A method to reduce ELISA serial dilution assay workload applied to SARS-CoV-2 and seasonal HCoVs

Objectives Assays using ELISA measurements on serially diluted serum samples have been heavily used to measure serum reactivity to SARS-CoV-2 antigens and are widely used in virology and elsewhere in biology. We test a method to reduce the workload of these assays, and measure reactivity of SARS-CoV-2 and HCoV antigens to human serum samples collected before and during the COVID-19 pandemic. Methods We apply Bayesian hierarchical modelling to ELISA measurements of human serum samples against SARS-CoV-2 and HCoV antigens. Results Inflection titers for SARS-CoV-2 full-length spike protein (S1S2), spike protein receptor-binding domain (RBD), and nucleoprotein (N) inferred from three spread-out dilutions correlated with those inferred from eight consecutive dilutions with an R2 value of 0.97 or higher. We confirm existing findings showing a small proportion of pre-pandemic human serum samples contain cross-reactive antibodies to SARS-CoV-2 S1S2 and N, and that SARS-CoV-2 infection increases serum reactivity to the beta-HCoVs OC43 and HKU1 S1S2. Conclusions In serial dilution assays, large savings in resources and/or increases in throughput can be achieved by reducing the number of dilutions measured and using Bayesian hierarchical modelling to infer inflection or endpoint titers. We have released software for conducting these types of analysis.

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Comparative analyses of SARS-CoV-2 binding (IgG, IgM, IgA) and neutralizing antibodies from human serum samples

10.1101/2020.08.10.243717 ◽

2020 ◽

Cited By ~ 4

Author(s):

Livia Mazzini ◽

Donata Martinuzzi ◽

Inesa Hyseni ◽

Giulia Lapini ◽

Linda Benincasa ◽

...

Keyword(s):

Human Serum ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

Binding Domain ◽

Spike Protein ◽

Wild Type Virus ◽

Receptor Binding Domain ◽

Serum Samples ◽

Neutralization Titer ◽

Human Serum Samples

ABSTRACTA newly identified coronavirus, named SARS-CoV-2, emerged in December 2019 in Hubei Province, China, and quickly spread throughout the world; so far, it has caused more than 18 million cases of disease and 700,000 deaths. The diagnosis of SARS-CoV-2 infection is currently based on the detection of viral RNA in nasopharyngeal swabs by means of molecular-based assays, such as real-time RT-PCR. Furthermore, serological assays aimed at detecting different classes of antibodies constitute the best surveillance strategy for gathering information on the humoral immune response to infection and the spread of the virus through the population, in order to evaluate the immunogenicity of novel future vaccines and medicines for the treatment and prevention of COVID-19 disease. The aim of this study was to determine SARS-CoV-2-specific antibodies in human serum samples by means of different commercial and in-house ELISA kits, in order to evaluate and compare their results first with one another and then with those yielded by functional assays using wild-type virus. It is important to know the level of SARS-CoV-2-specific IgM, IgG and IgA antibodies in order to predict population immunity and possible cross-reactivity with other coronaviruses and to identify potentially infectious subjects. In addition, in a small sub-group of samples, we performed a subtyping Immunoglobulin G ELISA. Our data showed an excellent statistical correlation between the neutralization titer and the IgG, IgM and IgA ELISA response against the receptor-binding domain of the spike protein, confirming that antibodies against this portion of the virus spike protein are highly neutralizing and that the ELISA Receptor-Binding Domain-based assay can be used as a valid surrogate for the neutralization assay in laboratories which do not have Biosecurity level-3 facilities.

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

Current Analytical Chemistry ◽

10.2174/1573411014666180730112905 ◽

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.

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Dual-readout test strips platform for portable and highly sensitive detection of alkaline phosphatase in human serum samples

Chinese Chemical Letters ◽

10.1016/j.cclet.2021.05.019 ◽

2021 ◽

Author(s):

Juanzu Liu ◽

Hongmin Meng ◽

Lin Zhang ◽

Shasha Li ◽

Juan Chen ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Human Serum ◽

Sensitive Detection ◽

Serum Samples ◽

Test Strips ◽

Human Serum Samples ◽

Highly Sensitive ◽

Highly Sensitive Detection

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

Journal of Endocrinology ◽

10.1677/joe.0.1260159 ◽

1990 ◽

Vol 126 (1) ◽

pp. 159-168 ◽

Cited By ~ 7

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

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

Clinical Microbiology and Infection ◽

10.1111/j.1469-0691.2008.02153.x ◽

2009 ◽

Vol 15 ◽

pp. 232-234 ◽

Cited By ~ 8

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

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

Microchemical Journal ◽

10.1016/j.microc.2016.07.004 ◽

2016 ◽

Vol 129 ◽

pp. 205-212 ◽

Cited By ~ 16

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

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

mSphere ◽

10.1128/msphere.00623-18 ◽

2018 ◽

Vol 3 (6) ◽

Cited By ~ 2

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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Cryogenic zone compression GC-HRTOFMS for the measurement of PCB-153 and DDE in 20 μL serum samples

Analytical Methods ◽

10.1039/c5ay00543d ◽

2016 ◽

Vol 8 (2) ◽

pp. 249-255 ◽

Cited By ~ 1

Author(s):

B. L'Homme ◽

J.-F. Focant

Keyword(s):

Human Serum ◽

Human Exposure ◽

Proof Of Concept ◽

Serum Samples ◽

Human Serum Samples

Human exposure to POPs is of concern and typical biomonitoring studies require large amounts of blood (5–75 mL) from participants. As a proof of concept, we developed a miniaturized method based on MEPS and CZC applied to GC-HRTOFMS for the measurement of markers of exposure (PCB-153, DDE) in 20 μL human serum samples.

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