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

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.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/162811/2/ajhb23394.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162811/1/ajhb23394_am.pd

Protein Evolution by Molecular Tinkering: Diversification of the Nuclear Receptor Superfamily from a Ligand-Dependent Ancestor

Phylogenetic reconstruction of the structure and function of the ancestor of the nuclear receptor protein family reveals how functional diversity evolves by subtle tinkering with an ancestral template

The utility of dried blood spot measurement of bone turnover markers in biological anthropology

ObjectivesBone is a dynamic organ under continual turnover influenced by life history stage, energy dynamics, diet, climate, and disease. Bone turnover data have enormous potential in biological anthropology for testing evolutionary and biocultural hypotheses, yet few studies have integrated these biomarkers. In the present article we systematically review the current availability, future viability, and applicability of measuring bone turnover markers (BTMs) in dried blood spot (DBS) samples obtained from finger prick whole blood.MethodsOur review considers clinical and public health relevance, biomarker stability in DBS, assay availability, and cost. We consider biomarkers of bone formation such as osteocalcin (bone matrix protein), PINP (N-terminal propeptide of type I collagen), and alkaline phosphatase (osteoblast enzyme), as well as biomarkers of bone resorption such as CTX (marker of collagen breakdown) and TRACP5b (tartrate-resistant acid phosphatase 5b; osteoclast enzyme).ResultsTwo BTMs have been validated for DBS: osteocalcin (formation) and TRACP5b (resorption). Prime candidates for future development are CTX and PINP, the formation and resorption markers used for clinical monitoring of response to osteoporosis treatment.ConclusionBTMs are a field-friendly technique for longitudinal monitoring of skeletal biology during growth, reproduction and aging, combining minimized risk to study participants with maximized ease of sample storage and transport. This combination allows new insights into the effects of energy availability, disease, and physical activity level on bone, and questions about bone gain and loss across life history and in response to environmental factors; these issues are important in human biology, paleoanthropology, bioarchaeology, and forensic anthropology.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/175199/1/ajhb23816.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/175199/2/ajhb23816_am.pd

A Nuclear DNA Phylogenetic Perspective on the Evolution of Echolocation and Historical Biogeography of Extant Bats (Chiroptera)

Bats (Order Chiroptera), the only mammals capable of powered flight and sophisticated laryngeal echolocation, represent one of the most species-rich and ubiquitous orders of mammals. However, phylogenetic relationships within this group are poorly resolved. A robust evolutionary tree of Chiroptera is essential for evaluating the phylogeny of echolocation within Chiroptera, as well as for understanding their biogeographical history. We generated 4 kb of sequence data from portions of four novel nuclear intron markers for multiple representatives of 17 of the 18 recognized extant bat families, as well as the putative bat family Miniopteridae. Three echolocation-call characters were examined by mapping them onto the combined topology: (1) high-duty cycle versus low-duty cycle, (2) high-intensity versus low-intensity call emission, and (3) oral versus nasal emission. Echolocation seems to be highly convergent, and the mapping of echolocation-call design onto our phylogeny does not appear to resolve the question of whether echolocation had a single or two origins. Fossil taxa may also provide insight into the evolution of bats; we therefore evaluate 195 morphological characters in light of our nuclear DNA phylogeny. All but 24 of the morphological characters were found to be homoplasious when mapped onto the supermatrix topology, while the remaining characters provided insufficient information to reconstruct the placement of the fossil bat taxa with respect to extant families. However, a morphological synapomorphy characterizing the Rhinolophoidea was identified and is suggestive of a separate origin of echolocation in this clade. Dispersal-Vicariance analysis together with a relaxed Bayesian clock were used to evaluate possible biogeographic scenarios that could account for the current distribution pattern of extant bat families. Africa was reconstructed as the center of origin of modern-day bat families

A dried blood spotâ based method to measure levels of tartrateâ resistant acid phosphatase 5b (TRACPâ 5b), a marker of bone resorption

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 fieldâ based populationâ level research. The present study addresses this gap by validating a fingerprick dried blood spot (fDBS) assay for tartrateâ resistant acid phosphatase 5b (TRACPâ 5b), a wellâ defined biomarker of bone resorption and osteoclast number.MethodsWe adapted a commercially available enzymeâ linked immunosorbent assay (ELISA) kit from MyBiosource for the quantitative determination of TRACPâ 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 TRACPâ 5b concentrations showed a linear relationship. There were no systematic differences in TRACPâ 5b levels in fDBS and vDBS, indicating no significant differences in TRACPâ 5b distribution between capillary and venous blood. Parallelism and spikeâ andâ recovery results indicated that matrix factors in DBS do not interfere with measurement of TRACPâ 5b levels from DBS using the validated kit. Intraâ 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 DBSâ based ELISA assay adds to the methodological toolkit available to human biologists and will facilitate research on bone turnover in population studies.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/149369/1/ajhb23240.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149369/2/ajhb23240_am.pd

Optimization of DNA Extraction from Dried Blood Spot Samples For Use In a Telomere Length Assay

Single pdf posterIn collaboration with colleagues at the World Health Organization (WHO), we are conducting a longitudinal study on global AGEing and adult health (SAGE). The SAGE project seeks to investigate patterns and determinants of aging in individuals around the world. Dried blood spots (DBS) are currently being collected from adults in six middle-income countries. These DBS will then be analyzed for a variety of biomarkers, including telomere length (TL)

Evolution of Minimal Specificity and Promiscuity in Steroid Hormone Receptors

Most proteins are regulated by physical interactions with other molecules; some are highly specific, but others interact with many partners. Despite much speculation, we know little about how and why specificity/promiscuity evolves in natural proteins. It is widely assumed that specific proteins evolved from more promiscuous ancient forms and that most proteins' specificity has been tuned to an optimal state by selection. Here we use ancestral protein reconstruction to trace the evolutionary history of ligand recognition in the steroid hormone receptors (SRs), a family of hormone-regulated animal transcription factors. We resurrected the deepest ancestral proteins in the SR family and characterized the structure-activity relationships by which they distinguished among ligands. We found that that the most ancient split in SR evolution involved a discrete switch from an ancient receptor for aromatized estrogens—including xenobiotics—to a derived receptor that recognized non-aromatized progestagens and corticosteroids. The family's history, viewed in relation to the evolution of their ligands, suggests that SRs evolved according to a principle of minimal specificity: at each point in time, receptors evolved ligand recognition criteria that were just specific enough to parse the set of endogenous substances to which they were exposed. By studying the atomic structures of resurrected SR proteins, we found that their promiscuity evolved because the ancestral binding cavity was larger than the primary ligand and contained excess hydrogen bonding capacity, allowing adventitious recognition of larger molecules with additional functional groups. Our findings provide an historical explanation for the sensitivity of modern SRs to natural and synthetic ligands—including endocrine-disrupting drugs and pollutants—and show that knowledge of history can contribute to ligand prediction. They suggest that SR promiscuity may reflect the limited power of selection within real biological systems to discriminate between perfect and “good enough.”</p

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

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 fieldâ based populationâ level research. The present study addresses this gap by validating a fingerprick dried blood spot (fDBS) assay for tartrateâ resistant acid phosphatase 5b (TRACPâ 5b), a wellâ defined biomarker of bone resorption and osteoclast number.MethodsWe adapted a commercially available enzymeâ linked immunosorbent assay (ELISA) kit from MyBiosource for the quantitative determination of TRACPâ 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 TRACPâ 5b concentrations showed a linear relationship. There were no systematic differences in TRACPâ 5b levels in fDBS and vDBS, indicating no significant differences in TRACPâ 5b distribution between capillary and venous blood. Parallelism and spikeâ andâ recovery results indicated that matrix factors in DBS do not interfere with measurement of TRACPâ 5b levels from DBS using the validated kit. Intraâ 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 DBSâ based ELISA assay adds to the methodological toolkit available to human biologists and will facilitate research on bone turnover in population studies.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/149369/1/ajhb23240.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149369/2/ajhb23240_am.pd

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

Abstract Background The prevalence of allergic and autoimmune conditions has been steadily increasing in wealthy nations over the past century. One hypothesis put forward to explain this is the Old Friends Hypothesis, which posits that increased hygiene, urbanization, and lifestyle changes have reduced our exposure to parasites and microbes that we co-evolved with, resulting in immune dysregulation. However, research in traditionally living populations, who are exposed to greater parasite and pathogen loads such as those encountered during our evolution, is limited, in part due to a lack of minimally invasive, field-friendly biomarkers of autoimmune disorders. We therefore developed an ELISA to assess positivity for thyroid peroxidase autoantibody (TPO-Ab), an indicator of autoimmune thyroid disease, based on dried blood spot (DBS) samples. Results We used the Accubind anti-thyroid peroxidase test system to screen our validation samples comprising matched fingerprick DBS, venous DBS, and plasma samples from 182 adults. After confirming that we had TPO-Ab-positive individuals in our validation sample (n = 12), we developed an indirect ELISA to measure TPO-Ab levels from one 3-mm DBS punch. The sensitivity and specificity of our assay for DBS samples ranged from 91.7–100% and 98.2–98.8%, respectively, using a cut-off value of ≥ 26 IU/mL. Intra-assay reliability for duplicate quality control DBS punches was 5.2%, while inter-assay reliability ranged from 11.5–24.4% for high, medium, and low DBS controls. Dilutional linearity ranged from 80 to 120%, and spike and recovery experiments indicated that the DBS matrix does not interfere with the detection of TPO-Ab. TPO-Ab levels remained stable in DBS samples stored at − 28 °C or − 80 °C, but decreased over time in DBS samples kept at 22 °C or at 37 °C. Conclusions We developed an in-house, kit-independent indirect ELISA assay to determine individuals’ TPO-Ab positivity based on dried blood spots, representing a cost-effective method with potential applications in a range of research settings.http://deepblue.lib.umich.edu/bitstream/2027.42/173958/1/40101_2020_Article_228.pd