%–112.7%, 81.7%–121.8%, 72.7%–93.5%, 79.8%–108.5%, 79.2%–109.4%, 76.3%–103.6%, and 80.6%–108.3% in pork, pig liver, fish, lamb, beef, milk, chicken, egg, and honey, respectively. The detection results of AIE-LFA were compatible with HPLC-MS/MS in detecting NOR in 135 real samples from nine types of animal-derived food. The developed AIE−LFA was sensitive and reliable for NOR detection in those real samples.

SARS-CoV-2 Coronavirus Nucleocapsid Antigen-Detecting Half-Strip Lateral Flow Assay Toward the Development of Point of Care Tests Using Commercially Available Reagents.

The SARS-CoV-2 pandemic has created an unprecedented need for rapid diagnostic testing to enable the efficient treatment and mitigation of COVID-19. The primary diagnostic tool currently employed is reverse transcription polymerase chain reaction (RT-PCR), which can have good sensitivity and excellent specificity. Unfortunately, implementation costs and logistical problems with reagents during the global SARS-CoV-2 pandemic have hindered its universal on demand adoption. Lateral flow assays (LFAs) represent a class of diagnostic that, if sufficiently clinically sensitive, may fill many of the gaps in the current RT-PCR testing regime, especially in low- and middle-income countries (LMICs). To date, many serology LFAs have been developed, though none meet the performance requirements necessary for diagnostic use cases, primarily due to the relatively long delay between infection and seroconversion. However, on the basis of previously reported results from SARS-CoV-1, antigen-based SARS-CoV-2 assays may have significantly better clinical sensitivity than serology assays. To date, only a very small number of antigen-detecting LFAs have been developed. Development of a half-strip LFA is a useful first step in the development of any LFA format. In this work, we present a half-strip LFA using commercially available antibodies for the detection of SARS-CoV-2. We have tested this LFA in buffer and measured an LOD of 0.65 ng/mL (95% CI of 0.53 to 0.77 ng/mL) ng/mL with recombinant antigen using an optical reader with sensitivity equivalent to a visual read. Further development, including evaluating the appropriate sample matrix, will be required for this assay approach to be made useful in a point of care setting, though this half-strip LFA may serve as a useful starting point for others developing similar tests.

Development of lateral flow assay combined with recombinase polymerase amplification for highly sensitive detection of Dickeya solani

Dickeya solani, one of the most significant bacterial pathogens, infects potato plants, resulting in severe economic damage. In this study, a lateral flow assay (LFA) combined with isothermal DNA amplification was developed for rapid, specific, and sensitive diagnosis of the potato blackleg disease caused by D. solani. Recombinase polymerase amplification (RPA) was chosen for this purpose. Five primer pairs specific to different regions of the D. solani genome were designed and screened. A primer pair providing correct recognition of the target sequence was aligned with the SOL-C region specific to D. solani and flanked by fluorescein (forward primer) and biotin (reverse primer). Lateral flow test strips were constructed to detect DNA amplicons. The RPA-LFA demonstrated a detection limit equal to 14,000 D. solani colony-forming units per gram of potato tuber. This assay provided sensitivity corresponding to the polymerase chain reaction (PCR) but was implemented at a fixed temperature (39 °C) over 30 min. No unspecific reactions with Pectobacterium, Clavibacter, and other Dickeya species were observed. Detection of latent infection of D. solani in the potato tubers by the developed RPA-LFA was verified by PCR. The obtained results confirmed that RPA-LFA has great potential for highly sensitive detection of latent infection.

Strategies for developing sensitive and specific nanoparticle-based lateral flow assays as point-of-care diagnostic device

• The multi-parameteric process of developing lateral flow assays (LFAs) is elucidated. • Novel capture/detector agent generation for improved target detection is presented. • Multiplexed detection is introduced, enabling increased throughput and efficiency. • Signal amplification methods bridge the performance of LFAs with lab techniques. • Prospects for more quantitative LFAs and broader applications are discussed. From a home-based pregnancy self-testing kit, lateral flow assays (LFAs) have proliferated and gained widespread utilization as point-of-care (POC) test kits. Their prevalence is due to their portability, rapid time-to-result, simplicity, stability, and cost-effectiveness. LFAs are well-suited for early detection of infectious diseases, which threatens public health and the economy, enabling timely medical intervention, and management of disease and treatment. This is vital for rural and resource-limited settings where well-equipped laboratories with well-trained personnel are scarce. Nevertheless, LFAs have certain limitations, such as moderate sensitivity and target throughput, as compared to lab-based technologies. Also, the development of LFA, although well-defined, is not straightforward. In this review article, we will elucidate the iterative process of LFA development, and discuss strategies for generating sensitive and specific capture/detector agents, multiplexed detection and signal amplification. An important starting point in LFA development is the clear definition/identification of design inputs and predicate. Newly engineered capture/detector agents, such as nanobodies and aptamers, should possess high binding affinity and functionality in body fluid samples for practical field application. Also, chemical methods, combined with engineering, have enabled multiplexing and signal amplification capabilities for higher target throughput and detection sensitivity. Furthermore, LFAs can be augmented or adapted to other platforms, such as smartphone, spectroscopy and electrochemistry, for quantitative measurements that can engender wider applications and adoption. Through these advances, LFA will transform into a genuinely versatile platform, capable of delivering accurate results that are similar to lab-based technologies, while retaining its advantage as a simple, portable, inexpensive and rapid test.

Development of Lateral Flow Assay for Point-of-Care Diagnosis of Trypanosomosis in Equines

Trypanosomosis, commonly known as “Surra” in Indian subcontinent caused by Trypanosoma evansi, is a worldwide parasitic disease of wild and domestic animals. The disease is transmitted mechanically by biting and interrupted feeding of infected animals by hematophagous flies. The diagnostic methods available for this disease require high-end instruments and skilled manpower and are time-consuming. In the present investigation, we attempted an immunoassay based on gold nanoparticles for diagnosis of T. evansi infection in equines. Antibodies to horse IgG were raised in rabbits and conjugated to gold nanoparticles after purification with the protein-A column. The soluble whole cell lysate antigen was prepared from purified T. evansi parasites and coated as test line on nitrocellulose membrane laminates, along with control line to detect anti-Trypanosoma antibodies. The experimental and field serum samples were tested and evaluated to determine the sensitivity and specificity of the assay. The antigen-specific antibodies were detected in experimental serum samples between 10 and 21 days post infection by the lateral flow assay (LFA). The LFA results showed 96.3% sensitivity and 93.9% specificity comparable with enzyme-linked immunosorbent assay. The study suggests that the LFA can be used as a point-of-care diagnostic method in the field, which does not require any specialized instrument for the detection of T. evansi infection in animals.

Small molecule detection with aptamer based lateral flow assays: Applying aptamer-C-reactive protein cross-recognition for ampicillin detection

Aptamer-based lateral flow assays (LFAs) are an emerging field of aptamer applications due to numerous potential applications. When compared to antibodies, potential advantages like cost effectiveness or lower batch to batch variations are evident. The development of LFAs for small molecules, however, is still challenging due to several reasons, primarily linked to target size and accessible interaction sites. In small molecule analysis, however, aptamers in many cases are preferable since immunogenicity is not required and they may exhibit even higher target selectivity. We report the first cross-recognition of a small molecule (ampicillin) and a protein (C-reactive protein), predicted by in-silico analysis, then experimentally confirmed - using two different aptamers. These features can be exploited for developing an aptamer-based LFA for label-free ampicillin detection, functioning also for analysis in milk extract. Most importantly, the principal setup denotes a novel, transferable and versatile general approach for detection of small molecules using competitive LFAs, unlikely to be generally realized by aptamer-DNA-binding otherwise.

Improving Lateral Flow Assay Performance Using Computational Modeling.

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.