Theoretical and experimental assessment of a novel method to establish the complete measurement range of the calorimeter and its limit of detection and quantification

Abstract Background Molecular assays are important tools for pathogen detection but need to be periodically re-evaluated with the discovery of additional genetic diversity that may cause assays to exclude target taxa or include non-target taxa. A single well-developed assay can find broad application across research, clinical, and industrial settings. Pathogen prevalence within a population is estimated using such assays and accurate results are critical for formulating effective public health policies and guiding future research. A variety of assays for the detection of Staphylococcus aureus are currently available. The utility of commercial assays for research is limited, given proprietary signatures and lack of transparent validation. Results In silico testing of existing peer-reviewed assays show that most suffer from a lack of sensitivity and specificity. We found no assays that were specifically designed and validated for quantitative use. Here we present a qPCR assay, SaQuant, for the detection and quantification of S. aureus as might be collected on sampling swabs. Sensitivity and specificity of the assay was 95.6 and 99.9 %, respectively, with a limit of detection of between 3 and 5 genome equivalents and a limit of quantification of 8.27 genome equivalents. The presence of DNA from non-target species likely to be found in a swab sample, did not impact qualitative or quantitative abilities of the assay. Conclusions This assay has the potential to serve as a valuable tool for the accurate detection and quantification of S. aureus collected from human body sites in order to better understand the dynamics of prevalence and transmission in community settings.

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Expanding Assay Dynamics: A Combined Competitive and Direct Assay System for the Quantification of Proteins in Multiplexed Immunoassays

Clinical Chemistry ◽

10.1373/clinchem.2007.099812 ◽

2008 ◽

Vol 54 (6) ◽

pp. 956-963 ◽

Cited By ~ 17

Author(s):

Michael Hartmann ◽

Monika Schrenk ◽

Anette Döttinger ◽

Sarah Nagel ◽

Johan Roeraade ◽

...

Keyword(s):

Dynamic Range ◽

Protein Microarray ◽

Measurement Range ◽

Multiplex Assay ◽

Competitive System ◽

Direct Immunoassay ◽

Sandwich Configuration ◽

Readout Channel ◽

Autoantibody Detection ◽

Detection And Quantification

Abstract Background: The concurrent detection and quantification of analytes that vary widely in concentration present a principal problem in multiplexed assay systems. Combining competitive and sandwich immunoassays permits coverage of a wide concentration range, and both highly abundant molecules and analytes present in low concentration can be quantified within the same assay. Methods: The use of different fluorescence readout channels allows the parallel use of a competitive system and a sandwich configuration. The 2 generated assay signals are combined and used to calculate the amount of analyte. The measurement range can be adjusted by varying the competitor concentration, and an extension of the assay system’s dynamic range is possible. Results: We implemented the method in a planar protein microarray–based autoimmune assay to detect autoantibodies against 13 autoantigens and to measure the concentration of a highly abundant protein, total human IgG, in one assay. Our results for autoantibody detection and IgG quantification agreed with results obtained with commercially available assays. The use of 2 readout channels in the protein microarray–based system reduced spot-to-spot variation and intraassay variation. Conclusions: By combining a direct immunoassay with a competitive system, analytes present in widely varying concentrations can be quantified within a single multiplex assay. Introducing a second readout channel for analyte quantification is an effective tool for spot-to-spot normalization and helps to lower intraassay variation.

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Determination of Triglycidyl Isocyanurate in Workplace Air

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph16224455 ◽

2019 ◽

Vol 16 (22) ◽

pp. 4455

Author(s):

Anna Jeżewska ◽

Joanna Kowalska

Keyword(s):

High Performance ◽

Limit Of Detection ◽

Crosslinking Agent ◽

Measurement Range ◽

Array Detector ◽

Phase System ◽

Limit Of Quantification ◽

Workplace Air ◽

Air Sample ◽

Triglycidyl Isocyanurate

Triglycidyl isocyanurate (TGIC) is a white solid in powder or granular form. TGIC does not occur naturally in the environment. It is intentionally manufactured and used as a crosslinking agent or hardener to produce polyester powder coatings. TGIC may cause genetic defects. This article presents the method of TGIC determination in workplace air using high-performance liquid chromatography (HPLC) with a diode-array detector (DAD). The method is based on the collection of TGIC present in the air on a polypropylene filter, extraction with acetonitrile, and chromatographic analysis of the solution obtained in this way. The determination was carried out in the reverse-phase system (mobile phase: acetonitrile: water) using an Ultra C18 column. The measurement range is 2 to 40 µg/m3 for a 720 liters air sample. Limit of detection (LOD) is 23 ng/m3 and limit of quantification (LOQ): 70 ng/m3. The method can be used for assessing occupational exposure to TGIC and associated risk to workers’ health.

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A Sensitive and Cost-Effective Chemiluminescence ELISA for Measurement of Amyloid-β 1-42 Peptide in Human Plasma

Journal of Alzheimer s Disease ◽

10.3233/jad-200861 ◽

2020 ◽

Vol 78 (3) ◽

pp. 1237-1244

Author(s):

Pankaj D. Mehta ◽

Bruce A. Patrick ◽

David L. Miller ◽

Patricia K. Coyle ◽

Thomas Wisniewski

Keyword(s):

Human Plasma ◽

Single Molecule ◽

Limit Of Detection ◽

Sandwich Elisa ◽

Amyloid Β ◽

Cost Effective ◽

Measurement Range ◽

Rabbit Monoclonal Antibody ◽

Low Levels ◽

The Brain

Background: Amyloid-β42 (Aβ42) is associated with plaque formation in the brain of patients with Alzheimer’s disease (AD). Studies have suggested the potential utility of plasma Aβ42 levels in the diagnosis, and in longitudinal study of AD pathology. Conventional ELISAs are used to measure Aβ42 levels in plasma but are not sensitive enough to quantitate low levels. Although ultrasensitive assays like single molecule array or immunoprecipitation-mass spectrometry have been developed to quantitate plasma Aβ42 levels, the high cost of instruments and reagents limit their use. Objective: We hypothesized that a sensitive and cost-effective chemiluminescence (CL) immunoassay could be developed to detect low Aβ42 levels in human plasma. Methods: We developed a sandwich ELISA using high affinity rabbit monoclonal antibody specific to Aβ42. The sensitivity of the assay was increased using CL substrate to quantitate low levels of Aβ42 in plasma. We examined the levels in plasma from 13 AD, 25 Down syndrome (DS), and 50 elderly controls. Results: The measurement range of the assay was 0.25 to 500 pg/ml. The limit of detection was 1 pg/ml. All AD, DS, and 45 of 50 control plasma showed measurable Aβ42 levels. Conclusion: This assay detects low levels of Aβ42 in plasma and does not need any expensive equipment or reagents. It offers a preferred alternative to ultrasensitive assays. Since the antibodies, peptide, and substrate are commercially available, the assay is well suited for academic or diagnostic laboratories, and has a potential for the diagnosis of AD or in clinical trials.

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Development of Quantitative Real-Time PCR Assays for Detection and Quantification of Surrogate Biological Warfare Agents in Building Debris and Leachate

Applied and Environmental Microbiology ◽

10.1128/aem.00779-07 ◽

2007 ◽

Vol 73 (20) ◽

pp. 6557-6565 ◽

Cited By ~ 38

Author(s):

Pascal E. Saikaly ◽

Morton A. Barlaz ◽

Francis L. de los Reyes

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Genomic Dna ◽

Dynamic Range ◽

Limit Of Detection ◽

Fate And Transport ◽

Biological Warfare ◽

Quantitative Real Time Pcr ◽

Pcr Assays ◽

Detection And Quantification

ABSTRACT Evaluation of the fate and transport of biological warfare (BW) agents in landfills requires the development of specific and sensitive detection assays. The objective of the current study was to develop and validate SYBR green quantitative real-time PCR (Q-PCR) assays for the specific detection and quantification of surrogate BW agents in synthetic building debris (SBD) and leachate. Bacillus atrophaeus (vegetative cells and spores) and Serratia marcescens were used as surrogates for Bacillus anthracis (anthrax) and Yersinia pestis (plague), respectively. The targets for SYBR green Q-PCR assays were the 16S-23S rRNA intergenic transcribed spacer (ITS) region and recA gene for B. atrophaeus and the gyrB, wzm, and recA genes for S. marcescens. All assays showed high specificity when tested against 5 ng of closely related Bacillus and Serratia nontarget DNA from 21 organisms. Several spore lysis methods that include a combination of one or more of freeze-thaw cycles, chemical lysis, hot detergent treatment, bead beat homogenization, and sonication were evaluated. All methods tested showed similar threshold cycle values. The limit of detection of the developed Q-PCR assays was determined using DNA extracted from a pure bacterial culture and DNA extracted from sterile water, leachate, and SBD samples spiked with increasing quantities of surrogates. The limit of detection for B. atrophaeus genomic DNA using the ITS and B. atrophaeus recA Q-PCR assays was 7.5 fg per PCR. The limits of detection of S. marcescens genomic DNA using the gyrB, wzm, and S. marcescens recA Q-PCR assays were 7.5 fg, 75 fg, and 7.5 fg per PCR, respectively. Quantification of B. atrophaeus vegetative cells and spores was linear (R 2 > 0.98) over a 7-log-unit dynamic range down to 101 B. atrophaeus cells or spores. Quantification of S. marcescens (R 2 > 0.98) was linear over a 6-log-unit dynamic range down to 102 S. marcescens cells. The developed Q-PCR assays are highly specific and sensitive and can be used for monitoring the fate and transport of the BW surrogates B. atrophaeus and S. marcescens in building debris and leachate.

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Performance Evaluation of CEA Assay on Beckman Coulter UniCel DxI 800

American Journal of Clinical Pathology ◽

10.1093/ajcp/aqz112.038 ◽

2019 ◽

Vol 152 (Supplement_1) ◽

pp. S20-S20

Author(s):

Qing Wei ◽

Robert Hardy ◽

Liyun Cao

Keyword(s):

Reference Range ◽

Limit Of Detection ◽

Measurement Range ◽

Surgical Removal ◽

Primary Colorectal Cancer ◽

Serum Cea ◽

The Mean ◽

Precision And Accuracy ◽

Patient Prognosis ◽

High Level

Abstract Carcinoembryonic antigen (CEA) is a glycoprotein normally found in embryonic entodermal epithelium. Increased levels may be found in patients with primary colorectal cancer or other malignancies. CEA levels are not useful in screening the general population for undetected cancers. However, CEA levels provide important information about patient prognosis, recurrence of tumors after surgical removal, and effectiveness of therapy. The aim of this study was to assess the analytical performance of CEA assay on a Beckman Coulter UniCel DxI 800. The linearity, sensitivity, reference range, precision, and accuracy were evaluated following CLSI guidelines. The within-run and between-run precisions were assessed by analyzing QC material at low and high levels of concentrations. The accuracy was assessed by comparison of 152 patient serum CEA on DXI with CEA on a previously established ADVIA Centaur System. The analytical measurement range was determined to be linear between 0.00 and 951.3 ng/mL with a slope of 0.976 and intercept of 0.075. The limit of blank (LOB) was determined as 0.03 ng/mL and the limit of detection (LOD) was 0.1 ng/mL. The reference range was verified as 0.0 to 3.0 ng/mL (nonsmokers) and 0.0 to 5.0 ng/mL (smokers). The within-run CVs for CEA were 4.4% at the low level of 2.239 ng/mL and 2.5% at the high level of 38.1 ng/mL. The between-run CVs at low and high levels were 4.4%, and 4.0%, respectively. Comparison of CEA on the Beckman Coulter UniCel DxI 800 with CEA on the ADVIA Centaur System of 152 patient samples showed the slope was 1.541 (95% CI, 1.515-1.567) with an intercept of 0.17 and a correlation coefficient of 0.9943 (Deming). The mean bias between Beckman and ADVIA was 6.49 (41.71%). In summary, our data demonstrate that CEA assay on the Beckman Coulter UniCel DxI 800 has good linearity and precision. There is also good correlation between CEA tested on the Beckman Coulter UniCel DxI 800 and on the ADVIA Centaur System with a positive bias.

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Carbon and Tin-Based Polyacrylonitrile Hybrid Architecture Solid Phase Microextraction Fiber for the Detection and Quantification of Antibiotic Compounds in Aqueous Environmental Systems

Molecules ◽

10.3390/molecules24091670 ◽

2019 ◽

Vol 24 (9) ◽

pp. 1670 ◽

Cited By ~ 4

Author(s):

Sandip Mondal ◽

Jialing Jiang ◽

Yin Li ◽

Gangfeng Ouyang

Keyword(s):

Solid Phase Microextraction ◽

Tin Dioxide ◽

Limit Of Detection ◽

Solid Phase ◽

Tap Water ◽

Coefficient Of Determination ◽

Lower Amount ◽

Custom Made ◽

Relative Standard ◽

Detection And Quantification

In this study, the detection and quantification of multiple classes of antibiotics in water matrices are proposed using a lab-made solid phase microextraction (SPME) fiber coupled with high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). The lab-made fiber was prepared using a graphene oxide (G), carbon nanotubes (C), and tin dioxide (T) composite, namely GCT, with polyacrylonitrile (PAN) as supporting material. The detected antibiotics were enrofloxacin, sulfathiazole, erythromycin, and trimethoprim. The custom-made fiber was found to be superior compared with a commercial C18 fiber. The excellent reproducibility and lower intra-fiber relative standard deviations (RSDs 1.8% to 6.8%) and inter-fiber RSDs (4.5% to 8.8%) made it an ideal candidate for the detection of traces of antibiotics in real environmental samples. The proposed validated method provides a satisfactory limit of detection and good linear ranges with higher (>0.99) coefficient of determination in the aqueous system. Application of the method was made in different real water systems such as river, pond and tap water using the standard spiking method. Excellent sensitivity, reproducibility, lower amount of sample detection and higher recovery was found in a real water sample. Therefore, the extraction method was successfully applied to the detection and quantification of multiple classes of antibiotics in different aqueous systems with satisfactory results.

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Development and validation of a HPLC-UV method for the simultaneous detection and quantification of paclitaxel and sulforaphane in lipid based self-microemulsifying formulation

Journal of Chromatographic Science ◽

10.1093/chromsci/bmz068 ◽

2019 ◽

Vol 57 (10) ◽

pp. 931-938 ◽

Cited By ~ 1

Author(s):

Mohammad M Kamal ◽

Sami Nazzal

Keyword(s):

Analytical Method ◽

Mobile Phase ◽

High Performance ◽

Limit Of Detection ◽

Simultaneous Detection ◽

Reversed Phase ◽

Limit Of Quantitation ◽

Development And Validation ◽

Simultaneous Delivery ◽

Detection And Quantification

Abstract Paclitaxel (PTX) and sulforaphane (SFN) are known anticancer molecules. Their activity was found to be potentiated when tested concurrently. Only recently, however, a novel SFN enabled PTX self-microemulsifying formulation (SMEDDS) was developed for their simultaneous delivery. This necessitated the development of an analytical method for the simultaneous detection and quantitation of PTX and SFN. In this study, a simple and sensitive isocratic high performance liquid chromatography-ultraviolet (HPLC-UV) analytical method was developed and validated per International Conference on Harmonization guidelines to satisfy this objective. Its application was demonstrated when quantifying the amount of PTX and SFN released from the SMEDDS in various dissolution media. The separation of the analytes was performed with the aid of a reversed phase C18 column at ambient temperature using a 60:40 mixture of acetonitrile and KH2PO4 buffer (pH 5.0) as the mobile phase. PTX and SFN peaks were detected at 202 nm with high resolution without interference from excipients. This method showed linearity within 2.5–100 μg/mL range with r2 > 0.999. The limit of detection and lower limit of quantitation were 0.1638 and 0.4964 μg/mL for PTX and 0.4419 and 1.3389 μg/mL for SFN, respectively. A total of 98–101% of the injected samples was recovered with RSD of 0.06–0.68% indicating the suitability of the method for the simultaneous detection and quantitation of the molecules in dissolution media.

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Sensitive Spectrophotometric Determinations of Paracetamol and Protriptyline HCl Using 3-Chloro-7-hydroxy-4-methyl-2H-chromen-2-one

ISRN Spectroscopy ◽

10.1155/2013/935819 ◽

2013 ◽

Vol 2013 ◽

pp. 1-6 ◽

Cited By ~ 1

Author(s):

Kumble Divya ◽

Badiadka Narayana ◽

Majal Sapnakumari

Keyword(s):

Charge Transfer ◽

Spectrophotometric Method ◽

Charge Transfer Complex ◽

Limit Of Detection ◽

Pharmaceutical Formulations ◽

Molar Absorptivity ◽

Pharmaceutical Samples ◽

Chromogenic Reagent ◽

Detection And Quantification

A new spectrophotometric method is developed for the determination of Paracetamol (PCT) and protriptyline HCl (PTP) in pure forms and in pharmaceutical formulations. The experiment involves the use of 3-chloro-7-hydroxy-4-methyl-2H-chromen-2-one as a novel chromogenic reagent for the determination of PCT and PTP. The method is based on the formation of charge transfer complex between the drugs and chromogenic reagent. Beer's law is obeyed in the concentration ranges 10.00–60.00 µg mL−1 for PCT at 545 nm and 40.00–160.00 µg mL−1 for PTP at 468 nm. The molar absorptivity, Sandell, sensitivity, and limit of detection and quantification are also calculated. The method has been successfully applied for the determination of both PCT and PTP in pharmaceutical samples with acceptable results.

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