A Microflow Cytometry-Based Agglutination Immunoassay for Point-of-Care Quantitative Detection of SARS-CoV-2 IgM and IgG

Abstract Introduction A fast (<5 min), time-resolved fluorescence resonance energy transfer (FRET)-based immunoassay was developed for the quantitative detection of infliximab (IFX) and biosimilars for use in therapeutic drug monitoring using only 20 µL of fingerstick whole blood or serum at the point-of-care. The Procise IFX assay and ProciseDx analyzer are CE-marked. Studies were performed to characterize analytical performance of the Procise IFX assay on the ProciseDx analyzer. Methods Analytical testing was performed by spiking known amounts of IFX into negative serum and whole blood specimens. Analytical sensitivity was determined using limiting concentrations of IFX. Linearity was determined by testing IFX across the assay range. Hook effect was assessed at IFX concentrations beyond levels expected to be found within a patient. Testing of assay precision, cross-reactivity and potential interfering substances, and biosimilars was performed. The Procise IFX assay was also compared head-to-head with another CE-marked assay: LISA-TRACKER infliximab ELISA test (Theradiag, France). The accuracy of the Procise IFX assay is established through calibrators and controls traceable to the WHO 1st International Standard for Infliximab (NIBSC code: 16/170). Results The Procise IFX assay shows a Limit of Blank, Limit of Detection, and Lower Limit of Quantitation (LLoQ) of 0.1, 0.2, and 1.1 µg/mL in serum and 0.6, 1.1, and 1.7 µg/mL in whole blood, respectively. The linear assay range was determined to be 1.7 to 77.2 µg/mL in serum and whole blood. No hook effect was observed at an IFX concentration of 200 µg/mL as the value reported as “>ULoQ”. Assay precision testing across 20 days with multiple runs and reagent lots showed an intra-assay coefficient of variation (CV) of 2.7%, an inter-assay CV of <2%, and a total CV of 3.4%. The presence of potentially interfering/cross-reacting substances showed minimal impact on assay specificity with %bias within ±8% of control. Testing of biosimilars (infliximab-dyyb and infliximab-abda) showed good recovery. A good correlation to the Theradiag infliximab ELISA was obtained for both serum (slope=1.01; r=0.99) and whole blood (slope=1.01; r=0.98) samples (Figure 1). Conclusion Results indicate that the Procise IFX assay is sensitive, specific, and precise yielding results within 5 minutes from both whole blood and serum without the operator needing to specify sample type. Additionally, it shows very good correlation to a comparator assay that takes several hours and sample manipulation to yield results. This makes the Procise IFX assay ideal for obtaining fast and accurate IFX quantitation, thus allowing for immediate drug level dosing decisions to be made by the physician during patient treatment.

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Rapid and quantitative detection of COVID-19 markers in micro-liter sized samples

10.1101/2020.04.20.052233 ◽

2020 ◽

Cited By ~ 5

Author(s):

Xiaotian Tan ◽

Cory Lin ◽

Jie Zhang ◽

Maung Kyaw Khaing Oo ◽

Xudong Fan

Keyword(s):

Point Of Care ◽

False Negative ◽

False Negative Rate ◽

Quantitative Detection ◽

Enzyme Linked Immunosorbent Assay ◽

High False Negative Rate ◽

Conventional Elisa ◽

Assay Time ◽

Specific Igg ◽

Lateral Flow Test

AbstractCOVID-19 pandemic has caused tens of thousands of deaths and is now a severe threat to global health. Clinical practice has demonstrated that the SARS-CoV-2 S1 specific antibodies and viral antigens can be used as diagnostic and prognostic markers of COVID-19. However, the popular point-of-care biomarker detection technologies, such as the lateral-flow test strips, provide only yes/no information and have very limited sensitivities. Thus, it has a high false negative rate and cannot be used for the quantitative evaluation of patient’s immune response. Conventional ELISA (enzyme-linked immunosorbent assay), on the other hand, can provide quantitative, accurate, and sensitive results, but it involves complicated and expensive instruments and long assay time. In addition, samples need to be sent to centralized labs, which significantly increases the turn-around time. Here, we present a microfluidic ELISA technology for rapid (15-20 minutes), quantitative, sensitive detection of SARS-CoV-2 biomarkers using SARS-CoV-2 specific IgG and viral antigen – S protein in serum. We also characterized various humanized monoclonal IgG, and identified a candidate with a high binding affinity towards SARS-CoV-2 S1 protein that can serve as the calibration standard of anti-SARS-CoV-2 S1 IgG in serological analyses. Furthermore, we demonstrated that our microfluidic ELISA platform can be used for rapid affinity evaluation of monoclonal anti-S1 antibodies. The microfluidic ELISA device is highly portable and requires less than 10 μL of samples for each channel. Therefore, our technology will greatly facilitate rapid and quantitative analysis of COVID-19 patients and vaccine recipients at point-of-care.

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Polystyrene Microsphere-Based Immunochromatographic Assay for Detection of Aflatoxin B1 in Maize

Biosensors ◽

10.3390/bios11060200 ◽

2021 ◽

Vol 11 (6) ◽

pp. 200

Author(s):

Jin Wang ◽

Xiangmei Li ◽

Xing Shen ◽

Ang Zhang ◽

Jinxiu Liu ◽

...

Keyword(s):

Aflatoxin B1 ◽

Limit Of Detection ◽

Point Of Care ◽

Immunochromatographic Assay ◽

Quantitative Detection ◽

Point Of Care Testing ◽

Polystyrene Microsphere ◽

Accuracy And Precision ◽

Wide Range ◽

Liquid Chromatography Tandem Mass

Aflatoxin B1 (AFB1), a mycotoxin, is hepatotoxic, carcinogenic, and nephrotoxic in humans and animals, and contaminate a wide range of maize. In this study, an immunochromatographic assay (ICA) based on polystyrene microspheres (PMs) was developed for sensitive and quantitative detection of AFB1 in maize. The amounts of PMs, the condition for activating carboxyl groups of PMs, the amount of monoclonal antibody (mAb), and the volume of the immune probe were optimized to enhance the performance PMs-ICA for point-of-care testing of AFB1 in maize. The PMs-ICA showed the cut-off value of 1 ng/mL in phosphate buffer (PB) and 6 µg/kg in maize samples, respectively. The quantitative limit of detection (qLOD) was 0.27 and 1.43 µg/kg in PB and maize samples, respectively. The accuracy and precision of the PMs-ICA were evaluated by analysis of spiked maize samples with recoveries of 96.0% to 107.6% with coefficients of variation below 10%. In addition, the reliability of PMs-ICA was confirmed by the liquid chromatography-tandem mass spectrometry method. The results indicated that the PMs-ICA could be used as a sensitive, simple, rapid point-of-care testing of AFB1 in maize.

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Detector-Free Photothermal Bar-Chart Microfluidic Chips (PT-Chips) for Visual Quantitative Detection of Biomarkers

10.26434/chemrxiv.14131514 ◽

2021 ◽

Author(s):

Wan Zhou ◽

Guanglei Fu [email protected] ◽

Xiujun Li

Keyword(s):

Whole Blood ◽

Limit Of Detection ◽

Point Of Care ◽

Clinical Diagnostics ◽

Detection Sensitivity ◽

Quantitative Detection ◽

Microfluidic Chips ◽

Analytical Instruments ◽

Photothermal Effects ◽

On Chip

The volumetric bar-chart microfluidic chips (V-Chips) driven by chemical reaction-generated gas provide a promising platform for point-of-care (POC) visual biomarker quantitation. However, multiple limitations are encountered in conventional V-Chips, such as costly and complex chip fabrication, complicated assembly, and imprecise controllability of gas production. Herein, we introduced nanomaterial-mediated photothermal effects to V-Chips, and for the first time developed a new type of V-Chip, photothermal bar-chart microfluidic chip (PT-Chip), for visual quantitative detection of biochemicals without any bulky and costly analytical instruments. Immunosensing signals were converted to visual readout signals via photothermal effects, the on-chip bar-chart movements, enabling quantitative biomarker detection on a low-cost polymer hybrid PT-Chip with on-chip scale rulers. Four different human serum samples containing prostate-specific antigen (PSA) as a model analyte were detected simultaneously using the PT-Chip, with the limit of detection of 2.1 ng/mL, meeting clinical diagnostic requirements. Although no conventional signal detectors were used, it achieved comparable detection sensitivity to absorbance measurements with a microplate reader. The PT-Chip was further validated by testing human whole blood without the color interference problem, demonstrating good analytical performance of our method even in complex matrixes and thus the potential to fill a gap in current clinical diagnostics that is incapable of testing whole blood. This new PT-Chip driven by nanomaterial-mediated photothermal effects opens a new horizon of microfluidic platforms for instrument-free diagnostics at the point of care.

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Detector-Free Photothermal Bar-Chart Microfluidic Chips (PT-Chips) for Visual Quantitative Detection of Biomarkers

10.26434/chemrxiv.14131514.v1 ◽

2021 ◽

Author(s):

Wan Zhou ◽

Guanglei Fu [email protected] ◽

Xiujun Li

Keyword(s):

Whole Blood ◽

Limit Of Detection ◽

Point Of Care ◽

Clinical Diagnostics ◽

Detection Sensitivity ◽

Quantitative Detection ◽

Microfluidic Chips ◽

Analytical Instruments ◽

Photothermal Effects ◽

On Chip

The volumetric bar-chart microfluidic chips (V-Chips) driven by chemical reaction-generated gas provide a promising platform for point-of-care (POC) visual biomarker quantitation. However, multiple limitations are encountered in conventional V-Chips, such as costly and complex chip fabrication, complicated assembly, and imprecise controllability of gas production. Herein, we introduced nanomaterial-mediated photothermal effects to V-Chips, and for the first time developed a new type of V-Chip, photothermal bar-chart microfluidic chip (PT-Chip), for visual quantitative detection of biochemicals without any bulky and costly analytical instruments. Immunosensing signals were converted to visual readout signals via photothermal effects, the on-chip bar-chart movements, enabling quantitative biomarker detection on a low-cost polymer hybrid PT-Chip with on-chip scale rulers. Four different human serum samples containing prostate-specific antigen (PSA) as a model analyte were detected simultaneously using the PT-Chip, with the limit of detection of 2.1 ng/mL, meeting clinical diagnostic requirements. Although no conventional signal detectors were used, it achieved comparable detection sensitivity to absorbance measurements with a microplate reader. The PT-Chip was further validated by testing human whole blood without the color interference problem, demonstrating good analytical performance of our method even in complex matrixes and thus the potential to fill a gap in current clinical diagnostics that is incapable of testing whole blood. This new PT-Chip driven by nanomaterial-mediated photothermal effects opens a new horizon of microfluidic platforms for instrument-free diagnostics at the point of care.

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Fully integrated rapid microfluidic device translated from conventional 96-well ELISA kit

Scientific Reports ◽

10.1038/s41598-021-81433-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

M. Jalal Uddin ◽

Nabil H. Bhuiyan ◽

Joon S. Shim

Keyword(s):

Microfluidic Device ◽

Troponin I ◽

Limit Of Detection ◽

Point Of Care ◽

Sample Volume ◽

Elisa Test ◽

Enzyme Linked Immunosorbent Assay ◽

Fully Integrated ◽

Assay Time ◽

On Chip

AbstractIn this work, a fully integrated active microfluidic device transforming a conventional 96-well kit into point-of-care testing (POCT) device was implemented to improve the performance of traditional enzyme-linked immunosorbent assay (ELISA). ELISA test by the conventional method often requires the collection of 96 samples for its operation as well as longer incubation time from hours to overnight, whereas our proposed device conducts ELISA immediately individualizing a 96-well for individual patients. To do that, a programmable and disposable on-chip pump and valve were integrated on the device for precise control and actuation of microfluidic reagents, which regulated a reaction time and reagent volume to support the optimized protocols of ELISA. Due to the on-chip pump and valve, ELISA could be executed with reduced consumption of reagents and shortening the assay time, which are crucial for conventional ELISA using 96-well microplate. To demonstrate highly sensitive detection and easy-to-use operation, this unconventional device was successfully applied for the quantification of cardiac troponin I (cTnI) of 4.88 pg/mL using a minimum sample volume of 30 µL with a shorter assay time of 15 min for each ELISA step. The limit of detection (LOD) thus obtained was significantly improved than the conventional 96-well platform.

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PERFORMANCE CHARACTERISTICS OF A FRET-BASED IMMUNOASSAY FOR QUANTITATION OF ADALIMUMAB ON A POINT-OF-CARE INSTRUMENT SYSTEM

Inflammatory Bowel Diseases ◽

10.1093/ibd/izaa347.134 ◽

2021 ◽

Vol 27 (Supplement_1) ◽

pp. S56-S57

Author(s):

Edgar Ong ◽

Ruo Huang ◽

Richard Kirkland ◽

Michael Hale ◽

Larry Mimms

Keyword(s):

Whole Blood ◽

Limit Of Detection ◽

Point Of Care ◽

Quantitative Detection ◽

Therapeutic Drug ◽

Analytical Sensitivity ◽

Sample Type ◽

Hook Effect ◽

Limit Of Quantitation ◽

Assay Precision

Abstract Introduction A fast (<5 min), time-resolved fluorescence resonance energy transfer (FRET)-based immunoassay was developed for the quantitative detection of adalimumab (ADL) and biosimilars for use in therapeutic drug monitoring using only 20 µL of fingerstick whole blood or serum at the point-of-care. The Procise ADL assay and the ProciseDx analyzer are CE-marked. Studies were performed to characterize analytical performance of the Procise ADL assay on the ProciseDx analyzer. Methods Analytical testing was performed by spiking known amounts of ADL into negative serum and whole blood specimens. Analytical sensitivity was determined using limiting concentrations of ADL. Linearity was determined by testing ADL across the assay range. Hook effect was assessed at ADL concentrations beyond levels expected to be found within a patient. Testing of assay precision, cross-reactivity and potential interfering substances, and biosimilars was performed. The Procise ADL assay was also compared head-to-head with another CE-marked assay: LISA-TRACKER adalimumab ELISA test (Theradiag, France). The accuracy of the Procise ADL assay is established through calibrators and controls traceable to the WHO 1st International Standard for Adalimumab (NIBSC code: 17/236). Results The Procise ADL assay shows a Limit of Blank, Limit of Detection, and Lower Limit of Quantitation (LLoQ) of 0.1, 0.2, and 0.6 µg/mL in serum and 0.5, 0.9, and 1.3 µg/mL in whole blood, respectively. The linear assay range was determined to be 1.3 to 51.5 µg/mL in serum and whole blood. No hook effect was observed at an ADL concentration of 200 µg/mL as the value reported as “>ULoQ”. Assay precision testing across 10 days with multiple runs and reagent lots showed an intra-assay coefficient of variation (CV) of 2.8%, an inter-assay CV of ≤1.5%, and a total CV of 3.5%. The presence of potentially interfering/cross-reacting substances showed minimal impact on assay specificity with %bias within ±7.4% of control. Testing of biosimilars (adalimumab-atto and adalimumab-xxxx) showed good recovery. A good correlation to the Theradiag adalimumab ELISA was obtained for both serum (slope=0.94; r=0.99) and whole blood (slope=1.13; r=0.98) samples (Figure 1). Conclusion Results indicate that the Procise ADL assay is sensitive, specific, and precise yielding results within 5 minutes from both whole blood and serum without the operator needing to specify sample type. Additionally, it shows good correlation to a comparator assay that takes several hours and sample manipulation to yield results. This makes the Procise ADL assay ideal for obtaining fast and accurate ADL quantitation, thus allowing for immediate drug level dosing decisions to be made by the physician during patient treatment.

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Assessing Immunity by Quantitative Measurement of SARS-CoV-2 IgG Antibodies in Fingerstick Samples

10.1101/2021.12.21.21268075 ◽

2021 ◽

Author(s):

Javier T. Garza ◽

Jacob Quick ◽

Dev Chatterjee ◽

Robert Patrick Garr ◽

Atul Varadhachary ◽

...

Keyword(s):

Clinical Performance ◽

Point Of Care ◽

Treatment Options ◽

Antibody Test ◽

Cross Reactivity ◽

International Standards ◽

Antibody Concentration ◽

Immune Protection ◽

Igg Antibodies ◽

Individual Level

COVID-19 has affected billions of people around the world directly or indirectly. The response to the pandemic has focused on preventing the spread of the disease and improving treatment options. Diagnostic technologies have played a key role in this response since the beginning of the pandemic. As vaccines and other treatments have been developed and deployed, interest in understanding and measuring the individual level of immune protection has increased. Historically, use of antibody titers to measure systemic immunity has been constrained by an incomplete understanding of the relationship between antibodies and immunity, the lack of international standards for antibody concentration to enable cross-study comparisons, and insufficient clinical data to allow for the development of robust antibody-immunity models. However, these constraints have recently shifted. With a deeper understanding of antibodies, the promulgation of WHO antibody standards, and the development of immunity models using datasets from multiple COVID-19 vaccine trials, certain types of quantitative antibody tests may now provide a way to monitor individual or community immunity against COVID-19. Specifically, tests that quantitate the concentration of anti-RBD IgG -antibodies that target the receptor binding domain of the S1 spike protein component of the SARS-CoV-2 virus- show promise as a useful and scalable measure of the COVID-19 immunity of both individuals and communities. However, to fulfill this promise, a rapid and easy-to-administer test is needed. To address this important clinical need, Brevitest deployed its point-of-care-capable technology platform that can run a rapid (<15 minute), quantitative antibody test with a sample of 10 µl of whole blood from a fingerstick. The test we validated on this platform measures the concentration of anti-RBD IgG in Binding Antibody Units per milliliter (BAU/mL) per WHO Reference Standard NIBSC 20/136. In this paper, we present studies used to characterize the Brevitest anti-RBD IgG assay and evaluate its clinical performance, lower limits of measurement, precision, linearity, interference, and cross-reactivity. The results demonstrate the ability of this assay to measure a patient's anti-RBD IgG concentration. This information, together with models developed from recent COVID-19 vaccine clinical trials, can provide a means of assessing the current level of immune protection of an individual or community against COVID-19 infection.

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Electronic and electrochemical viral detection for point-of-care use: A systematic review

PLoS ONE ◽

10.1371/journal.pone.0258002 ◽

2021 ◽

Vol 16 (9) ◽

pp. e0258002

Author(s):

Solen Monteil ◽

Alexander J. Casson ◽

Samuel T. Jones

Keyword(s):

Systematic Review ◽

Field Effect ◽

Electrochemical Impedance ◽

Field Effect Transistors ◽

Limit Of Detection ◽

Point Of Care ◽

Evidence Synthesis ◽

Quantitative Detection ◽

Original Research ◽

Median Response

Detecting viruses, which have significant impact on health and the economy, is essential for controlling and combating viral infections. In recent years there has been a focus towards simpler and faster detection methods, specifically through the use of electronic-based detection at the point-of-care. Point-of-care sensors play a particularly important role in the detection of viruses. Tests can be performed in the field or in resource limited regions in a simple manner and short time frame, allowing for rapid treatment. Electronic based detection allows for speed and quantitative detection not otherwise possible at the point-of-care. Such approaches are largely based upon voltammetry, electrochemical impedance spectroscopy, field effect transistors, and similar electrical techniques. Here, we systematically review electronic and electrochemical point-of-care sensors for the detection of human viral pathogens. Using the reported limits of detection and assay times we compare approaches both by detection method and by the target analyte of interest. Compared to recent scoping and narrative reviews, this systematic review which follows established best practice for evidence synthesis adds substantial new evidence on 1) performance and 2) limitations, needed for sensor uptake in the clinical arena. 104 relevant studies were identified by conducting a search of current literature using 7 databases, only including original research articles detecting human viruses and reporting a limit of detection. Detection units were converted to nanomolars where possible in order to compare performance across devices. This approach allows us to identify field effect transistors as having the fastest median response time, and as being the most sensitive, some achieving single-molecule detection. In general, we found that antigens are the quickest targets to detect. We also observe however, that reports are highly variable in their chosen metrics of interest. We suggest that this lack of systematisation across studies may be a major bottleneck in sensor development and translation. Where appropriate, we use the findings of the systematic review to give recommendations for best reporting practice.

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Rapid Detection of Shellfish Major Allergen Tropomyosin Using Superparamagnetic Nanoparticle-Based Lateral Flow Immunoassay

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.311-313.436 ◽

2011 ◽

Vol 311-313 ◽

pp. 436-445 ◽

Cited By ~ 4

Author(s):

Liang Shi ◽

Xi Chang Wang ◽

Yuan Liu ◽

Ying Lu

Keyword(s):

Limit Of Detection ◽

Point Of Care ◽

High Specificity ◽

Major Allergen ◽

Lateral Flow ◽

Lateral Flow Immunoassay ◽

Quantitative Detection ◽

Point Of Care Test ◽

Western Blot Method ◽

Food Species

In this study, a competitive assay format using superparamagnetic nanoparticle-based lateral flow immunoassay (LFIA) was developed for rapid, quantitative detection of shellfish major allergen tropomyosin (Tm). Sartorius CN140 nitrocellulose membrane and 0.05mg/mL Tm immobilized in the test line (T line) were optimized in order to improve the performance of the LFIA system. Calibration curves for Tm under PBS-T diluents and carp muscle extraction diluents were established. Limit of detection (LOD) for Tm calibrated by carp muscle matrix was 12.4ng/mL with a work range of 0.01 to 20μg/mL. According to magnetic signals change with the time of sample flowing on the strip, the qualitative time of the LFIA was about 10min, while the quantitative time of the LFIA was about 25min. 30 food species were detected separately by the LFIA and Western blot method to evaluate the specificity of the LFIA. Overall relative agreement of the two methods was 96.7% (29/30). Moreover, intra- and inter-assay precisions of the LFIA for Tm detection were <10.20% and <12.34%, respectively. The average recovery range in different food matrices was 80.3~111.8%, within a reasonable range. Our data confirmed that the superparamagnetic nanoparticle-based LFIA method developed in this study is rapid, simple, high specificity and capable of quantitative test. Consequently, the LFIA has the potential application in the field of point-of-care test of shellfish major allergen Tm.

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