Enzyme immunoassay for SARS-CoV-2 antibodies in dried blood spot samples: A minimally-invasive approach to facilitate community- and population-based screening

AbstractBackgroundSerological testing for SARS-CoV-2 IgG antibodies is needed to document the community prevalence and distribution of the virus, particularly since many individuals have mild symptoms and cannot access molecular diagnostic testing of naso-pharyngeal swabs. However, the requirement for serum/plasma limits serological testing to clinical settings where it is feasible to collect and process venous blood. To address this problem we developed a serological test for SARS-CoV-2 IgG antibodies that requires only a single drop of capillary whole blood, collected from a simple finger prick and dried on filter paper (dried blood spot, DBS).MethodsEnzyme linked immunosorbent assay (ELISA) was optimized to detect SARS-CoV-2 IgG antibodies against the receptor-binding domain (RBD) of the spike protein. DBS samples were eluted overnight and transferred to a 96-well plate coated with antigen, and anti-human IgG-HRP was used to generate signal in proportion to bound antibody. DBS samples spiked with anti-SARS IgG antibody, and samples from known positive and negative cases, were compared to evaluate assay performance.ResultsAnalysis of samples with known concentrations of anti-SARS IgG produced the expected pattern of dose-response. Optical density (OD) values were significantly elevated for known positive cases in comparison with samples from unexposed individuals.DiscussionDBS ELISA provides a minimally-invasive alternative to venous blood collection that combines the convenience of sample collection in the home or non-clinical setting with the quantitation of ELISA in the lab. Serological testing for SARS-CoV-2 IgG antibodies in DBS samples should facilitate research across a wide range of community- and population-based settings on seroprevalence, predictors and duration of antibody responses, as well as correlates of protection from reinfection, each of which is critically important for pandemic control.

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High seroprevalence for SARS-CoV-2 among household members of essential workers detected using a dried blood spot assay

10.1101/2020.06.01.20119602 ◽

2020 ◽

Cited By ~ 1

Author(s):

Thomas W. McDade ◽

Elizabeth M. McNally ◽

Aaron S. Zelikovich ◽

Richard D’Aquila ◽

Brian Mustanski ◽

...

Keyword(s):

Receptor Binding ◽

Venous Blood ◽

Binding Domain ◽

Spike Protein ◽

Dried Blood Spot ◽

Receptor Binding Domain ◽

Serological Testing ◽

Igg Antibodies ◽

Household Members ◽

Blood Spot

AbstractObjectiveSerological testing is needed to investigate the extent of transmission of SARS-CoV-2 from front-line essential workers to their household members. However, the requirement for serum/plasma limits serological testing to clinical settings where it is feasible to collect and process venous blood. To address this problem we developed a serological test for SARS-CoV-2 IgG antibodies that requires only a single drop of finger stick capillary whole blood, collected in the home and dried on filter paper (dried blood spot, DBS).MethodsAn ELISA to the receptor binding domain of the SARS-CoV-2 spike protein was optimized to quantify IgG antibodies in DBS. Samples were self-collected from a community sample of 232 participants enriched with health care workers, including 30 known COVID-19 cases and their household members.ResultsAmong 30 individuals sharing a household with a virus-confirmed case of COVID-19, 80% were seropositive. Of 202 community individuals without prior confirmed acute COVID-19 diagnoses, 36% were seropositive. Of documented convalescent COVID-19 cases from the community, 29 of 30 (97%) were seropositive for IgG antibodies to the receptor binding domain.ConclusionDBS ELISA provides a minimally-invasive alternative to venous blood collection. Early analysis suggests a high rate of transmission among household members. High rates of seroconversion were also noted following recovery from infection. Serological testing for SARSCoV-2 IgG antibodies in DBS samples can facilitate seroprevalence assessment in community settings to address epidemiological questions, monitor duration of antibody responses, and assess if antibodies against the spike protein correlate with protection from reinfection.

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A surrogate virus neutralization test to quantify antibody-mediated inhibition of SARS-CoV-2 in finger stick dried blood spot samples

Scientific Reports ◽

10.1038/s41598-021-94653-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Amelia E. Sancilio ◽

Richard T. D’Aquila ◽

Elizabeth M. McNally ◽

Matthew P. Velez ◽

Michael G. Ison ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Venous Blood ◽

Host Cells ◽

Dried Blood Spot ◽

Blood Collection ◽

Widespread Application ◽

Live Virus ◽

Dilution Series ◽

Wide Range ◽

Blood Spot

AbstractThe spike protein of SARS-CoV-2 engages the human angiotensin-converting enzyme 2 (ACE2) receptor to enter host cells, and neutralizing antibodies are effective at blocking this interaction to prevent infection. Widespread application of this important marker of protective immunity is limited by logistical and technical challenges associated with live virus methods and venous blood collection. To address this gap, we validated an immunoassay-based method for quantifying neutralization of the spike-ACE2 interaction in a single drop of capillary whole blood, collected on filter paper as a dried blood spot (DBS) sample. Samples are eluted overnight and incubated in the presence of spike antigen and ACE2 in a 96-well solid phase plate. Competitive immunoassay with electrochemiluminescent label is used to quantify neutralizing activity. The following measures of assay performance were evaluated: dilution series of confirmed positive and negative samples, agreement with results from matched DBS-serum samples, analysis of results from DBS samples with known COVID-19 status, and precision (intra-assay percent coefficient of variation; %CV) and reliability (inter-assay; %CV). Dilution series produced the expected pattern of dose–response. Agreement between results from serum and DBS samples was high, with concordance correlation = 0.991. Analysis of three control samples across the measurement range indicated acceptable levels of precision and reliability. Median % surrogate neutralization was 46.9 for PCR confirmed convalescent COVID-19 samples and 0.1 for negative samples. Large-scale testing is important for quantifying neutralizing antibodies that can provide protection against COVID-19 in order to estimate the level of immunity in the general population. DBS provides a minimally-invasive, low cost alternative to venous blood collection, and this scalable immunoassay-based method for quantifying inhibition of the spike-ACE2 interaction can be used as a surrogate for virus-based assays to expand testing across a wide range of settings and populations.

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Validation of dried blood spot sample modifications to two commercially available COVID-19 IgG antibody immunoassays

Bioanalysis ◽

10.4155/bio-2020-0289 ◽

2020 ◽

Author(s):

Theodore T Zava ◽

David T Zava

Keyword(s):

Venous Blood ◽

Dried Blood Spot ◽

Blood Collection ◽

Igg Antibodies ◽

Antibody Testing ◽

Igg Antibody ◽

Nucleocapsid Proteins ◽

Antibody Assays ◽

Finger Stick ◽

Blood Spot

Aim: Coronavirus disease 2019 antibody testing often relies on venous blood collection, which is labor-intensive, inconvenient and expensive compared with finger-stick capillary dried blood spot (DBS) collection. The purpose of our work was to determine if two commercially available anti-severe acute respiratory syndrome coronavirus 2 enzyme-linked immunosorbent assays for IgG antibodies against spike S1 subunit and nucleocapsid proteins could be validated for use with DBS. Materials & methods: Kit supplied reagents were used to extract DBS, and in-house DBS calibrators were included on every run. Results: Positive/negative concordance between DBS and serum was 100/99.3% for the spike S1 subunit assay and 100/98% for the nucleocapsid assay. Conclusion: Validation of the DBS Coronavirus disease 2019 IgG antibody assays demonstrated that serum and DBS can produce equivalent results with minimal kit modifications.

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A surrogate virus neutralization test to quantify antibody-mediated inhibition of SARS-CoV-2 in finger stick dried blood spot samples

10.1101/2021.02.14.21251709 ◽

2021 ◽

Author(s):

Amelia Sancilio ◽

Richard D’Aquila ◽

Elizabeth M. McNally ◽

Matt E Velez ◽

Michael G. Ison ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Venous Blood ◽

Host Cells ◽

Dried Blood Spot ◽

Blood Collection ◽

Widespread Application ◽

Live Virus ◽

Dilution Series ◽

Wide Range ◽

Blood Spot

ABSTRACTBackgroundThe spike protein of SARS-CoV-2 engages the human angiotensin-converting enzyme 2 (ACE2) receptor to enter host cells, and neutralizing antibodies are effective at blocking this interaction to prevent infection. Widespread application of this important marker of protective immunity is limited by logistical and technical challenges associated with live virus methods and venous blood collection. To address this gap, we validated an immunoassay-based method for quantifying neutralization of the spike-ACE2 interaction in a single drop of capillary whole blood, collected on filter paper as a dried blood spot (DBS) sample.MethodsSamples are eluted overnight and incubated in the presence of spike antigen and ACE2 in a 96-well solid phase plate. Competitive immunoassay with electrochemiluminescent label is used to quantify neutralizing activity. The following measures of assay performance were evaluated: dilution series of confirmed positive and negative samples, agreement with results from matched DBS-serum samples, analysis of results from DBS samples with known COVID-19 status, and precision (intra-assay percent coefficient of variation; %CV) and reliability (inter-assay; %CV).ResultsDilution series produced the expected pattern of dose-response. Agreement between results from serum and DBS samples was high, with concordance correlation = 0.991. Analysis of three control samples across the measurement range indicated acceptable levels of precision and reliability. Median % neutralization was 46.9 for PCR confirmed convalescent COVID-19 samples and 0.1 for negative samples.ConclusionsLarge-scale testing is important for quantifying neutralizing antibodies that can provide protection against COVID-19 in order to estimate the level of immunity in the general population. DBS provides a minimally-invasive, low cost alternative to venous blood collection, and this scalable immunoassay-based method for quantifying neutralization of the spike-ACE2 interaction can be used as a surrogate for virus-based assays to expand testing across a wide range of settings and populations.

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Candidate reference method for determination of vitamin D from dried blood spot samples

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2019-0397 ◽

2020 ◽

Vol 58 (5) ◽

pp. 817-827 ◽

Cited By ~ 2

Author(s):

Rosita Zakaria ◽

Katrina J. Allen ◽

Jennifer J. Koplin ◽

Peter Roche ◽

Ronda F. Greaves

Keyword(s):

Vitamin D ◽

Internal Standard ◽

Reference Method ◽

Population Level ◽

Population Based ◽

Dried Blood Spot ◽

Coefficient Of Determination ◽

Plasma Vitamin ◽

Blood Spot

AbstractBackgroundThe current millennium has seen an explosion in vitamin D testing with the overarching aim of requests to clinically stratify patients as replete or deficient in vitamin D. At a population level, dried blood spot (DBS) sampling offers a less invasive and more practical application for assessment of vitamin D status. We have therefore aimed to develop a sensitive and robust DBS vitamin D method that is traceable to serum for use in population-based studies.MethodsBlood spots, calibrators and controls were prepared by punching a 3.2 mm DBS from filter paper and placed into a 96-well micro-plate. The DBS disk was eluted with a combination of water-methanol and internal standard (ISTD) solution followed by supported-liquid extraction and derivatisation. The extract was analysed by liquid-chromatography tandem-mass spectrometry in positive electrospray-ionisation mode with 732.5 > 673.4 and 738.4 > 679.4 m/z ion-transitions for derivatised vitamin D and the ISTD, respectively. Vitamin D results were made traceable to the National Institute of Standards and Technology reference material through the inclusion of Chromsystems vitamin D calibrators.Results25-Hydroxy-vitamin D3 and its related ISTD were detected at a retention time of 7 min. The seven-point calibration-curve consistently demonstrated a coefficient of determination of 0.99 with an experimentally determined reportable range of 0.5–376 nmol/L. Method validation studies using DBS samples demonstrated 12.9% between-assay imprecision at 45 nmol/L, 84% average recovery and high correlation with plasma vitamin D (correlation coefficient = 0.86).ConclusionsWe have successfully developed an analytical method for vitamin D quantitation from DBSs which will be applied to our population-based vitamin D research study. This approach improves traceability of DBS results and potentially could be used broadly for other DBS measurands that require comparison to serum/plasma for their interpretation.

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Fully automated dried blood spot sample handling and extraction for serological testing of SARS‐CoV‐2 antibodies

Drug Testing and Analysis ◽

10.1002/dta.2946 ◽

2020 ◽

Author(s):

Stefan Gaugler ◽

Pierre‐Edouard Sottas ◽

Katharina Blum ◽

Marc Luginbühl

Keyword(s):

Dried Blood Spot ◽

Serological Testing ◽

Sample Handling ◽

Blood Spot

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Assessment of a dried blood spot C-reactive protein method to identify disease flares in rheumatoid arthritis patients

Scientific Reports ◽

10.1038/s41598-020-77826-0 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Leon G. D’Cruz ◽

Kevin G. McEleney ◽

Chris Cochrane ◽

Kyle B. C. Tan ◽

Priyank Shukla ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Joint Damage ◽

Reference Method ◽

Detection Sensitivity ◽

Dried Blood Spot ◽

Enzyme Linked Immunosorbent Assay ◽

C Reactive Protein ◽

Reactive Protein ◽

Deming Regression ◽

Blood Spot

AbstractRheumatoid arthritis (RA) is characterised by painful, stiff and swollen joints. RA features sporadic ‘flares’ or inflammatory episodes—mostly occurring outside clinics—where symptoms worsen and plasma C-reactive protein (CRP) becomes elevated. Poor control of inflammation results in higher rates of irreversible joint damage, increased disability, and poorer quality of life. Flares need to be accurately identified and managed. A method comparison study was designed to assess agreement between CRP values obtained by dried blood spot (DBS) versus conventional venepuncture sampling. The ability of a weekly DBS sampling and CRP test regime to detect flare outside the clinic was also assessed. Matched venepuncture and finger lancet DBS samples were collected from n = 100 RA patients with active disease at baseline and 6 weeks (NCT02809547). A subset of n = 30 RA patients submitted weekly DBS samples over the study period. Patient demographics, including self-reported flares were recorded. DBS sample CRP measurements were made by enzyme-linked immunosorbent assay, and venepuncture samples by a reference immunoturbometric assay. Data was compared between sample types by Bland–Altman and weighted Deming regression analyses. Flare detection sensitivity and specificity were compared between ‘minimal’ baseline and 6 week sample CRP data and the ‘continuous’ weekly CRP data. Baseline DBS ELISA assay CRP measures yielded a mean positive bias of 2.693 ± 8.640 (95% limits of agreement − 14.24 to 19.63%), when compared to reference assay data. Deming regression revealed good agreement between the DBS ELISA method and reference assay data, with baseline data slope of 0.978 and intercept -0.153. The specificity of ‘continuous’ area under the curve (AUC) CRP data (72.7%) to identify flares, was greater than ‘minimal’ AUC CRP data (54.5%). This study indicates reasonable agreement between DBS and the reference method, especially at low to mid-range CRP values. Importantly, longitudinal CRP measurement in RA patients helps to clearly identify flare and thus could assist in remote monitoring strategies and may facilitate timely therapeutic intervention.Trial registration: The Remote Arthritis Disease Activity MonitoR (RADAR) study was registered on 22/06/2016 at ClinicalTrials.gov Identifier: NCT02809547. https://clinicaltrials.gov/ct2/show/NCT02809547.

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Comparison of Glycated Hemoglobin Results Based on At-Home and In-Lab Dried Blood Spot Sampling to Routine Venous Blood Sampling In-Lab in Adult Patients With Type 1 or Type 2 Diabetes

Canadian Journal of Diabetes ◽

10.1016/j.jcjd.2017.10.053 ◽

2018 ◽

Vol 42 (4) ◽

pp. 426-432.e1 ◽

Cited By ~ 1

Author(s):

Thomas G. Elliott ◽

Kent C. Dooley ◽

Mira Zhang ◽

Harlan S.D. Campbell ◽

Darby J.S. Thompson

Keyword(s):

Type 2 Diabetes ◽

Glycated Hemoglobin ◽

Venous Blood ◽

Adult Patients ◽

Blood Sampling ◽

Dried Blood Spot ◽

Venous Blood Sampling ◽

Blood Spot

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Dried Blood Spot Analysis Suitable for Therapeutic Drug Monitoring of Voriconazole, Fluconazole, and Posaconazole

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00707-13 ◽

2013 ◽

Vol 57 (10) ◽

pp. 4999-5004 ◽

Cited By ~ 36

Author(s):

Kim C. M. van der Elst ◽

Lambert F. R. Span ◽

Kai van Hateren ◽

Karin M. Vermeulen ◽

Tjip S. van der Werf ◽

...

Keyword(s):

Therapeutic Drug Monitoring ◽

Drug Monitoring ◽

Sampling Method ◽

Fungal Infections ◽

Venous Blood ◽

Blood Sampling ◽

Dried Blood Spot ◽

Therapeutic Drug ◽

Venous Blood Sampling ◽

Blood Spot

ABSTRACTInvasive aspergillosis and candidemia are important causes of morbidity and mortality in immunocompromised and critically ill patients. The triazoles voriconazole, fluconazole, and posaconazole are widely used for the treatment and prophylaxis of these fungal infections. Due to the variability of the pharmacokinetics of the triazoles among and within individual patients, therapeutic drug monitoring is important for optimizing the efficacy and safety of antifungal treatment. A dried blood spot (DBS) analysis was developed and was clinically validated for voriconazole, fluconazole, and posaconazole in 28 patients. Furthermore, a questionnaire was administered to evaluate the patients' opinions of the sampling method. The DBS analytical method showed linearity over the concentration range measured for all triazoles. Results for accuracy and precision were within accepted ranges; samples were stable at room temperature for at least 12 days; and different hematocrit values and blood spot volumes had no significant influence. The ratio of the drug concentration in DBS samples to that in plasma was 1.0 for voriconazole and fluconazole and 0.9 for posaconazole. Sixty percent of the patients preferred DBS analysis as a sampling method; 15% preferred venous blood sampling; and 25% had no preferred method. There was significantly less perception of pain with the DBS sampling method (P= 0.021). In conclusion, DBS analysis is a reliable alternative to venous blood sampling and can be used for therapeutic drug monitoring of voriconazole, fluconazole, and posaconazole. Patients were satisfied with DBS sampling and had less pain than with venous sampling. Most patients preferred DBS sampling to venous blood sampling.

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