scholarly journals A Paper-based Immunofluorescent Device for the Detection of SARS-CoV-2 Humanized Antibody

2021 ◽  
Author(s):  
Surasak Kasetsirikul ◽  
Muhammad Umer ◽  
Narshone Soda ◽  
Kamalalayam Rajan Sreejith ◽  
Muhammad J. A. Shiddiky ◽  
...  
Keyword(s):  
Human Serum ◽  
Point Of Care ◽  
Serum Samples ◽  
Igg Antibody ◽  
Healthy Human ◽  
Specific Antibodies ◽  
Human Serum Samples ◽  
Humanized Antibody ◽  
Relative Standard ◽  
Humanized Antibodies

We report on an immunofluorescent paper-based assay for the detection of severe acute respiratory symptom coronavirus 2 (SARS-CoV-2) humanized antibody. The paper-based device was fabricated by using lamination technique for easy and optimized handling. Our approach utilises a two-step strategy that involves (i) initial coating of the paper-electrode with recombinant SARS-CoV-2 nucleocapsid antigen to capture the target SARS-CoV-2 specific antibodies, and (ii) subsequent detection of SARS-CoV-2 antibodies using fluorophore-conjugated IgG antibody. The fluorescence readout was observed with fluorescence microscopy. The images were processed and quantified using a MATLAB program. The assay can selectively detect SARS-CoV-2 humanized antibodies spiked in PBS and healthy human serum samples with the relative standard deviation of approximately 6.4% (for n = 3). It has broad dynamic ranges (1 ng to 50 ng/µL in PBS and 5 to 100 ng/µL in human serum samples) for SARS-CoV-2 humanized antibodies with the detection limits of 2 ng/µL (0.025 IU/mL) and 10 ng/µL (0.125 IU/mL) in PBS and human serum samples, respectively. We believe that our assay has the potential to be used as a simple, rapid, and inexpensive paper-based diagnostic device with a portable fluorescent reader to provide point-of-care diagnosis. This assay can be used for rapid examination of a large batch of samples toward clinical screening of SARS-CoV-2 specific antibodies as a confirmed infected active case or to evaluate the immune response to a SARS-CoV-2 vaccine.

scholarly journals Molecular Markers in Acute and Chronic Phases of Human Toxoplasmosis: Determination of Immunoglobulin G Avidity by Western Blotting

2000 ◽  
Vol 7 (3) ◽  
pp. 384-389 ◽  
Author(s):  
P. T. Marcolino ◽  
D. A. O. Silva ◽  
P. G. Leser ◽  
M. E. Camargo ◽  
J. R. Mineo
Keyword(s):  
Human Serum ◽  
Immunoglobulin G ◽  
Chronic Infection ◽  
High Avidity ◽  
Serum Samples ◽  
Modified Technique ◽  
Igg Antibody ◽  
Recent Infection ◽  
Human Serum Samples

ABSTRACT We characterized antigenic markers recognized by human serum samples from patients presenting with acute and chronic toxoplasmosis by the determination of immunoglobulin G (IgG) antibody avidity by a Western blot modified technique (avidity immunoblotting) that includes the dissociation of the antigen-antibody interaction with 6 or 8 M urea solutions. Human serum samples from 20 patients presenting with recent infection and from 20 patients with chronic infection were analyzed. It was observed that bands p16, p32, p38, p40, p43, p54, p60, p66, and p97 were more frequently recognized by low-avidity IgG in recent infection and by high-avidity IgG in chronic toxoplasmosis. From these antigenic bands, p38 can be characterized as an optimal antigenic marker of low avidity for recent forms of toxoplasmosis due to a significant decrease of their frequencies (from 80 to 0%) after treatment with 6 M urea solutions. The p30 antigen was not considered a good marker to distinguish acute from chronic infection since corresponding IgG antibodies were determined to have high avidity in both phases of the infection. Thus, the avidity immunoblotting assay proved to be a useful tool for determining antigenic markers of recent and chronic phases ofToxoplasma gondii infection.

Single molecule array (Simoa) assay with optimal antibody pairs for cytokine detection in human serum samples

The Analyst ◽  
2015 ◽  
Vol 140 (18) ◽  
pp. 6277-6282 ◽  
Author(s):  
Danlu Wu ◽  
Milena Dumont Milutinovic ◽  
David R. Walt
Keyword(s):  
Human Serum ◽  
Single Molecule ◽  
Serum Samples ◽  
Healthy Human ◽  
Human Serum Samples ◽  
Cytokine Profiles ◽  
Cytokine Detection

By employing optimal antibody pairs, ultrasensitive Simoa assays were developed to measure ten cytokine profiles in healthy human serum samples.

A Novel Ionic Liquid-Based Liquid-Liquid Microextraction Combined with High Performance Liquid Chromatography for Simultaneous Determination of Eight Vitamin E Isomers in Human Serum

2020 ◽  
Vol 103 (4) ◽  
pp. 989-996
Author(s):  
Shuo Yin ◽  
Yi Yang ◽  
Jing Zhang ◽  
Yongxin Li ◽  
Ling Wu ◽  
...  
Keyword(s):  
Ionic Liquid ◽  
Vitamin E ◽  
Human Serum ◽  
Simultaneous Determination ◽  
Serum Samples ◽  
Fluorescence Detector ◽  
Human Serum Samples ◽  
Relative Standard ◽  
Liquid Microextraction

Abstract Background Vitamin E deficiencies are prevalent around the world and have become one of the major public health issues. It is necessary to determine their levels in human serum for routine clinical practice. Objective In this study, a simple and green ionic liquid-based (IL)vortex-assisted (VA) liquid–liquid microextraction (LLME) combined with HPLC was developed for simultaneous determination of eight vitamin E isomers in human serum. Methods The IL, 1-octyl-methylimidazolium trifluoromethanesulfonate ([OMIM]OTf), was added into the diluted sample and vortexed to form a cloudy solution. After centrifugation, the IL phase was collected for HPLC analysis. The separation was accomplished on a Phenomenex Luna-C18 column (250 mm × 4.6 mm, 5 μm) and the column temperature was 30°C. The mobile phase was methanol/acetonitrile (80 + 20, v/v) and the flow rate was 0.7 mL/min. A fluorescence detector was used for the simultaneous detection of eight vitamin E isomers, and the detection wavelength was set at 290/327 nm. The LLME procedure can be completed within 10 min without using any organic solvent. The parameters affecting the extraction efficiencies were optimized, including the type and volume of the ILs, dispersive solvent, vortex time, and salt addition. Results Under the optimal conditions, limits of detection were 0.857–4.16 ng/mL. Acceptable recoveries ranging from 80.1% to 103% were achieved, with relative standard deviations less than 13.0%. The proposed method was successfully applied to the detection of eight vitamin E isomers in human serum samples. Conclusions This method is simple, fast, environment-friendly, cheap, and has similar linear ranges, sensitivities, accuracy, and precision as those reported chromatographic methods. Highlights The IL, [OMIM]OTf, was chosen as the green extractant of LLME for vitamin E extraction because of its strong adsorption property for vitamin E isomers. An IL-VA-LLME method has been developed for the analysis of 8 vitamin E isomers. The established method was successfully applied to the analysis of 8 vitamin E isomers in human serum samples.

Simultaneous Monitoring of Febuxostat and Uric Acid in Human Serum Samples Using the Direct Square-Wave Voltammetric Method

2019 ◽  
Vol 15 (6) ◽  
pp. 678-684
Author(s):  
Biljana Nigović ◽  
Jakov Vlak
Keyword(s):  
Uric Acid ◽  
Human Serum ◽  
Oxidase Inhibitor ◽  
Serum Samples ◽  
Boron Doped Diamond ◽  
Fast Analysis ◽  
Xanthine Oxidase Inhibitor ◽  
Voltammetric Method ◽  
Square Wave ◽  
Human Serum Samples

Background: High uric acid serum level, hyperuricemia, is now associated with many diseases such as gout, chronic kidney disease, hypertension, coronary artery disease and diabetes. Febuxostat is a novel selective xanthine oxidase inhibitor approved for the treatment of hyperuricemia. Objective: The aim of this study was to develop a first analytical method for the simultaneous determination of febuxostat and uric acid. Methods: An unmodified boron-doped diamond electrode provided concurrent quantitation of drug at low levels and uric acid, which has clinical significance in the diagnosis and therapy of hyperuricemia, at relatively high concentrations. The direct square-wave voltammetric method was applied to the analysis of both analytes in human serum samples. Results: Under the optimized conditions, the linear response of peak current on febuxostat concentration was achieved in the range from 7.5 × 10-7 to 3 × 10-5 M, while uric acid showed two linear ranges of 5 × 10-6 - 5 × 10-5 M and 5 × 10-5 - 2 × 10-4 M. The method was successfully utilised for quantification of both analytes in human serum samples. Good recoveries were obtained without interference from common inorganic cations and anions as well as glucose, dopamine, ascorbic and folic acids at concentrations expected in physiological conditions. Conclusion: The great benefits of developed method are fast analysis (only 7.5 s for run), low cost and simplicity of performance.

A rat granulosa cell plasminogen activator bioassay for FSH in human serum

1990 ◽  
Vol 126 (1) ◽  
pp. 159-168 ◽  
Author(s):  
A. N. Thakur ◽  
R. Coles ◽  
A. Sesay ◽  
B. Earley ◽  
H. S. Jacobs ◽  
...  
Keyword(s):  
Human Serum ◽  
Plasminogen Activator ◽  
Granulosa Cell ◽  
Serum Samples ◽  
Glycoprotein Hormone ◽  
Assay Sensitivity ◽  
Serum Factors ◽  
Human Serum Samples ◽  
Heat Treated ◽  
Fsh Bioactivity

ABSTRACT A previously described in-vitro rat granulosa cell plasminogen activator bioassay for FSH has been modified and applied in the assay of human serum. This modified method consists of exposing the diethylstilboestrol-stimulated granulosa cells from 25- to 26-day-old rats to FSH or test substance for 3·5 h in wells coated with 125I-labelled fibrinogen and treated with thrombin. Following stimulation with FSH, the dose-related production of plasminogen activator was measured as the degree of 125I-labelled fibrinolysis in the presence of added plasminogen. Using the urinary FSH/LH bioassay reference preparation as the assay standard, the useful range of the assay was 0·3–15IU/l, with an assay sensitivity of 0·3 IU/l. As determined using purified glycoprotein hormone preparations, the assay was highly specific for FSH. The minor degree of FSH bioactivity measured in some of the hormone preparations was accounted for by the amount of FSH contamination in these preparations. To abolish interference caused by unknown serum factors, we heat-treated the serum samples for 15 min at 56 °C before the assay. The results indicated that neither immunoreactivity nor bioactivity was affected by this treatment. Furthermore, heat-treated human sera gave responses parallel to the standard curve at the three dose levels (2, 4 and 8 μl) studied. We used this bioassay to estimate the FSH-like bioactivity in 15 human serum samples. The estimates of immunoreactive FSH in these samples correlated well with the corresponding FSH bioactivity (r = 0·745, n = 15 and P < 0·05). The results indicate that with this sensitive and rapid (completed within 24 h) bioassay, it should be possible to measure FSH bioactivity in heat-treated human serum samples. Journal of Endocrinology (1990) 126, 159–168

scholarly journals Evaluation of PCR-based assay in human serum samples for diagnosis of fatal cases of spotted fever group rickettsiosis

2009 ◽  
Vol 15 ◽  
pp. 232-234 ◽  
Author(s):  
E.M Mendes do Nascimento ◽  
S. Colombo ◽  
T.K. Nagasse-Sugahara ◽  
R.N. Angerami ◽  
M.R. Resende ◽  
...  
Keyword(s):  
Human Serum ◽  
Spotted Fever ◽  
Spotted Fever Group ◽  
Serum Samples ◽  
Human Serum Samples

Simultaneous determination of ascorbic and uric acids and dopamine in human serum samples using three-way calibration with data from square wave voltammetry

2016 ◽  
Vol 129 ◽  
pp. 205-212 ◽  
Author(s):  
Adrian Marcelo Granero ◽  
Gastón Darío Pierini ◽  
Sebastián Noel Robledo ◽  
María Susana Di Nezio ◽  
Héctor Fernández ◽  
...  
Keyword(s):  
Human Serum ◽  
Simultaneous Determination ◽  
Square Wave Voltammetry ◽  
Serum Samples ◽  
Square Wave ◽  
Human Serum Samples ◽  
Wave Voltammetry

scholarly journals Development and Validation of 13-plex Luminex-Based Assay for Measuring Human Serum Antibodies to Streptococcus pneumoniae Capsular Polysaccharides

mSphere ◽  
2018 ◽  
Vol 3 (4) ◽  
pp. e00128-18 ◽  
Author(s):  
Danka Pavliakova ◽  
Peter C. Giardina ◽  
Soraya Moghazeh ◽  
Shite Sebastian ◽  
Maya Koster ◽  
...  
Keyword(s):  
Human Serum ◽  
Lower Limit ◽  
Immunosorbent Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibodies ◽  
Igg Antibody ◽  
Correlate Of Protection ◽  
Immune Correlate ◽  
Relative Standard ◽  
Limit Of Quantitation

ABSTRACT A Luminex-based direct immunoassay (dLIA) platform has been developed to replace the standardized pneumococcal enzyme-linked immunosorbent assay platform. The multiplex dLIA simultaneously measures the concentration of serum immunoglobulin G (IgG) antibodies specific for pneumococcal capsular polysaccharide (PnPS) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. The assay uses poly-l-lysine (PLL)-conjugated PnPS, chemically coupled to spectrally distinct Luminex microspheres. Assay validation experiments were performed using residual human serum samples obtained from 13-valent pneumococcal conjugate vaccine (13vPnC) clinical studies. Assay results are expressed as IgG antibody concentrations in micrograms per milliliter using the international reference serum, 007sp. The lower limit of quantitation (LLOQ) for all serotypes covered in the 13-plex dLIA fell within the range of 0.002 to 0.038 µg/ml serum IgG. The difference between the lower limit and upper limit of the assay range was >500-fold for all serotypes, and assay variability was <20% relative standard deviation (RSD) for all serotypes. IgG antibody measurements were shown to be serotype-specific (some cross-reactivity was observed only between the structurally related serotypes 6A and 6B as well as 19A and 19F), and no interference was observed between the serotypes when the assay was performed in the 13-plex format compared to the singleplex assays. The 13-plex dLIA platform developed by Pfizer Inc. generates up to 143 test results in a single 96-well plate and is a suitable replacement of the enzyme-linked immunosorbent assay (ELISA) platform for evaluating vaccine clinical trials. IMPORTANCE The pneumococcal enzyme-linked immunosorbent assay (ELISA) measures IgG antibodies in human serum, and it is an important assay that supports licensure of pneumococcal vaccines. The immune correlate of protection, 0.35 µg/ml of IgG antibodies, was determined by the ELISA method. Pfizer has developed a new Luminex-based assay platform to replace the ELISA. These papers describe the important work of (i) validating the Luminex-based assay and (ii) bridging the immune correlate of protection (0.35 µg/ml IgG) to equivalent values reported by the Luminex platform.

scholarly journals A Novel, Rapid, and Low-Volume Assay for Therapeutic Drug Monitoring of Posaconazole and Other Long-Chain Azole-Class Antifungal Drugs

mSphere ◽  
2018 ◽  
Vol 3 (6) ◽  
Author(s):  
Gregory R. Wiedman ◽  
Yanan Zhao ◽  
David S. Perlin
Keyword(s):  
Liquid Chromatography ◽  
Therapeutic Drug Monitoring ◽  
Human Serum ◽  
Drug Monitoring ◽  
Fungal Infections ◽  
Antifungal Drugs ◽  
Therapeutic Drug ◽  
Serum Samples ◽  
Human Serum Samples ◽  
Long Chain

ABSTRACT Clinicians need a better way to accurately monitor the concentration of antimicrobials in patient samples. In this report, we describe a novel, low-sample-volume method to monitor the azole-class antifungal drug posaconazole, as well as certain other long-chain azole-class antifungal drugs in human serum samples. Posaconazole represents an important target for therapeutic drug monitoring (TDM) due to its widespread use in treating invasive fungal infections and well-recognized variability of pharmacokinetics. The current “gold standard” requires trough and peak monitoring through high-pressure liquid chromatography (HPLC) or liquid chromatography-tandem mass spectroscopy (LC-MS/MS). Other methods include bioassays that use highly susceptible strains of fungi in culture plates or 96-well formats to monitor concentrations. Currently, no method exists that is both highly accurate in detecting free drug concentrations and is also rapid. Herein, we describe a new method using reduced graphene oxide (rGO) and a fluorescently labeled aptamer, which can accurately assess clinically relevant concentrations of posaconazole and other long-chain azole-class drugs in little more than 1 h in a total volume of 100 µl. IMPORTANCE This work describes an effective assay for TDM of long-chain azole-class antifungal drugs that can be used in diluted human serum samples. This assay will provide a quick, cost-effective method for monitoring concentrations of drugs such as posaconazole that exhibit well-documented pharmacokinetic variability. Our rGO-aptamer assay has the potential to improve health care for those struggling to treat fungal infections in rural or resource-limited setting.

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