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פותח על ידי קלירמאש פתרונות בע"מ -
Capture-Layer Lateral Flow Immunoassay: A New Platform Validated in the Detection and Quantification of Dengue NS1
Year:
2020
Source of publication :
ACS Omega
Authors :
ילצוב, יבגני
;
.
Volume :
5
Co-Authors:

Axelrod, T. - A Department of Biotechnology Engineering, Faculty of Engineering Science, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel.
Marks, R.S. - Department of Biotechnology Engineering, Faculty of Engineering Science, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel; National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel;  Ilse Katz Center for Meso and Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel

Facilitators :
From page:
10433
To page:
10440
(
Total pages:
8
)
Abstract:

The lateral flow immunoassay (LFIA) is the most successful point-of-care testing (POCT) method to date. In the case of clinical biomarkers that require quantification, it remains a challenge to quantitate those biomarkers using the lateral flow immunoassay remains a challenge due to the cost of the reader and possibly the type of marker used. In the present work, a new concept of a platform LFIA device configuration is proposed in which different, aligned membrane components, some already existing in the classical lateral flow immunoassay, and the others created with special new functions in the present device. As the sample containing the target analyte passes through the aforementioned membranes, the target analyte will initially interact with a target-specific antibody-conjugated to horseradish peroxidase (HRP). Thereafter, the newly formed immunocomplex will diffuse through a proprietary capture membrane (that ensures that the nontarget-bound antibodies do not continue further and thus remain "captured" to that specific area). This is done by having the target molecules (or components thereof) immobilized onto the said capture layer. The target-bound immunocomplexes will then be allowed by the system configuration to continue further to the last layer, where the signal will be generated and quantified. Thus, in the absence of the target analyte in the sample, the free antibodies will be filtered at the capture layer by preimmobilized analyte molecules, thus preventing a false positive signal to occur. We validated the concept in the detection of dengue NS1 protein in view of making a triage test. The sample containing NS1 will first meet HRP-conjugated NS1-specific antibodies and become attached, thus producing an NS1-specific antibody-HRP immunocomplex. The sample then flows through the blocking layer, where the immunocomplex is unchallenged and thus allowed to reach the last "absorbent" pad, incorporating the substrate for the HRP marker. In the case of a positive test, a signal is generated, that is proportional to the amount of immunocomplexes (and therefore the NS1 concentration), and then analyzed and measured at the absorbent pad. Any unbound anti-NS1 antibody will be stopped at the blocking matrix by preimmobilized NS1, so there will be no false positive. As this study is the initial study of a novel configuration, much of the work comprised of optimization steps, such as determining the required NS1 membrane-immobilization concentration and the required target-specific capture antibody concentration. Our immunoassay was tested with spiked buffer and serum samples to mimic the clinical conditions, with a range of NS1 concentrations, and was found, at this time, to be fivefold more sensitive than a gold standard enzyme-linked immunosorbent assay (ELISA) test (5 ng mL-1) performed in our laboratory. This method shows another form of LFIA that has the potential to be quantitative (at least semiquantitative), albeit not solving the reader cost; however, unlike the regular LFIA, we do not use nanobeads but instead enzymes, allowing, in theory, greater sensitivity, while retaining the one-step procedure. The test is accurate and has low production costs.

Note:
Related Files :
Biopolymers
immunology
layers
membrane
peptides and proteins
עוד תגיות
תוכן קשור
More details
DOI :
10.1021/acsomega.0c00367
Article number:
0
Affiliations:
Database:
סקופוס
Publication Type:
מאמר
;
.
Language:
אנגלית
Editors' remarks:
ID:
47964
Last updated date:
02/03/2022 17:27
Creation date:
26/05/2020 17:56
You may also be interested in
Scientific Publication
Capture-Layer Lateral Flow Immunoassay: A New Platform Validated in the Detection and Quantification of Dengue NS1
5

Axelrod, T. - A Department of Biotechnology Engineering, Faculty of Engineering Science, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel.
Marks, R.S. - Department of Biotechnology Engineering, Faculty of Engineering Science, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel; National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel;  Ilse Katz Center for Meso and Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel

Capture-Layer Lateral Flow Immunoassay: A New Platform Validated in the Detection and Quantification of Dengue NS1

The lateral flow immunoassay (LFIA) is the most successful point-of-care testing (POCT) method to date. In the case of clinical biomarkers that require quantification, it remains a challenge to quantitate those biomarkers using the lateral flow immunoassay remains a challenge due to the cost of the reader and possibly the type of marker used. In the present work, a new concept of a platform LFIA device configuration is proposed in which different, aligned membrane components, some already existing in the classical lateral flow immunoassay, and the others created with special new functions in the present device. As the sample containing the target analyte passes through the aforementioned membranes, the target analyte will initially interact with a target-specific antibody-conjugated to horseradish peroxidase (HRP). Thereafter, the newly formed immunocomplex will diffuse through a proprietary capture membrane (that ensures that the nontarget-bound antibodies do not continue further and thus remain "captured" to that specific area). This is done by having the target molecules (or components thereof) immobilized onto the said capture layer. The target-bound immunocomplexes will then be allowed by the system configuration to continue further to the last layer, where the signal will be generated and quantified. Thus, in the absence of the target analyte in the sample, the free antibodies will be filtered at the capture layer by preimmobilized analyte molecules, thus preventing a false positive signal to occur. We validated the concept in the detection of dengue NS1 protein in view of making a triage test. The sample containing NS1 will first meet HRP-conjugated NS1-specific antibodies and become attached, thus producing an NS1-specific antibody-HRP immunocomplex. The sample then flows through the blocking layer, where the immunocomplex is unchallenged and thus allowed to reach the last "absorbent" pad, incorporating the substrate for the HRP marker. In the case of a positive test, a signal is generated, that is proportional to the amount of immunocomplexes (and therefore the NS1 concentration), and then analyzed and measured at the absorbent pad. Any unbound anti-NS1 antibody will be stopped at the blocking matrix by preimmobilized NS1, so there will be no false positive. As this study is the initial study of a novel configuration, much of the work comprised of optimization steps, such as determining the required NS1 membrane-immobilization concentration and the required target-specific capture antibody concentration. Our immunoassay was tested with spiked buffer and serum samples to mimic the clinical conditions, with a range of NS1 concentrations, and was found, at this time, to be fivefold more sensitive than a gold standard enzyme-linked immunosorbent assay (ELISA) test (5 ng mL-1) performed in our laboratory. This method shows another form of LFIA that has the potential to be quantitative (at least semiquantitative), albeit not solving the reader cost; however, unlike the regular LFIA, we do not use nanobeads but instead enzymes, allowing, in theory, greater sensitivity, while retaining the one-step procedure. The test is accurate and has low production costs.

Scientific Publication
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