The intra-individual variation in plasma A8 levels was determined in 48 unrelated fasting individuals during 4 separate visits over a 6-week period (40). not been fully investigated. To glean further insights into the role of A8 in lipid and glucose homeostasis, we developed a robust, sensitive, and specific sandwich ELISA. We used this assay to measure circulating A8 levels in a large, multiracial/multiethnic population, the Dallas Heart Study (DHS). Here, we report the relationship between circulating levels of A8, genetic variations, and metabolic features in a population-based study. Methods Antibodies and Purified Proteins A list of the names and sources for the reagents, instruments, software, and other materials used in the studies described in this paper is provided in Table 1. Recombinant full-length human A8 was cloned into pCold Trigger Factor to generate an A8 expression construct with a hexa-histidine tag at the C-terminus. The recombinant protein was expressed in bacteria and purified with Ni-NTA agarose. The purified protein was prepared using the Sigma Adjuvant System (50 g) and injected into New Zealand Black (NZB) mice (n = 2) 7 times. Mouse anti-human A8 monoclonal antibody (mAb) was prepared by fusing SP2-mIL6 mouse myeloma cells with splenic B lymphocytes from the NZB mice. Supernatants from cultured hybridomas were screened by ELISA, and positive clones were confirmed by immunoblotting. Two hybridomas, designated IgG-34B4 (subclass 1, light chain ) and the other IgG-42C1 (subclass 2a, ), were subcloned by serial dilution 3 times and purified by gravity-flow affinity chromatography on Protein G Sepharose 4 Fast Flow columns. Table 1. Reagents, instruments, software, and other materials used in this study for 10 minutes at 4 C and stored at ?80 C. Prior to use, plasma samples from all subjects were thawed on ice, diluted 8-fold in 0.9% NaCl and Laemmli SDS reducing sample buffer, and then incubated at 95 C for 4 minutes. Diluted plasma was size-fractionated by SDS-PAGE (12%) and transferred to a nitrocellulose membrane. Membranes were blocked in PBST with 5% fat-free milk for 60 minutes at room temperature before addition of primary antibodies diluted in PBST with 5% fat-free milk. After overnight incubation with the primary Ab at 4 C, membranes were washed 5 Piceatannol times for 5 minutes in PBST and incubated with secondary antibody in PBST with 1.25% fat-free milk (1:10 000 dilution). Following a 60-minute incubation at room temperature, membranes were washed again 5 times with PBST and visualized using SuperSignal-enhanced chemiluminescence substrate. Gel images were captured on Blue X-Ray films and LI-COR Odyssey Fc, and band intensities were analyzed using LI-COR Image Studio Lite version 5.2.5. Exome-Wide Association Study Genomic DNA extracted from circulating leukocytes of DHS participants was used to perform whole-exome sequencing at the Regeneron Genetics Center (Tarrytown, Piceatannol New York) as described (42, 43). After excluding variants that failed our quality control criteria (missing rate of > 5%, deviated from Hardy-Weinberg equilibrium with a < 1.0 10?6, Piceatannol were outside of the capture region or had a minor allele count < 5), a total of 346 167 bi-allelic variants were tested for association with circulating A8 PSEN2 levels. Our primary analysis was performed in DHS-1 participants with whole-exome sequencing, plasma A8 levels and covariate data available (n = 3457). Plasma A8 levels were transformed using a rank-based, inverse normal transformation and tested for association with sequence variants using linear regression with adjustment for age, sex, 4 principal components of genetic ancestry, and body mass index (BMI), using PLINK version 1.9b (33). The reported values were calculated assuming an additive genetic model. Statistical Methods Baseline characteristics were compared between racial/ethnic groups using Kruskal-Wallis test (continuous traits) and Fishers exact test (categorical variables). The correlations between A8 levels and continuous phenotypes were assessed using Spearmans rank correlation coefficient. Partial correlations were computed after adjusting both traits for age, sex, self-reported race/ethnicity and BMI, and correlating the residuals. Independent predictors of A8 levels were assessed using a multiple linear regression model adjusted for age, sex and self-reported race/ethnicity. The association of A8 levels with type 2 diabetes was tested using logistic regression, adjusted for age, sex, self-reported race/ethnicity and BMI. The association of rs2278426 A8(R59W) genotype.