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Abstract ObjectivesA number of basic questions about bone biology have not been answered, including population differences in bone turnover. In part, this stems from the lack of validated minimally invasive biomarker techniques to measure bone formation and resorption in field‐based population‐level research. The present study addresses this gap by validating a fingerprick dried blood spot (fDBS) assay for tartrate‐resistant acid phosphatase 5b (TRACP‐5b), a well‐defined biomarker of bone resorption and osteoclast number. MethodsWe adapted a commercially available enzyme‐linked immunosorbent assay (ELISA) kit from MyBiosource for the quantitative determination of TRACP‐5b levels in serum and plasma for use with DBS. We used a rigorous process of assay modification and validation, including the use of a matched set of 189 adult plasma, fDBS, and venous DBS (vDBS) samples; parameters evaluated included precision, reliability, and analyte stability. ResultsPlasma and DBS TRACP‐5b concentrations showed a linear relationship. There were no systematic differences in TRACP‐5b levels in fDBS and vDBS, indicating no significant differences in TRACP‐5b distribution between capillary and venous blood. Parallelism and spike‐and‐recovery results indicated that matrix factors in DBS do not interfere with measurement of TRACP‐5b levels from DBS using the validated kit. Intra‐ and interassay CVs were 5.0% and 12.1%, respectively. DBS samples should preferably be stored frozen but controlled room temperature storage for up to a month may be acceptable. ConclusionsThis DBS‐based ELISA assay adds to the methodological toolkit available to human biologists and will facilitate research on bone turnover in population studies.
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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
Abstract The 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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- Award ID(s):
- 2035114
- PAR ID:
- 10330112
- Date Published:
- Journal Name:
- Scientific Reports
- Volume:
- 11
- Issue:
- 1
- ISSN:
- 2045-2322
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract ObjectivesInvestigating factors that contribute to bone loss and accretion across populations in remote settings is challenging, particularly where diagnostic tools are scarce. To mitigate this challenge, we describe validation of a commercial ELISA assay to measure osteocalcin, a biomarker of bone formation, from dried blood spots (DBS). MethodsWe validated the Osteocalcin Human SimpleStep ELISA kit from Abcam (ab1951214) using 158 matched plasma and DBS samples. Passing‐Bablok regression analysis assessed the relationships between plasma and DBS osteocalcin concentrations. Dilutional linearity and spike and recovery experiments determined if the DBS matrix interfered with osteocalcin measurement, and intra‐ and inter‐assay coefficients of variation (CVs) were calculated. Limit of detection, analyte stability, and specific forms of osteocalcin measured by the kit were also investigated. ResultsMean plasma osteocalcin value was 218.2 ng/mL (range 64.6‐618.1 ng/mL). Linear relationships existed between plasma and DBS concentrations of osteocalcin, with no apparent bias in plasma vs DBS concentrations. There was no apparent interference of the DBS matrix with measurement of osteocalcin in DBS. Intra‐assay CV for DBS was ~8%, while average inter‐assay CV was 14.8%. Limit of detection was 0.34 ng/mL. Osteocalcin concentrations were stable in DBS stored at −28°C and room temperature, but not those stored at 37°C. This ELISA kit detects total osteocalcin. ConclusionsOsteocalcin, a bone formation biomarker, can be measured from DBS. Combined with a previously validated DBS assay for TRACP‐5b, a bone resorption biomarker, these assays have the potential to help researchers disentangle the many factors contributing to bone strength.more » « less
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1038/s41598-021-94653-z
