Enzyme-Linked Immunoassay-Based Quantitative Measurement of Apolipoprotein B (ApoB) in Dried Blood Spots, a Biomarker of Cardiovascular Disease Risk

Abstract

Apolipoprotein B (ApoB) is a strong predictor of cardiovascular disease, which remains the leading cause of mortality in both higher and lower income countries. Here, we adapted an enzyme-linked immunosorbent assay (ELISA) development kit for quantitative determination of ApoB levels in serum and plasma for use with dried blood spots (DBS). After confirming the dilution linearity of the assay for DBS, we measured ApoB in 208 venous DBS samples. Then, using Passing-Bablok regression analysis and Spearman rank correlation analysis, we evaluated the correspondence in ApoB values between matched plasma and finger-prick DBS samples from 40 individuals who had ApoB values spanning the range of ApoB values observed in the 208 vDBS samples. We also evaluated assay precision and recovery, the effects of hematocrit, number of freeze-thaw cycles, and different storage temperatures on ApoB levels in DBS. There was a strong, significant correlation between plasma and DBS ApoB levels with little bias. Assay precision and recovery were within the range recommended by the U.S. government's industry guidelines for bioanalytical assay validation. The assay was not affected by the DBS matrix or physiological hematocrit levels. This DBS-based ELISA assay will facilitate population-scale assessment of cardiovascular risk in previously unexplored populations.

Validation of an enzyme‐linked immunoassay assay for osteocalcin, a marker of bone formation, in dried blood spots

“…The apparent stability of osteocalcin in DBS at room temperature is noteworthy, as osteocalcin has been shown to be unstable in vitro both at room temperature and at 4°C (Banfi & Daverio, ; Garnero et al, ). This suggests that the filter paper matrix protected against degradation, as has been demonstrated previously for other analytes such as TRACP‐5b, hydrolases, and esterases (Eick et al, ; Freeman et al, ). Finally, the results we obtained for the carboxylated osteocalcin standard from Anaspec suggest that the Abcam assay targets both carboxylated and undercarboxylated forms of full‐length osteocalcin (ie, total osteocalcin; no information is provided by the kit manufacturer regarding the specificity of the kit for carboxylated or uncarboxlyated osteocalcin), indicating that the osteocalcin values obtained using this kit can be used as a biomarker of bone formation.…”

“…However, when the vDBS and fDBS values were converted to their plasma‐equivalent values and compared, no obvious bias was observed in the Bland‐Altman plot (Figure D). It is unclear what to attribute the higher fDBS than vDBS values to; we previously observed slightly higher levels of TRACP‐5b in vDBS than fDBS, and attributed this to the generally smaller diameters of the fDBS than vDBS and correspondingly lower analyte concentrations (Eick et al, ; George & Moat, ), but we observed the opposite pattern here.…”

“…One hundred fifty‐eight matched fingerprick DBS (fDBS), venous blood DBS (vDBS), and plasma samples collected from a convenience sample of adults in Eugene/Springfield, Oregon between November 2014 and February 2015 were used to validate the osteocalcin ELISA following an established protocol (Eick, Kowal, Barrett, Thiele, & Snodgrass, ). Ninety‐one participants were female, with an age range of 18 to 72 years, median age of 26 years, and average age of 31.6 years.…”

A dried blood spot‐based method to measure levels of tartrate‐resistant acid phosphatase 5b (TRACP‐5b), a marker of bone resorption

“…Validation samples comprised matched fingerprick DBS (fDBS), venous DBS (vDBS), and plasma samples ('Eugene200 Validation Set') collected from a convenience sample of 189 adults (≥18 years) from the Eugene/ Springfield, Oregon (USA) area between November 2014 and February 2015 as described in detail previously (Eick, Kowal, Barrett, Thiele, & Snodgrass, 2017). All samples were collected in the evening between 5 PM and 9 PM, but exact time of collection was not recorded and, therefore, not considered in the analyses.…”

Development and validation of an ELISA for a biomarker of thyroid dysfunction, thyroid peroxidase autoantibodies (TPO-Ab), in dried blood spots

“…Validation samples comprised matched fingerprick DBS (fDBS), venous DBS (vDBS), and plasma samples ("Eugene200 Validation Set" or E2V2) collected from a convenience sample of 182 adults (M to F 76:106; ≥ 18 years) from the Eugene/Springfield, Oregon (USA) area between November 2014 and February 2015 as described in detail previously [16]. IRB approval for this study was obtained from the Committee for the Protection of Human Subjects, University of Oregon (protocol # 7062016.007), and informed consent was obtained from all participants.…”