scholarly journals Evaluation of a novel multiplexed assay for determining IgG levels and functional activity to SARS-CoV-2

2020 ◽  
Author(s):  
Marina Johnson ◽  
Helen R. Wagstaffe ◽  
Kimberly C. Gilmour ◽  
Annabelle Lea Mai ◽  
Joanna Lewis ◽  
...  
Keyword(s):  
Solid Phase ◽  
Simultaneous Detection ◽  
Serum Samples ◽  
Meso Scale Discovery ◽  
Assay Sensitivity ◽  
Rigorous Evaluation ◽  
Specificity And Sensitivity ◽  
Igg Binding ◽  
Control Serum ◽  
Multiplexed Immunoassay

AbstractBackgroundThe emergence of SARS-CoV-2 has led to the development of new serological assays that could aid in diagnosis and evaluation of seroprevalence to inform an understanding of the burden of COVID-19 disease. Many available tests lack rigorous evaluation and therefore results may be misleading.ObjectivesThe aim of this study was to assess the performance of a novel multiplexed immunoassay for the simultaneous detection of antibodies against SARS-CoV-2 trimeric spike (S), spike receptor binding domain (RBD), spike N terminal domain and nucleocapsid antigen and a novel pseudo-neutralisation assay.MethodsA multiplexed solid-phase chemiluminescence assay (Meso Scale Discovery) was evaluated for the simultaneous detection of IgG binding to four SARS-CoV-2 antigens and the quantification of antibody-induced ACE-2 binding inhibition (pseudo-neutralisation assay). Sensitivity was evaluated with a total of 196 COVID-19 serum samples (169 confirmed PCR positive and 27 anti-nucleocapsid IgG positive) from individuals with mild symptomatic or asymptomatic disease. Specificity was evaluated with 194 control serum samples collected from adults prior to December 2019.ResultsThe specificity and sensitivity of the binding IgG assay was highest for S protein with a specificity of 97.4% and sensitivity of 96.2% for samples taken 14 days and 97.9% for samples taken 21 days following the onset of symptoms. IgG concentration to S and RBD correlated strongly with percentage inhibition measured by the pseudo-neutralisation assay.ConclusionExcellent sensitivity for IgG detection was obtained over 14 days since onset of symptoms for three SARS-CoV-2 antigens (S, RBD and N) in this multiplexed assay which can also measure antibody functionality.

scholarly journals Simultaneous Detection of Eight Analytes in Human Serum by Two Commercially Available Platforms for Multiplex Cytokine Analysis

2007 ◽  
Vol 15 (1) ◽  
pp. 42-48 ◽  
Author(s):  
Gary Toedter ◽  
Karen Hayden ◽  
Carrie Wagner ◽  
Carrie Brodmerkel
Keyword(s):  
Simultaneous Detection ◽  
Tissue Level ◽  
Careful Examination ◽  
Serum Samples ◽  
Meso Scale Discovery ◽  
Necrosis Factor Alpha ◽  
Monocyte Chemoattractant Protein 1 ◽  
Inflammatory Protein ◽  
Cytokine Analysis ◽  
Endothelial Growth Factor

ABSTRACT The accurate detection and quantitation of cytokines in serum are important in the study of disease mechanisms, pathogenesis, and treatment. Serum cytokines can reflect processes that are occurring at the cellular or tissue level and thus provide a means of indirectly monitoring these processes. Multiplex detection of cytokines allows the simultaneous measurement of multiple cytokines in a sample, increasing the efficiency of measuring the cytokines while reducing the serum sample volumes required for the testing. Two commercially available multiplex platforms were evaluated (Pierce SearchLight and Meso Scale Discovery), using multiplexes capable of simultaneously detecting eight cytokines. The cytokines analyzed in this study were gamma interferon, vascular endothelial growth factor, tumor necrosis factor alpha, interleukin-6 (IL-6), macrophage inflammatory protein 1β, monocyte chemoattractant protein 1, IL-12p40, and IL-4. The range of quantitation of the platforms, the recovery of spiked cytokines, and the detection of the cytokines in serum samples from subjects with ulcerative colitis, Crohn's disease, rheumatoid arthritis, and psoriasis were examined. The findings showed that the detection of the cytokines was highly dependent upon the platform, with the consistency of the detection of cytokines across platforms being dependent upon the cytokine being analyzed. A careful examination of platform assay performance must be made prior to utilizing multiplex platforms in a study. While some cytokines will give similar patterns of results across platforms, others will be highly variable. The use of the same platform within a study or across studies where data will be compared is advised.

scholarly journals All-Optical Planar Polymer Waveguide-Based Biosensor Chip Designed for Smartphone-Assisted Detection of Vitamin D

Sensors ◽  
2020 ◽  
Vol 20 (23) ◽  
pp. 6771
Author(s):  
Johanna-Gabriela Walter ◽  
Lourdes S. M. Alwis ◽  
Bernhard Roth ◽  
Kort Bremer
Keyword(s):  
Large Scale ◽  
Simultaneous Detection ◽  
Serum Samples ◽  
Plasmonic Sensor ◽  
Assay Sensitivity ◽  
Lab On Chip ◽  
Polymer Waveguide ◽  
Hydroxyvitamin D ◽  
All Optical ◽  
Biosensor Chip

An all-optical plasmonic sensor platform designed for smartphones based on planar-optical waveguide structures integrated in a polymer chip is reported for the first time. To demonstrate the applicability of the sensor system for biosensing purposes, the detection of 25-hydroxyvitamin D (25OHD) in human serum samples using an AuNP-enhanced aptamer-based assay was demonstrated. With the aid of the developed assay sensitivity of 0.752 pixel/nM was achieved for 25OHD concentrations ranging from 0–100 nM. The waveguide structure of the sensor enables miniaturisation and parallelisation, thus, demonstrates the potential for simultaneous detection of various analytes including biomarkers. The entire optical arrangement can be integrated into a single polymer chip which allows for large scale and cost-efficient sensor fabrication. The broad utilization and access of smartphone electronics make the proposed design most attractive for its wider use in lab-on-chip applications.

scholarly journals Quantitative PCR-Enhanced Immunoassay for Measurement of Enteroviral Immunoglobulin M Antibody and Diagnosis of Aseptic Meningitis

2005 ◽  
Vol 12 (2) ◽  
pp. 235-241 ◽  
Author(s):  
Amal Elfaitouri ◽  
Nahla Mohamed ◽  
Jan Fohlman ◽  
Robert Aspholm ◽  
Gun Frisk ◽  
...  
Keyword(s):  
Aseptic Meningitis ◽  
Solid Phase ◽  
Immunoglobulin M ◽  
Serum Samples ◽  
Serum Dilution ◽  
Group 4 ◽  
Control Serum ◽  
Group 3 ◽  
Group 2 ◽  
Capture Technique

ABSTRACT A PCR-enhanced immunoassay (PIA) to detect enterovirus (EV) immunoglobulin M (IgM) for diagnosis of recent EV infection was recently developed. This test was compared with another EV IgM capture technique, the solid-phase reverse immunosorbent test (SPRIST). Fourteen of 43 serum samples from aseptic meningitis patients were positive by PIA, whereas 10 were positive by SPRIST. One of 39 control serum samples was weakly positive by PIA. A single-serum-dilution real-time PCR-based PIA for EV IgM (quantitative PIA [QPIA]) was also developed and evaluated against PIA, SPRIST, an EV IgM radioimmunoassay (RIA), and clinical data. A mixture of 12 EVs was used as the antigen. Results from investigating four groups of serum samples were as follows. (i) The nine PIA-positive serum samples in group 1 were all positive by QPIA. (ii) Group 2 consisted of 59 serum samples from aseptic meningitis patients. Nineteen of 30 serum samples (63%) taken at hospital admission were positive by QPIA. Of these, 17 were positive in EV PCR. (iii) None of the 30 control serum samples in group 3 were positive by QPIA. (iv) For the 24 serum samples in group 4, of which 11 were positive and 13 were negative by RIA, the QPIA results were completely concordant. The sensitivity and specificity of QPIA for diagnosis of EV infection were 70 and 80%, respectively. QPIA provides a rational strategy for the detection of EV IgM, allows the use of viral antigens with minimal purification, and needs no virus-specific reagents apart from those in the PCR. QPIA is a generally applicable method for the detection of viral IgM in IgM capture assays.

scholarly journals Determination of Tilmicosin in Bovine and Porcine Sera by Liquid Chromatography

1997 ◽  
Vol 80 (6) ◽  
pp. 1183-1190 ◽  
Author(s):  
John W Moran ◽  
James M Turner ◽  
Mark R Coleman
Keyword(s):  
Limit Of Detection ◽  
Solid Phase ◽  
Reversed Phase ◽  
Gradient System ◽  
Serum Samples ◽  
Porcine Serum ◽  
Microbiological Methods ◽  
Control Serum ◽  
Relative Standard ◽  
Limit Of Quantitation

Abstract A liquid chromatographic (LC) assay is described for determining tilmicosin in bovine and porcine blood sera. Tilmicosin is isolated from the serum matrix and purified by solid-phase extraction with C18 sorbent. Sample is analyzed by LC using a gradient system with a phenyl reversed-phase column that separates tilmicosin from the matrix in 30 min. Tilmicosin is measured by UV absorbance at 280 nm. Validation of assay included evaluation of accuracy, precision, linearity, specificity, sensitivity, range, and sample stability. The method has a limit of quantitation of 0.1 ppm and a validated range of 0.1 to 10.0 ppm. Recoveries were 91–95% for bovine serum and 85–93% porcine serum. The limit of detection was 0.05 (μg/mL. Limits of detection and quantitation were based on 3 and 6 times the baseline noise of control serum samples, respectively. Relative standard deviations of precision samples (n = 6) were 2% or less for both sera. The method has better specificity and analysis time than previous microbiological methods for tilmicosin in sera.

scholarly journals A Novel Multiplex RT-PCR Assay for Simultaneous Detection of Dengue and Chikungunya Viruses

2020 ◽  
Vol 21 (21) ◽  
pp. 8281
Author(s):  
Mohammed Alimul Islam ◽  
Mohamed E. El Zowalaty ◽  
Sumaiya Islam ◽  
Mohiuddin Sharif ◽  
Md. Rajibur Rahman ◽  
...  
Keyword(s):  
Simultaneous Detection ◽  
Cross Reactivity ◽  
Single Step ◽  
Epidemiological Surveillance ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Serum Samples ◽  
Viral Genomes ◽  
Specificity And Sensitivity ◽  
Field Samples

The goal of the study was to develop a specific, sensitive, and cost-effective molecular RT-PCR diagnostic assay for the rapid and simultaneous detection of the serotypes of dengue virus (DENV) and Chikungunya virus (CHIKV) from sera of suspected febrile patients. A single-tube, single-step multiplex RT-PCR (mRT-PCR) assay was designed for the detection of viral genomes from clinical and field samples. Specificity and sensitivity of the mRT-PCR assay were evaluated against six different combinations using two reverse transcriptases (AMV-RT and RT-Ace) and three DNA polymerases (LA-Taq, rTaq, and Tth). Among the six combinations, the AMV-RT and LA-Taq combination was more specific and sensitive than other enzyme combinations for detecting viral genomes of DENV-1, DENV-2, DENV-3, and DENV-4 (p < 0.01), and for detecting viral genomes of CHIKV (p < 0.05). The detection limits of the mRT-PCR were 10 focus forming units (FFU) for CHIKV and 1 FFU, 20 FFU, 0.1 FFU, and 10 FFU for DENV-1, DENV-2, DENV-3, and DENV-4, respectively. The primers used for the mRT-PCR did not show any cross-reactivity among the serotypes of DENV or CHIKV. Specificity and sensitivity of the newly developed mRT-PCR were validated using serum samples collected from febrile patients during dengue outbreaks in Bangladesh. The sensitivity for serotype detection of DENV and CHIKV was superior to the virus isolation method and the antigen detection method using the Dengue NS1-Ag assay. This novel mRT-PCR method can be used for molecular epidemiological surveillance of DENV and CHIKV in epidemic and endemic countries.

scholarly journals Sphingolipid Analysis Indicate Lactosylceramide as a Potential Biomarker of Inflammatory Bowel Disease in Children

Biomolecules ◽  
2020 ◽  
Vol 10 (7) ◽  
pp. 1083
Author(s):  
Aleksandra Filimoniuk ◽  
Agnieszka Blachnio-Zabielska ◽  
Monika Imierska ◽  
Dariusz Marek Lebensztejn ◽  
Urszula Daniluk
Keyword(s):  
Inflammatory Bowel Disease ◽  
Bowel Disease ◽  
High Performance ◽  
Area Under The Curve ◽  
Study Groups ◽  
Diagnostic Value ◽  
Serum Samples ◽  
Specificity And Sensitivity ◽  
Potential Biomarker ◽  
Inflammatory Bowel

An altered ceramide composition in patients with inflammatory bowel disease (IBD) has been reported recently. The aim of this study was to evaluate the concentrations of sphingolipids in the serum of treatment-naive children with newly diagnosed IBD and to determine the diagnostic value of the tested lipids in pediatric IBD. The concentrations of sphingolipids in serum samples were evaluated using a quantitative method, an ultra-high-performance liquid chromatography-tandem mass spectrometry in children with Crohn’s disease (CD) (n=34), ulcerative colitis (UC) (n = 39), and controls (Ctr) (n = 24). Among the study groups, the most significant differences in concentrations were noted for C16:0-LacCer, especially in children with CD compared to Ctr or even to UC. Additionally, the relevant increase in C20:0-Cer and C18:1-Cer concentrations were detected in both IBD groups compared to Ctr. The enhanced C24:0-Cer level was observed only in UC, while C18:0-Cer only in the CD group. The highest area under the curve (AUC), specificity, and sensitivity were determined for C16:0-LacCer in CD diagnosis. Our results suggest that the serum LacC16-Cer may be a potential biomarker that distinguishes children with IBD from healthy controls and differentiates IBD subtypes. In addition, C20:0-Cer and C18:0-Cer levels also seem to be closely connected with IBD.

scholarly journals UHPLC-ESI-MS/MS Quantification of Relevant Substrates and Metabolites of the Kynurenine Pathway Present in Serum and Peritoneal Fluid from Gastric Cancer Patients—Method Development and Validation

2021 ◽  
Vol 22 (13) ◽  
pp. 6972
Author(s):  
Ilona Sadok ◽  
Katarzyna Jędruchniewicz ◽  
Karol Rawicz-Pruszyński ◽  
Magdalena Staniszewska
Keyword(s):  
Gastric Cancer ◽  
Cancer Patients ◽  
Peritoneal Fluid ◽  
Method Development ◽  
Solid Phase ◽  
Kynurenine Pathway ◽  
Serum Samples ◽  
Esi Ms ◽  
Gastric Cancer Patients ◽  
Development And Validation

Metabolites and enzymes involved in the kynurenine pathway (KP) are highly promising targets for cancer treatment, including gastrointestinal tract diseases. Thus, accurate quantification of these compounds in body fluids becomes increasingly important. The aim of this study was the development and validation of the UHPLC-ESI-MS/MS methods for targeted quantification of biologically important KP substrates (tryptophan and nicotinamide) and metabolites(kynurenines) in samples of serum and peritoneal fluid from gastric cancer patients. The serum samples were simply pretreated with trichloroacetic acid to precipitate proteins. The peritoneal fluid was purified by solid-phase extraction before analysis. Validation was carried out for both matrices independently. Analysis of the samples from gastric cancer patients showed different accumulations of tryptophan and its metabolites in different biofluids of the same patient. The protocols will be used for the evaluation of tryptophan and kynurenines in blood and peritoneal fluid to determine correlation with the clinicopathological status of gastric cancer or the disease’s prognosis.

scholarly journals Serum MicroRNA Signature as a Diagnostic and Therapeutic Marker in Patients with Psoriatic Arthritis

2020 ◽  
Vol 47 (12) ◽  
pp. 1760-1767
Author(s):  
Sarah M. Wade ◽  
Trudy McGarry ◽  
Siobhan C. Wade ◽  
Ursula Fearon ◽  
Douglas J. Veale
Keyword(s):  
Psoriatic Arthritis ◽  
Characteristic Curve ◽  
High Specificity ◽  
Serum Samples ◽  
Rna Molecules ◽  
Serum Mirna ◽  
Serum Microrna ◽  
Specificity And Sensitivity ◽  
European League Against Rheumatism ◽  
Noninvasive Markers

ObjectiveMicroRNA (miRNA) are small endogenous regulatory RNA molecules that have emerged as potential therapeutic targets and biomarkers in autoimmunity. Here, we investigated serum miRNA levels in patients with psoriatic arthritis (PsA) and further assessed a serum miRNA signature in therapeutic responder versus nonresponder PsA patients.MethodsSerum samples were collected from healthy controls (HC; n = 20) and PsA patients (n = 31), and clinical demographics were obtained. To examine circulatory miRNA in serum from HC and PsA patients, a focused immunology miRNA panel was analyzed utilizing a miRNA Fireplex assay (FirePlex Bioworks Inc.). MiRNA expression was further assessed in responders versus nonresponders according to the European League Against Rheumatism response criteria.ResultsSix miRNA (miR-221-3p, miR-130a-3p, miR-146a-5p, miR-151-5p, miR-26a-5p, and miR-21-5p) were significantly higher in PsA compared to HC (all P < 0.05), with high specificity and sensitivity determined by receiver-operating characteristic curve analysis. Analysis of responder versus nonresponders demonstrated higher baseline levels of miR-221-3p, miR-130a-3p, miR-146a-5p, miR-151-5p, and miR-26a-5p were associated with therapeutic response.ConclusionThis study identified a 6-serum microRNA signature that could be attractive candidates as noninvasive markers for PsA and may help to elucidate the disease pathogenesis.

scholarly journals Development of Sensitive Immunoassays for Free and Total Human Glandular Kallikrein 2

2004 ◽  
Vol 50 (9) ◽  
pp. 1607-1617 ◽  
Author(s):  
Ville Väisänen ◽  
Susann Eriksson ◽  
Kaisa K Ivaska ◽  
Hans Lilja ◽  
Martti Nurmi ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Detection Limit ◽  
Solid Phase ◽  
Specific Antigen ◽  
Control Group ◽  
Serum Samples ◽  
Human Kallikrein 2 ◽  
Kallikrein 2 ◽  
Capture Antibody ◽  
Total Psa

Abstract Background: Free and total human kallikrein 2 (hK2) might improve the discrimination between prostate cancer and benign prostatic hyperplasia. Concentrations of hK2 are 100-fold lower than concentrations of prostate-specific antigen (PSA); therefore, an hK2 assay must have a low detection limit and good specificity. Methods: PSA- and hK2-specific monoclonal antibodies were used in solid-phase, two-site immunofluorometric assays to detect free and total hK2. The total hK2 assay used PSA-specific antibodies to block nonspecific signal. The capture antibody of the free hK2 assay did not cross-react with PSA. To determine the hK2 concentrations in the male bloodstream, total hK2 was measured in a control group consisting of 426 noncharacterized serum samples. Free and total hK2 were measured in plasma from 103 patients with confirmed prostate cancer. Results: All 426 males in the control group had a total hK2 concentration above the detection limit of 0.0008 μg/L. The median total hK2 concentration was 0.022 μg/L (range, 0.0015–0.37 μg/L). hK2 concentrations were 0.1–58% of total PSA (median, 3.6%). hK2 concentrations were similar in men 41–50 and 51–60 years of age. The ratio of hK2 to PSA steadily decreased from 5–30% at PSA &lt;1 μg/L to 1–2% at higher PSA concentrations. In 103 patients with prostate cancer, the median hK2 concentration in plasma was 0.079 μg/L (range, 0.0015–16.2 μg/L). The median free hK2 concentration was 0.070 (range, 0.005–12.2) μg/L. The proportion of free to total hK2 varied from 17% to 131% (mean, 85%). Conclusions: The wide variation in the free-to-total hK2 ratio suggests that hK2 in blood plasma is not consistently in the free, noncomplexed form in patients with prostate cancer. The new assay is sufficiently sensitive to be used to study the diagnostic accuracies of free and total hK2 for prostate cancer.

scholarly journals Ribavirin Quantification in Combination Treatment of Chronic Hepatitis C

2003 ◽  
Vol 47 (1) ◽  
pp. 124-129 ◽  
Author(s):  
Sylvie Larrat ◽  
Françoise Stanke-Labesque ◽  
Agnès Plages ◽  
Jean-Pierre Zarski ◽  
Germain Bessard ◽  
...  
Keyword(s):  
Chronic Hepatitis ◽  
Hepatitis C ◽  
Chronic Hepatitis C ◽  
Combination Treatment ◽  
High Performance ◽  
Solid Phase ◽  
Ammonium Phosphate ◽  
Serum Samples ◽  
Coefficients Of Variation ◽  
Immunodeficiency Virus

ABSTRACT Ribavirin in combination with alpha 2 interferon is the consensus treatment for chronic hepatitis C. However, recent preliminary pharmacological studies have suggested that the bioavailability of ribavirin displays great interindividual variability. In order to monitor serum ribavirin levels during combination treatment, we developed and validated a quantitative assay using an approach adaptable for routine hospital laboratories. The method involved solid-phase extraction on phenyl boronic acid cartridges followed by high-performance liquid chromatography with a C18-bonded silica column and a mobile phase containing 10 mM ammonium phosphate buffer (with the pH adjusted to 2.5) and UV detection (207 nm). The sensitivity, recovery, linearity of the calibration curve, intra- and interassay accuracies, precision, and stability at 4°C were consistent with its use in the laboratory routine. In addition, other nucleoside analogues sometimes used with ribavirin in patients coinfected with hepatitis C virus (HCV) and human immunodeficiency virus did not interfere with the quantification of ribavirin levels. The ribavirin concentration was quantified in 24 serum samples from patients with chronic hepatitis C treated with a combination of ribavirin and alpha 2 interferon. The mean serum ribavirin concentration was 2.67 ± 1.06 μg/ml (n = 24) at week 12 of treatment (W12) and 3.24 ± 1.35 μg/ml (n = 24) at week 24 of treatment (W24). In addition, ribavirin concentrations displayed high interindividual variabilities: the coefficients of variation of the serum ribavirin concentrations adjusted to the administered dose were 44 and 48% at W12 and W24, respectively. Moreover, the ribavirin concentration was higher in patients with a sustained virological response (n = 11) than in patients with treatment failure (n = 13), with significant intergroup differences at W12 (P = 0.030) and W24 (P = 0.049). The present study describes a simple analytical method for the quantification of ribavirin in human serum that could be a useful tool for the monitoring of ribavirin concentrations in HCV-infected patients in order to improve the efficacy and safety of therapy with ribavirin plus interferon.

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