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

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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Therapeutic Drug Monitoring: Performance of a FRET-Based Point-Of-Care Immunoassay for the Quantitation of Infliximab and Adalimumab in Blood

10.26434/chemrxiv.13277321.v1 ◽

2020 ◽

Author(s):

Edgar Ong ◽

Ruo Huang ◽

Richard Kirkland ◽

Stefan Westin ◽

Jared Salbato ◽

...

Keyword(s):

Therapeutic Drug Monitoring ◽

Drug Monitoring ◽

Whole Blood ◽

Point Of Care ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Drug Level ◽

Analytical Performance ◽

Quantitative Detection ◽

Therapeutic Drug

Two fast (<5 min), time-resolved fluorescence resonance energy transfer (FRET)-based immunoassays (Procise IFX™ and Procise ADL™) were developed for the quantitative detection of infliximab (IFX), adalimumab (ADL), and their respective biosimilars for use in therapeutic drug monitoring (TDM) using 20 µL of finger prick whole blood at the point-of-care or whole blood/serum in a central lab. Studies were performed to characterize analytical performance of the Procise IFX and the Procise ADL assays on the ProciseDx™ analyzer. The Procise IFX and Procise ADL assays both showed good analytical performance with respect to sensitivity, specificity, linearity, and precision suitable for routine clinical use as well as excellent correlation to current commercial ELISA IFX and ADL measurement methods. Results indicated that the Procise IFX and Procise ADL assays are sensitive, specific, and precise yielding results in less than 5 minutes from either whole blood or serum. This indicates the Procise IFX and Procise ADL assays are useful for obtaining fast and accurate IFX or ADL quantitation, thus avoiding delays inherent to current methods and enabling immediate drug level dosing decisions to be made during a single patient visit.

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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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P384 Therapeutic drug monitoring: performance of the first lateral flow-based Point of Care test for the quantification of infliximab in a finger prick

Journal of Crohn s and Colitis ◽

10.1093/ecco-jcc/jjab076.508 ◽

2021 ◽

Vol 15 (Supplement_1) ◽

pp. S396-S397

Author(s):

A Ametzazurra ◽

J Pascual ◽

L Del Rio ◽

A Maguregui ◽

D Nagore ◽

...

Keyword(s):

Therapeutic Drug Monitoring ◽

Drug Monitoring ◽

Whole Blood ◽

Limit Of Detection ◽

Point Of Care ◽

Package Insert ◽

Lateral Flow ◽

Therapeutic Drug ◽

Limit Of Quantification ◽

Finger Prick

Abstract Background Promonitor Quick IFX is a lateral flow test (LFT) for the quantification of infliximab (IFX) in human whole blood (finger prick or venous) or serum in 20 minutes. This LFT is based on a sandwich immunoassay to quantify either the reference IFX or biosimilars. This abstract describes the studies performed to establish the analytical specifications of the product. Methods Clinical and Laboratory Standards Institute (CLSI) guidelines were followed for the evaluation of the analytical specifications of the LFT in whole blood and serum matrices: Linearity (EP-06-A), Detection capability (EP17-A2), Interfering substances (EP07, 3rd Edition), Intermediate precision (EP05-A3) and Bias evaluation study for Biosimilars (EP10-A3). Results were obtained in combination with the automated portable reader PQreader. A Datamatrix provided with each Promonitor Quick IFX kit contains the calibration information required for the PQreader to measure the Control and Test lines and report the IFX concentration. Results The linear assay range was determined to be 1-58 µg/mL in whole-blood and 0.6-67 µg/mL in serum according to the processes indicated in the Package Insert. The Limit of Blank is 0.8 μg/mL, the Limit of Detection and Lower Limit of Quantification (LLoQ) are 1.1 μg/mL, and the Upper Limit of Quantification (ULoQ) is 15.4 μg/mL. There was no effect on assay performance when each of the following substances were added to samples with 0, 3, and 7 μg/mL of IFX: Haemoglobin (>1000 mg/dL), Bilirubin (>40 mg/dL), Triglycerides (>1500 mg/dL), HAMA (160 AU/mL), Rheumatoid factor (200 IU/mL), EDTA (5.4 mg/mL), Heparin (51 U/mL), Citrate (11.4%), Vedolizumab (60 μg/mL) and Adalimumab (20.25 μg/mL). Repeatability and within-device precision results obtained for the positive samples are shown in the table below. The negative samples showed a negative result in all the measurements. A bias study showed that Promonitor Quick IFX can quantify CT-P13, SB2 and GP1111 biosimilars throughout the measurement range with a maximum bias of 14%. Conclusion Promonitor Quick IFX is the first LFT available for true Point of Care testing of patients treated with IFX with just a finger prick sample. It provides quick turnaround time to facilitate therapeutic drug monitoring and aid immediate decision making in the doctor office or hospitals with an excellent analytical performance.

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Therapeutic Drug Monitoring in Buprenorphine/Naloxone Treatment for Opioid Use Disorder: Clinical Feasibility and Optimizing Assay Precision

Pharmacopsychiatry ◽

10.1055/a-1083-6842 ◽

2020 ◽

Vol 53 (03) ◽

pp. 115-121

Author(s):

Hesham Farouk Elarabi ◽

Nael Hasan ◽

John Marsden ◽

Doaa Radwan ◽

Abdu Adem ◽

...

Keyword(s):

Therapeutic Drug Monitoring ◽

Drug Monitoring ◽

Maintenance Treatment ◽

Limit Of Detection ◽

Opioid Use Disorder ◽

Therapeutic Drug ◽

Opioid Use ◽

Limit Of Quantitation ◽

Trough Concentrations ◽

Assay Precision

Abstract Introduction Compliance with sublingual buprenorphine/naloxone (SL-BUP/NX) is associated with higher abstinence from illicit opioid use. Therapeutic drug monitoring (TDM) has been recommended for adherence monitoring of buprenorphine (BUP) maintenance treatment for opioid use disorder (OUD), but to date there have been no reported clinical applications. In this TDM feasibility study, we investigated BUP assay precision in 15 adults with OUD who had been stabilized on buprenorphine/naloxone. Methods Using solid phase extraction, BUP recovery was contrasted at 100 mMol and 1 Molar of acetic acid wash solution. Precision was determined by applying the condition generating highest recovery using 0.2 ng/mL and 10 ng/mL standards. Four blood samples were drawn to examine the BUP peak and trough plasma concentrations, and BUP elimination rate was estimated. BUP recovery was examined again in a random sample and contrasted with the concentration predicted applying first-order kinetics. Results Higher BUP recovery was achieved with 1 Molar wash (94.3%; p=0.05). Precision ranged from 15–20%. The estimated limit of detection (LoD) and limit of quantitation (LoQ) were 0.02 and 0.069 ng/mL, respectively. BUP peak and trough concentrations were successfully examined, and BUP trough concentrations were replicated confirming steady state. BUP concentrations were predicted at a variance of −7.20% to 1.54 %. Conclusions TDM for BUP maintenance treatment of OUD is feasible, and simple adjustment of the assay conditions enhances BUP recovery.

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Therapeutic Drug Monitoring: Performance of a FRET-Based Point-Of-Care Immunoassay for the Quantitation of Infliximab and Adalimumab in Blood

10.26434/chemrxiv.13277321 ◽

2020 ◽

Author(s):

Edgar Ong ◽

Ruo Huang ◽

Richard Kirkland ◽

Stefan Westin ◽

Jared Salbato ◽

...

Keyword(s):

Therapeutic Drug Monitoring ◽

Drug Monitoring ◽

Whole Blood ◽

Point Of Care ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Drug Level ◽

Analytical Performance ◽

Quantitative Detection ◽

Therapeutic Drug

Two fast (<5 min), time-resolved fluorescence resonance energy transfer (FRET)-based immunoassays (Procise IFX™ and Procise ADL™) were developed for the quantitative detection of infliximab (IFX), adalimumab (ADL), and their respective biosimilars for use in therapeutic drug monitoring (TDM) using 20 µL of finger prick whole blood at the point-of-care or whole blood/serum in a central lab. Studies were performed to characterize analytical performance of the Procise IFX and the Procise ADL assays on the ProciseDx™ analyzer. The Procise IFX and Procise ADL assays both showed good analytical performance with respect to sensitivity, specificity, linearity, and precision suitable for routine clinical use as well as excellent correlation to current commercial ELISA IFX and ADL measurement methods. Results indicated that the Procise IFX and Procise ADL assays are sensitive, specific, and precise yielding results in less than 5 minutes from either whole blood or serum. This indicates the Procise IFX and Procise ADL assays are useful for obtaining fast and accurate IFX or ADL quantitation, thus avoiding delays inherent to current methods and enabling immediate drug level dosing decisions to be made during a single patient visit.

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Therapeutic Drug Monitoring: Performance of a FRET-Based Point-Of-Care Immunoassay for the Quantitation of Infliximab and Adalimumab in Blood

10.26434/chemrxiv.13277321.v2 ◽

2020 ◽

Author(s):

Edgar Ong ◽

Ruo Huang ◽

Richard Kirkland ◽

Stefan Westin ◽

Jared Salbato ◽

...

Keyword(s):

Therapeutic Drug Monitoring ◽

Drug Monitoring ◽

Whole Blood ◽

Point Of Care ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Drug Level ◽

Analytical Performance ◽

Quantitative Detection ◽

Therapeutic Drug

Two fast (<5 min), time-resolved fluorescence resonance energy transfer (FRET)-based immunoassays (Procise IFX™ and Procise ADL™) were developed for the quantitative detection of infliximab (IFX), adalimumab (ADL), and their respective biosimilars for use in therapeutic drug monitoring (TDM) using 20 µL of finger prick whole blood at the point-of-care or whole blood/serum in a central lab. Studies were performed to characterize analytical performance of the Procise IFX and the Procise ADL assays on the ProciseDx™ analyzer. The Procise IFX and Procise ADL assays both showed good analytical performance with respect to sensitivity, specificity, linearity, and precision suitable for routine clinical use as well as excellent correlation to current commercial ELISA IFX and ADL measurement methods. Results indicated that the Procise IFX and Procise ADL assays are sensitive, specific, and precise yielding results in less than 5 minutes from either whole blood or serum. This indicates the Procise IFX and Procise ADL assays are useful for obtaining fast and accurate IFX or ADL quantitation, thus avoiding delays inherent to current methods and enabling immediate drug level dosing decisions to be made during a single patient visit.

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Quantitative Detection of Clostridium perfringens in the Broiler Fowl Gastrointestinal Tract by Real-Time PCR

Applied and Environmental Microbiology ◽

10.1128/aem.71.7.3911-3916.2005 ◽

2005 ◽

Vol 71 (7) ◽

pp. 3911-3916 ◽

Cited By ~ 71

Author(s):

Mark G. Wise ◽

Gregory R. Siragusa

Keyword(s):

Gastrointestinal Tract ◽

Real Time ◽

Clostridium Perfringens ◽

Real Time Pcr ◽

Limit Of Detection ◽

Quantitative Detection ◽

Necrotic Enteritis ◽

Analytical Sensitivity ◽

Quantitative Real Time Pcr ◽

Pcr Inhibitor

ABSTRACT Strains of Clostridium perfringens are a frequent cause of food-borne disease and gas gangrene and are also associated with necrotic enteritis in chickens. To detect and quantify the levels of C. perfringens in the chicken gastrointestinal tract, a quantitative real-time PCR assay utilizing a fluorogenic, hydrolysis-type probe was developed and utilized to assay material retrieved from the broiler chicken cecum and ileum. Primers and probe were selected following an alignment of 16S rDNA sequences from members of cluster I of the genus Clostridium, and proved to be specific for C. perfringens. The assay could detect approximately 50 fg of C. perfringens genomic DNA and approximately 20 cells in pure culture. Measurements of the analytical sensitivity determined with spiked intestinal contents indicated that the consistent limit of detection with ileal samples was approximately 102 CFU/g of ileal material, but only about 104 CFU/g of cecal samples. The decreased sensitivity with the cecal samples was due to the presence of an unidentified chemical PCR inhibitor(s) in the cecal DNA purifications. The assay was utilized to rapidly detect and quantify C. perfringens levels in the gut tract of broiler chickens reared without supplementary growth-promoting antibiotics that manifested symptoms of necrotic enteritis. The results illustrated that quantitative real-time PCR correlates well with quantification via standard plate counts in samples taken from the ileal region of the gastrointestinal tract.

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Whole Blood Immunoassay Based on Centrifugal Bead Sedimentation

Clinical Chemistry ◽

10.1373/clinchem.2011.162206 ◽

2011 ◽

Vol 57 (5) ◽

pp. 753-761 ◽

Cited By ~ 44

Author(s):

Ulrich Y Schaff ◽

Greg J Sommer

Keyword(s):

Whole Blood ◽

Single Channel ◽

Limit Of Detection ◽

Point Of Care ◽

Low Cost ◽

Microfluidic System ◽

Blood Samples ◽

Reactive Protein ◽

Immunoassay Technique ◽

Microfluidic Immunoassay

BACKGROUND Centrifugal “lab on a disk” microfluidics is a promising avenue for developing portable, low-cost, automated immunoassays. However, the necessity of incorporating multiple wash steps results in complicated designs that increase the time and sample/reagent volumes needed to run assays and raises the probability of errors. We present proof of principle for a disk-based microfluidic immunoassay technique that processes blood samples without conventional wash steps. METHODS Microfluidic disks were fabricated from layers of patterned, double-sided tape and polymer sheets. Sample was mixed on-disk with assay capture beads and labeling antibodies. Following incubation, the assay beads were physically separated from the blood cells, plasma, and unbound label by centrifugation through a density medium. A signal-laden pellet formed at the periphery of the disk was analyzed to quantify concentration of the target analyte. RESULTS To demonstrate this technique, the inflammation biomarkers C-reactive protein and interleukin-6 were measured from spiked mouse plasma and human whole blood samples. On-disk processing (mixing, labeling, and separation) facilitated direct assays on 1-μL samples with a 15-min sample-to-answer time, <100 pmol/L limit of detection, and 10% CV. We also used a unique single-channel multiplexing technique based on the sedimentation rate of different size or density bead populations. CONCLUSIONS This portable microfluidic system is a promising method for rapid, inexpensive, and automated detection of multiple analytes directly from a drop of blood in a point-of-care setting.

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