scholarly journals Ultrasensitive Detection of SARS-CoV-2 Spike Proteins Using the Thio-NAD Cycling Reaction: A Preliminary Study before Clinical Trials

2021 ◽  
Vol 9 (11) ◽  
pp. 2214
Author(s):  
Yuta Kyosei ◽  
Mayuri Namba ◽  
Daiki Makioka ◽  
Ayumi Kokubun ◽  
Satoshi Watabe ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Ultraviolet B ◽  
Detection Sensitivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antigen Test ◽  
Specific Test ◽  
Nucleocapsid Proteins ◽  
Global Pandemic ◽  
Testing Accuracy ◽  
Preliminary Study

To help control the global pandemic of coronavirus disease 2019 (COVID-19), we developed a diagnostic method targeting the spike protein of the virus that causes the infection, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We applied an ultrasensitive method by combining a sandwich enzyme-linked immunosorbent assay (ELISA) and the thio-nicotinamide adenine dinucleotide (thio-NAD) cycling reaction to quantify spike S1 proteins. The limit of detection (LOD) was 2.62 × 10−19 moles/assay for recombinant S1 proteins and 2.6 × 106 RNA copies/assay for ultraviolet B-inactivated viruses. We have already shown that the ultrasensitive ELISA for nucleocapsid proteins can detect ultraviolet B-inactivated viruses at the 104 RNA copies/assay level, whereas the nucleocapsid proteins of SARS-CoV-2 are difficult to distinguish from those in conventional coronaviruses and SARS-CoV. Thus, an antigen test for only the nucleocapsid proteins is insufficient for virus specificity. Therefore, the use of a combination of tests against both spike and nucleocapsid proteins is recommended to increase both the detection sensitivity and testing accuracy of the COVID-19 antigen test. Taken together, our present study, in which we incorporate S1 detection by combining the ultrasensitive ELISA for nucleocapsid proteins, offers an ultrasensitive, antigen-specific test for COVID-19.

scholarly journals Proposal of De Novo Antigen Test for COVID-19: Ultrasensitive Detection of Spike Proteins of SARS-CoV-2

Diagnostics ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 594 ◽  
Author(s):  
Yuta Kyosei ◽  
Mayuri Namba ◽  
Sou Yamura ◽  
Rikiya Takeuchi ◽  
Noriko Aoki ◽  
...  
Keyword(s):  
De Novo ◽  
Limit Of Detection ◽  
False Negative ◽  
Cost Effective ◽  
Detection Sensitivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antigen Test ◽  
Virus Rna ◽  
Antigen Tests ◽  
Spike Proteins

Polymerase chain reaction (PCR)-based antigen tests are technically difficult, time-consuming, and expensive, and may produce false negative results requiring follow-up confirmation with computed tomography. The global coronavirus disease 2019 (COVID-19) pandemic has increased the demand for accurate, easy-to-use, rapid, and cost-effective antigen tests for clinical application. We propose a de novo antigen test for diagnosing COVID-19 using the combination of sandwich enzyme-linked immunosorbent assay and thio-nicotinamide adenine dinucleotide (thio-NAD) cycling. Our test takes advantage of the spike proteins specific to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. The limit of detection of our test was 2.3 × 10−18 moles/assay. If the virus has ~25 spike proteins on its surface, our method should detect on the order of 10−20 moles of virus/assay, corresponding to ~104 copies of the virus RNA/assay. The detection sensitivity approaches that of PCR-based assays because the average virus RNA load used for PCR-based assays is ~105 copies per oro- or naso-pharyngeal swab specimen. To our knowledge, this is the first ultrasensitive antigen test for SARS-CoV-2 spike proteins that can be performed with an easy-to-use microplate reader. Sufficient sensitivity can be achieved within 10 min of thio-NAD cycling. Our antigen test allows for rapid, cost-effective, specific, ultrasensitive, and simultaneous multiple measurements of SARS-CoV-2, and has broad application for the diagnosis for COVID-19.

scholarly journals A rapid and highly sensitive biomarker detection platform based on a temperature-responsive liposome-linked immunosorbent assay

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Runkai Hu ◽  
Keitaro Sou ◽  
Shinji Takeoka
Keyword(s):  
Immunosorbent Assay ◽  
Limit Of Detection ◽  
Specific Antigen ◽  
Fluorescence Emission ◽  
Detection Sensitivity ◽  
Sensitive Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Temperature Responsive ◽  
Highly Sensitive ◽  
Highly Sensitive Detection

Abstract The enzyme-linked immunosorbent assay (ELISA) is widely used in various fields to detect specific biomarkers. However, ELISA tests have limited detection sensitivity (≥ 1 pM), which is insufficiently sensitive for the detection of small amounts of biomarkers in the early stages of disease or infection. Herein, a method for the rapid and highly sensitive detection of specific antigens, using temperature-responsive liposomes (TLip) containing a squaraine dye that exhibits fluorescence at the phase transition temperature of the liposomes, was developed. A proof-of-concept study using biotinylated TLip and a streptavidin-immobilized microwell plate showed that the TLip bound to the plate via specific molecular recognition could be distinguished from unbound TLip within 1 min because of the difference in the heating time required for the fluorescence emission of TLip. This system could be used to detect prostate specific antigen (PSA) based on a sandwich immunosorbent assay using detection and capture antibodies, in which the limit of detection was as low as 27.6 ag/mL in a 100-μL PSA solution, 0.97 aM in terms of molar concentration. The present temperature-responsive liposome-linked immunosorbent assay provides an advanced platform for the rapid and highly sensitive detection of biomarkers for use in diagnosis and biological inspections.

scholarly journals Analytical Validation of a COVID-19 qRT-PCR Detection Assay Using a 384-well Format and Three Extraction Methods

2020 ◽  
Author(s):  
Andrew C. Nelson ◽  
Benjamin Auch ◽  
Matthew Schomaker ◽  
Daryl M. Gohl ◽  
Patrick Grady ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Extraction Methods ◽  
Detection Sensitivity ◽  
Acid Extraction ◽  
Analytical Validation ◽  
Qrt Pcr ◽  
Global Pandemic ◽  
Detection Assay ◽  
Effective Public Health ◽  
Local Laboratory

AbstractThe COVID-19 global pandemic is an unprecedented health emergency. Insufficient access to testing has hampered effective public health interventions and patient care management in a number of countries. Furthermore, the availability of regulatory-cleared reagents has challenged widespread implementation of testing. We rapidly developed a qRT-PCR SARS-CoV-2 detection assay using a 384-well format and tested its analytic performance across multiple nucleic acid extraction kits. Our data shows robust analytic accuracy on residual clinical biospecimens. Limit of detection sensitivity and specificity was confirmed with currently available commercial reagents. Our methods and results provide valuable information for other high-complexity laboratories seeking to develop effective, local, laboratory-developed procedures with high-throughput capability to detect SARS-CoV-2.

scholarly journals Photooxidation-induced fluorescence amplification system for an ultra-sensitive enzyme-linked immunosorbent assay (ELISA)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Youhee Heo ◽  
Kwanwoo Shin ◽  
Min Cheol Park ◽  
Ji Yoon Kang
Keyword(s):  
Limit Of Detection ◽  
Slight Modification ◽  
Detection Sensitivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Protein Biomarkers ◽  
Amplex Red ◽  
Rate Of Increase ◽  
Amplification Rate ◽  
Amplification System ◽  
Fluorescence Amplification

AbstractThis report suggests a method of enhancing the sensitivity of chemifluorescence-based ELISA, using photooxidation-induced fluorescence amplification (PIFA). The PIFA utilized autocatalytic photooxidation of the chemifluorescent substrate, 10-acetyl 3,7-dihydroxyphenoxazine (ADHP, Amplex Red) to amplify the fluorescent product resorufin, initially oxidized by horse radish peroxidase (HRP). As the amplification rate is proportional to the initial level of resorufin, the level of antigen labeled by HRP is quantified by analyzing the profile of fluorescence intensity. The normalized profile was interpolated into an autocatalysis model, and the rate of increase at half-maximum time was quantified by the use of an amplification index (AI). The lower limit of detection, for resorufin or HRP, was less than one-tenth that of the plate reader. It requires only slight modification of the fluorescence reader and is fully compatible with conventional or commercial ELISA. When it is applied to a commercial ELISA kit for the detection of amyloid beta, it is verified that the PIFA assay enhanced the detection sensitivity by more than a factor of 10 and was compatible with a conventional 96-well ELISA assay kit. We anticipate this PIFA assay to be used in research for the detection of low levels of proteins and for the early diagnosis of various diseases with rare protein biomarkers, at ultra-low (pg/mL) concentrations.

scholarly journals Improved detection sensitivity of an antigen test for SARS-CoV-2 nucleocapsid proteins with thio-NAD cycling

2021 ◽  
Author(s):  
Yuta Kyosei ◽  
Mayuri Namba ◽  
Sou Yamura ◽  
Satoshi Watabe ◽  
Teruki Yoshimura ◽  
...  
Keyword(s):  
Detection Sensitivity ◽  
Antigen Test ◽  
Nucleocapsid Proteins

scholarly journals Rapid, Quantitative, and High-Sensitivity Detection of Anti-Phospholipase A2 Receptor Antibodies Using Novel Cdse/Zns-Based Fluorescence Immunosorbent Assay

2020 ◽  
Author(s):  
Chenxi Li ◽  
Manyun Qian ◽  
Qiaozhen Hong ◽  
Xiaohong Xin ◽  
Zichun Sun ◽  
...  
Keyword(s):  
Phospholipase A2 ◽  
Immunosorbent Assay ◽  
Limit Of Detection ◽  
High Sensitivity ◽  
Detection Sensitivity ◽  
Response To Treatment ◽  
Enzyme Linked Immunosorbent Assay ◽  
The Novel ◽  
Serum Samples ◽  
Phospholipase A2 Receptor

Abstract Autoantibodies against M-type phospholipase A2 receptor (PLA2R) are specific biomarkers for idiopathic membranous nephropathy (IMN) and their quantification has been helpful to monitor disease activity. In this study, we describe a highly sensitive and rapid quantum dots-based immunochromatography assay (QD-ICA) for quantifying PLA2R autoantibodies. Serum samples from 135 biopsy-confirmed patients with nephrotic syndrome were analyzed for PLA2R autoantibodies using the novel QD-ICA as well as enzyme-linked immunosorbent assay (ELISA). The detection sensitivity and specificity of QD-ICA (80.9 and 100%, respectively) exceeded those of ELISA (72.1 and 98.5%, respectively). The optimum cut-off value of QD-ICA was 18.18 RU/mL and limit of detection was 2.86 relative units/mL. The novel QD-ICA outperforms ELISA in detecting PLA2R autoantibodies, with shorter detection time, fewer steps, smaller equipment size, and broader testing application, suggesting its capability to improve IMN diagnosis and monitor patient response to treatment.

scholarly journals On-Site Detection of Carcinoembryonic Antigen in Human Serum

Biosensors ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 392
Author(s):  
Tohid Mahmoudi ◽  
Mohammad Pourhassan-Moghaddam ◽  
Behnaz Shirdel ◽  
Behzad Baradaran ◽  
Eden Morales-Narváez ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Human Serum ◽  
Carcinoembryonic Antigen ◽  
Limit Of Detection ◽  
Lateral Flow ◽  
Detection Sensitivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Care Providers ◽  
Lateral Flow Strips ◽  
Visual Limit

Real-time connectivity and employment of sustainable materials empowers point-of-care diagnostics with the capability to send clinically relevant data to health care providers even in low-resource settings. In this study, we developed an advantageous kit for the on-site detection of carcinoembryonic antigen (CEA) in human serum. CEA sensing was performed using cellulose-based lateral flow strips, and colorimetric signals were read, processed, and measured using a smartphone-based system. The corresponding immunoreaction was reported by polydopamine-modified gold nanoparticles in order to boost the signal intensity and improve the surface blocking and signal-to-noise relationship, thereby enhancing detection sensitivity when compared with bare gold nanoparticles (up to 20-fold in terms of visual limit of detection). Such lateral flow strips showed a linear range from 0.05 to 50 ng/mL, with a visual limit of detection of 0.05 ng/mL and an assay time of 15 min. Twenty-six clinical samples were also tested using the proposed kit and compared with the gold standard of immunoassays (enzyme linked immunosorbent assay), demonstrating an excellent correlation (R = 0.99). This approach can potentially be utilized for the monitoring of cancer treatment, particularly at locations far from centralized laboratory facilities.

scholarly journals Rapid, quantitative, and high-sensitivity detection of anti-phospholipase A2 receptor antibodies using a novel CdSe/ZnS-based fluorescence immunosorbent assay

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chenxi Li ◽  
Manyun Qian ◽  
Qiaozhen Hong ◽  
Xiaohong Xin ◽  
Zichun Sun ◽  
...  
Keyword(s):  
Phospholipase A2 ◽  
Immunosorbent Assay ◽  
Limit Of Detection ◽  
Characteristic Curve ◽  
Detection Sensitivity ◽  
Response To Treatment ◽  
Enzyme Linked Immunosorbent Assay ◽  
The Novel ◽  
Serum Samples ◽  
Phospholipase A2 Receptor

AbstractAutoantibodies against M-type phospholipase A2 receptor (PLA2R) serve as specific biomarkers for idiopathic membranous nephropathy (IMN), and its quantification helps monitor disease activity. In this study, we describe a rapid and highly sensitive quantum dots-based immunochromatography assay (QD-ICA) for quantifying PLA2R autoantibodies. Serum samples from 135 biopsy-confirmed patients with nephrotic syndrome were analyzed for PLA2R autoantibodies using the novel QD-ICA as well as commercialized enzyme-linked immunosorbent assay (ELISA). Areas under the receiver operating characteristic curve (AUC-ROC) of QD-ICA were significantly greater than those of ELISA (91.1% [95% CI 85.9–96.3%] and 83.9% [95% CI 76.5–91.2%] respectively; p < 0.01). The detection sensitivity and specificity of QD-ICA (80.9% [95% CI 69.2–89.0%] and 100% [95% CI 93.2–100.0%], respectively) exceeded those of ELISA (72.1% [95% CI 59.7–81.9%] and 98.5% [95% CI 90.9–100.0%], respectively). The optimum cut-off value of QD-ICA was 18.18 relative units (RU)/mL, and the limit of detection was 2.86 RU/mL. The novel QD-ICA outperforms ELISA in detecting PLA2R autoantibodies, with shorter detection time, fewer steps, smaller equipment size, and broader testing application, suggesting its capability to improve IMN diagnosis and monitor patient response to treatment.

Ferromagnetic Resonance Biosensor for Homogeneous and Volumetric Detection of DNA

2017 ◽  
Author(s):  
Bo Tian ◽  
Peter Svedlindh ◽  
Mattias Strömberg ◽  
Erik Wetterskog
Keyword(s):  
Magnetic Nanoparticles ◽  
Ferromagnetic Resonance ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Rolling Circle Amplification ◽  
Resonance Field ◽  
Isothermal Amplification ◽  
Detection Sensitivity ◽  
Serum Samples ◽  
Rolling Circle

In this work, we demonstrate for the first time, a ferromagnetic resonance (FMR) based homogeneous and volumetric biosensor for magnetic label detection. Two different isothermal amplification methods, <i>i.e.</i>, rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP) are adopted and combined with a standard electron paramagnetic resonance (EPR) spectrometer for FMR biosensing. For RCA-based FMR biosensor, binding of RCA products of a synthetic Vibrio cholerae target DNA sequence gives rise to the formation of aggregates of magnetic nanoparticles. Immobilization of nanoparticles within the aggregates leads to a decrease of the net anisotropy of the system and a concomitant increase of the resonance field. A limit of detection of 1 pM is obtained with an average coefficient of variation of 0.16%, which is superior to the performance of other reported RCA-based magnetic biosensors. For LAMP-based sensing, a synthetic Zika virus target oligonucleotide is amplified and detected in 20% serum samples. Immobilization of magnetic nanoparticles is induced by their co-precipitation with Mg<sub>2</sub>P<sub>2</sub>O<sub>7</sub> (a by-product of LAMP) and provides a detection sensitivity of 100 aM. The fast measurement, high sensitivity and miniaturization potential of the proposed FMR biosensing technology makes it a promising candidate for designing future point-of-care devices.<br>

scholarly journals A verified genomic reference sample for assessing performance of cancer panels detecting small variants of low allele frequency

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Wendell Jones ◽  
Binsheng Gong ◽  
Natalia Novoradovskaya ◽  
Dan Li ◽  
Rebecca Kusko ◽  
...  
Keyword(s):  
Quality Control ◽  
Allele Frequency ◽  
Liquid Biopsy ◽  
Limit Of Detection ◽  
Reference Sample ◽  
Digital Pcr ◽  
Detection Sensitivity ◽  
Analytical Accuracy ◽  
And Performance ◽  
Reference Samples

Abstract Background Oncopanel genomic testing, which identifies important somatic variants, is increasingly common in medical practice and especially in clinical trials. Currently, there is a paucity of reliable genomic reference samples having a suitably large number of pre-identified variants for properly assessing oncopanel assay analytical quality and performance. The FDA-led Sequencing and Quality Control Phase 2 (SEQC2) consortium analyze ten diverse cancer cell lines individually and their pool, termed Sample A, to develop a reference sample with suitably large numbers of coding positions with known (variant) positives and negatives for properly evaluating oncopanel analytical performance. Results In reference Sample A, we identify more than 40,000 variants down to 1% allele frequency with more than 25,000 variants having less than 20% allele frequency with 1653 variants in COSMIC-related genes. This is 5–100× more than existing commercially available samples. We also identify an unprecedented number of negative positions in coding regions, allowing statistical rigor in assessing limit-of-detection, sensitivity, and precision. Over 300 loci are randomly selected and independently verified via droplet digital PCR with 100% concordance. Agilent normal reference Sample B can be admixed with Sample A to create new samples with a similar number of known variants at much lower allele frequency than what exists in Sample A natively, including known variants having allele frequency of 0.02%, a range suitable for assessing liquid biopsy panels. Conclusion These new reference samples and their admixtures provide superior capability for performing oncopanel quality control, analytical accuracy, and validation for small to large oncopanels and liquid biopsy assays.

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