Antibody Screening Results for Anti-Nucleocapsid Antibodies Towards the Development of a SARS-CoV-2 Nucleocapsid Protein Antigen Detecting Lateral Flow Assay

2021 ◽  
Author(s):  
David Cate ◽  
Helen Hsieh ◽  
Veronika Glukhova ◽  
Joshua D Bishop ◽  
H Gleda Hermansky ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Point Of Care ◽  
Lateral Flow ◽  
Screening Methods ◽  
Antibody Screening ◽  
Liquid Handling ◽  
Large Numbers ◽  
Accurate Performance ◽  
Further Development ◽  
Assay Conditions

<p></p><p>The global COVID-19 pandemic has created an urgent demand for large numbers of inexpensive, accurate, rapid, point-of-care diagnostic tests. Analyte-based assays are suitably inexpensive and can be rapidly mass-produced, but for sufficiently accurate performance they require highly optimized antibodies and assay conditions. We used an automated liquid handling system, customized to handle arrays of lateral flow immunoassay (LFA) tests in a high-throughput screen, to identify anti-nucleocapsid antibodies that will perform optimally in an LFA. We tested 1021 anti-nucleocapsid antibody pairs as LFA capture and detection reagents with the goal of highlighting pairs that have the greatest affinity for unique epitopes of the nucleocapsid protein of SARS-CoV-2 within the LFA format. In contrast to traditional antibody screening methods (e.g., ELISA, bio-layer interferometry), the method described here integrates real-time reaction kinetics with transport in, and immobilization directly onto, nitrocellulose. We have identified several candidate antibody pairs that are suitable for further development of an LFA for SARS-CoV-2.</p><p></p>

scholarly journals Antibody Screening Results for Anti-Nucleocapsid Antibodies Towards the Development of a SARS-CoV-2 Nucleocapsid Protein Antigen Detecting Lateral Flow Assay

2021 ◽  
Author(s):  
David Cate ◽  
Helen Hsieh ◽  
Veronika Glukhova ◽  
Joshua D Bishop ◽  
H Gleda Hermansky ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Point Of Care ◽  
Lateral Flow ◽  
Screening Methods ◽  
Antibody Screening ◽  
Liquid Handling ◽  
Large Numbers ◽  
Accurate Performance ◽  
Further Development ◽  
Assay Conditions

<p></p><p>The global COVID-19 pandemic has created an urgent demand for large numbers of inexpensive, accurate, rapid, point-of-care diagnostic tests. Analyte-based assays are suitably inexpensive and can be rapidly mass-produced, but for sufficiently accurate performance they require highly optimized antibodies and assay conditions. We used an automated liquid handling system, customized to handle arrays of lateral flow immunoassay (LFA) tests in a high-throughput screen, to identify anti-nucleocapsid antibodies that will perform optimally in an LFA. We tested 1021 anti-nucleocapsid antibody pairs as LFA capture and detection reagents with the goal of highlighting pairs that have the greatest affinity for unique epitopes of the nucleocapsid protein of SARS-CoV-2 within the LFA format. In contrast to traditional antibody screening methods (e.g., ELISA, bio-layer interferometry), the method described here integrates real-time reaction kinetics with transport in, and immobilization directly onto, nitrocellulose. We have identified several candidate antibody pairs that are suitable for further development of an LFA for SARS-CoV-2.</p><p></p>

Antibody Screening Results for Anti-Nucleocapsid Antibodies Towards the Development of a SARS-CoV-2 Nucleocapsid Protein Antigen Detecting Lateral Flow Assay

2020 ◽  
Author(s):  
David Cate ◽  
Helen Hsieh ◽  
Veronika Glukhova ◽  
Joshua D Bishop ◽  
H Gleda Hermansky ◽  
...  
Keyword(s):  
Diagnostic Tests ◽  
Nucleocapsid Protein ◽  
Point Of Care ◽  
Lateral Flow ◽  
Screening Methods ◽  
Antibody Screening ◽  
Liquid Handling ◽  
Accurate Performance ◽  
Further Development ◽  
Assay Conditions

<p>The global COVID-19 pandemic has created an urgent demand for accurate rapid point of care diagnostic tests. Antigen-based assays are suitably inexpensive and can be rapidly mass-produced, but sufficiently accurate performance requires highly-optimized antibodies and assay conditions. An automated liquid handling system, customized to handle lateral flow immunoassay (LFA) arrays, was used for high-throughput antibody screening of anti-nucleocapsid antibodies that will perform optimally on an LFA. Six hundred seventy-three anti-nucleocapsid antibody pairs were tested as both capture and detection reagents with the goal of finding those pairs that have the greatest affinity for unique epitopes of the nucleocapsid protein of SARS-CoV-2 while also performing optimally in an LFA format. In contrast to traditional antibody screening methods (e.g. ELISA, bio-layer interferometry), the methods described here integrate real-time LFA reaction kinetics and binding directly on nitrocellulose. We have identified several candidate antibody pairs that are suitable for further development of an LFA for SARS-CoV-2. </p>

Development of a Smartphone Based Reader for the Quantitative Analysis of Lateral Flow Assays

2018 ◽  
Vol 941 ◽  
pp. 2522-2527
Author(s):  
Sylvio Schneider ◽  
Martina Selig ◽  
Verena Keil ◽  
Matthias Lehmann ◽  
Andreas H. Foitzik ◽  
...  
Keyword(s):  
Quantitative Analysis ◽  
Medical Devices ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Thrombotic Event ◽  
Lateral Flow ◽  
Proof Of Concept ◽  
Lateral Flow Assays ◽  
Further Development ◽  
D Dimer

Smartphones are developing into all-purposes devices. In the present work, the employment/application of smartphones as medical devices in home care and point-of-care (POC) diagnostics are investigated in the analysis of Lateral Flow Assays (LFA). A smartphone-based LFA reader was developed for the quantitative analysis of D-Dimer – a biomarker indicating e.g. thrombotic event or danger of embolism.The proof-of-concept has been shown with multiple smartphones in establishing: (I) Optimal dimensions of the LFA cell of 72.11mm distance of smartphone to D-Dimer test leading to a coefficients of variances (CV) between 0.8% and 4.2%. (II) Inter-device investigations: CVs around 13.5%; a limit of detection (LOD) of 100ng/ml (DDU) D-Dimer. (III) Inter-smartphone investigations: CV about 16%, a limit of detection (LOD) at 66.4ng/ml (DDU). (IV) Calibrations: CV and LOD of three smartphones are comparable to the commercial available LFA reader. Further development to put the multiple smartphone-based LFA reader on the market.

scholarly journals Antibody Screening Results for Anti-Nucleocapsid Antibodies Toward the Development of a Lateral Flow Assay to Detect SARS-CoV-2 Nucleocapsid Protein

ACS Omega ◽  
2021 ◽  
Author(s):  
David M. Cate ◽  
Joshua D. Bishop ◽  
Helen V. Hsieh ◽  
Veronika A. Glukhova ◽  
Luis F. Alonzo ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Antibody Screening

scholarly journals Lateral Flow Serodiagnosis in the Double-Antigen Sandwich Format: Theoretical Consideration and Confirmation of Advantages

Sensors ◽  
2020 ◽  
Vol 21 (1) ◽  
pp. 39
Author(s):  
Dmitriy V. Sotnikov ◽  
Anatoly V. Zherdev ◽  
Boris B. Dzantiev
Keyword(s):  
Specific Antibody ◽  
Large Scale ◽  
Point Of Care ◽  
Lateral Flow ◽  
Limiting Factor ◽  
Point Of Care Testing ◽  
Nonspecific Immunoglobulins ◽  
The Mathematical Model ◽  
Assay Conditions

Determination of the presence in the blood of antibodies specific to the causative agent of a particular disease (serodiagnosis) is an effective approach in medical analytical chemistry. Serodiagnostics performed in the lateral flow immunoassay format (immunochromatography) meet the modern requirements for point-of-care testing and are supported by existing technologies of large-scale diagnostic tests production, thus increasing the amount of attention in a tense epidemiological situation. For traditional lateral flow serodiagnostics formats, a large number of nonspecific immunoglobulins in the sample significantly reduces the degree of detectable binding. To overcome these limitations, an assay based on the formation of immobilized antigen-specific antibody-labeled antigen complexes detection was proposed. However, the requirements for its implementation, providing maximum sensitivity, have not been established. This article describes the mathematical model for the above assay. The influence of the ratio of reagent concentrations on the analysis results is considered. It is noted that the formation of specific antibody complexes with several labeled antigens is the main limiting factor in reducing the detection limit, and methods are proposed to minimize this factor. Recommendations for the choice of the assay conditions, following from the analysis of the model, are confirmed experimentally.

scholarly journals A SARS-CoV-2 Coronavirus Nucleocapsid Protein Antigen-Detecting Lateral Flow Assay

2020 ◽  
Author(s):  
Ben D Grant ◽  
Caitlin E Anderson ◽  
Spencer H Garing ◽  
Luis F Alonzo ◽  
John R Williford ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Lateral Flow ◽  
Rapid Diagnostics ◽  
Rt Pcr ◽  
Efficient Detection ◽  
Lateral Flow Assays ◽  
Optical Reader ◽  
Polymerase Chain

<p></p><p>Inexpensive, simple, rapid diagnostics are necessary for efficient detection, treatment and mitigation of COVID‑19. Currently, the primary diagnostic tool being utilized is reverse transcription polymerase chain reaction (RT-PCR). RT-PCR delivers results with good sensitivity and excellent specificity, but is expensive, prone to access challenges and is often slowed by transport to centralized testing laboratories. Antigen-based assays are inexpensive and can be rapidly mass-produced and deployed, with lateral flow assays (LFAs) being the most common inexpensive antigen test. To date, few antigen-detecting LFAs for COVID-19 have been commercialized. Herein, we present an open source LFA using commercially available antibodies and materials for the detection of SARS-CoV-2. Using an optical reader with comparable sensitivity to a visual read, the LFA yielded a Limit of Detection (LOD) of 23 TCID<sub>50</sub>/mL (95% CI of 9.1 to 37 TCID<sub>50</sub>/mL), equivalent to 1.4x10<sup>5</sup> copies/mL (95% CI of 5.5x10<sup>4</sup> to 2.3x10<sup>5</sup> copies/mL) irradiated virus in pooled nasal matrix. This LOD meets the criteria suggested by WHO for diagnosis of acute SARS-CoV-2 infection in a point of care format. A clinical evaluation and further testing is ongoing.</p><p></p>

scholarly journals Towards Lateral Flow Quantitative Assays: Detection Approaches

Biosensors ◽  
2019 ◽  
Vol 9 (3) ◽  
pp. 89 ◽  
Author(s):  
Urusov ◽  
Zherdev ◽  
Dzantiev
Keyword(s):  
Point Of Care ◽  
Visual Assessment ◽  
Quantitative Information ◽  
Lateral Flow ◽  
Advanced Manufacturing ◽  
Global Trend ◽  
Lateral Flow Assays ◽  
Qualitative Conclusion ◽  
Lateral Flow Tests ◽  
Further Development

Point-of-care (POC) or bedside analysis is a global trend in modern diagnostics. Progress in POC testing has largely been provided by advanced manufacturing technology for lateral flow (immunochromatographic) test strips. They are widely used to rapidly and easily control a variety of biomarkers of infectious diseases and metabolic and functional disorders, as well as in consumer protection and environmental monitoring. However, traditional lateral flow tests rely on visual assessment and qualitative conclusion, which limit the objectivity and information output of the assays. Therefore, there is a need for approaches that retain the advantages of lateral flow assays and provide reliable quantitative information about the content of a target compound in a sample mixture. This review describes the main options for detecting, processing, and interpreting immunochromatographic analysis results. The possibilities of modern portable detectors that register colored, fluorescent, magnetic, and conductive labels are discussed. Prospects for further development in this direction are also examined.

scholarly journals A SARS-CoV-2 Coronavirus Nucleocapsid Protein Antigen-Detecting Lateral Flow Assay

2020 ◽  
Author(s):  
Ben D Grant ◽  
Caitlin E Anderson ◽  
Spencer H Garing ◽  
Luis F Alonzo ◽  
John R Williford ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Lateral Flow ◽  
Rapid Diagnostics ◽  
Rt Pcr ◽  
Efficient Detection ◽  
Lateral Flow Assays ◽  
Optical Reader ◽  
Polymerase Chain

<p></p><p>Inexpensive, simple, rapid diagnostics are necessary for efficient detection, treatment and mitigation of COVID‑19. Currently, the primary diagnostic tool being utilized is reverse transcription polymerase chain reaction (RT-PCR). RT-PCR delivers results with good sensitivity and excellent specificity, but is expensive, prone to access challenges and is often slowed by transport to centralized testing laboratories. Antigen-based assays are inexpensive and can be rapidly mass-produced and deployed, with lateral flow assays (LFAs) being the most common inexpensive antigen test. To date, few antigen-detecting LFAs for COVID-19 have been commercialized. Herein, we present an open source LFA using commercially available antibodies and materials for the detection of SARS-CoV-2. Using an optical reader with comparable sensitivity to a visual read, the LFA yielded a Limit of Detection (LOD) of 23 TCID<sub>50</sub>/mL (95% CI of 9.1 to 37 TCID<sub>50</sub>/mL), equivalent to 1.4x10<sup>5</sup> copies/mL (95% CI of 5.5x10<sup>4</sup> to 2.3x10<sup>5</sup> copies/mL) irradiated virus in pooled nasal matrix. This LOD meets the criteria suggested by WHO for diagnosis of acute SARS-CoV-2 infection in a point of care format. A clinical evaluation and further testing is ongoing.</p><p></p>

Gold-Aptamer-Nanoconstructs Engineered to Detect Conserved Enteroviral Nucleic Acid Sequences

2019 ◽  
Author(s):  
Veeren Chauhan ◽  
Mohamed M Elsutohy ◽  
C Patrick McClure ◽  
Will Irving ◽  
Neil Roddis ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
In Silico ◽  
Point Of Care ◽  
Lateral Flow ◽  
Nucleic Acid Sequence ◽  
In Silico Screening ◽  
Lateral Flow Assays ◽  
Life Threatening ◽  
Software And Hardware ◽  
Nucleic Acid Sequences

<p>Enteroviruses are a ubiquitous mammalian pathogen that can produce mild to life-threatening disease. Bearing this in mind, we have developed a rapid, accurate and economical point-of-care biosensor that can detect a nucleic acid sequences conserved amongst 96% of all known enteroviruses. The biosensor harnesses the physicochemical properties of gold nanoparticles and aptamers to provide colourimetric, spectroscopic and lateral flow-based identification of an exclusive enteroviral RNA sequence (23 bases), which was identified through in silico screening. Aptamers were designed to demonstrate specific complementarity towards the target enteroviral RNA to produce aggregated gold-aptamer nanoconstructs. Conserved target enteroviral nucleic acid sequence (≥ 1x10<sup>-7</sup> M, ≥1.4×10<sup>-14</sup> g/mL), initiates gold-aptamer-nanoconstructs disaggregation and a signal transduction mechanism, producing a colourimetric and spectroscopic blueshift (544 nm (purple) > 524 nm (red)). Furthermore, lateral-flow-assays that utilise gold-aptamer-nanoconstructs were unaffected by contaminating human genomic DNA, demonstrated rapid detection of conserved target enteroviral nucleic acid sequence (< 60 s) and could be interpreted with a bespoke software and hardware electronic interface. We anticipate our methodology will translate in-silico screening of nucleic acid databases to a tangible enteroviral desktop detector, which could be readily translated to related organisms. This will pave-the-way forward in the clinical evaluation of disease and complement existing strategies at overcoming antimicrobial resistance.</p>

A Point of Care Lateral Flow Assay for Rapid and Colorimetric Detection of Interleukin 6 and Perspectives in Bedside Diagnostics

2020 ◽  
Author(s):  
Carla Eiras
Keyword(s):  
Interleukin 6 ◽  
Limit Of Detection ◽  
Inflammatory Diseases ◽  
Point Of Care ◽  
Colorimetric Detection ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Aggregation Assay ◽  
Before And After ◽  
Bedside Diagnosis

Interleukin-6 (IL-6) is a multifunctional cytokine and high bloodstream levels of which have been associated with severe inflammatory diseases, such as dengue fever, sepsis, various cancers, and visceral leishmaniasis (VL). Rapid tests for the quantification of IL-6 would be of great assistance for the bedside diagnosis and treatment of diseases such as VL. We have developed a lateral flow assay (LFA) for rapid and colorimetric IL-6 detection, consisting of anti-IL-6 antibodies conjugated to gold nanoparticles (AuNPs). The optimal concentration of anti-IL-6 used in the conjugate was determined to be 800.0 μg/mL, based on an aggregation assay using LFA. A linear relationship between IL-6 standard concentration and color intensity was observed after 20 min, with a linear range between 1.25 ng/mL and 9,000 ng/mL. The limit of detection for this method was estimated a t0.38 ng/mL. The concentration of IL-6 in five patients with severe VL was measured using LFA, and the results were consistent with those obtained using the cytometric bead array (CBA) method. A thorough analysis of the LFA membranes’ surface morphology, before and after sample contact, was performed using atomic force microscopy (AFM).The prototype described here is still being tested and improved, but this LFA will undoubtedly be of great help in the clinical quantification of IL-6.

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