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Validation of Human ApoB and ApoAI Immunoturbidity Assays for Non-human Primate Dyslipidemia and Atherosclerosis Research

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Abstract

Emerging evidence suggests apolipoprotein B (apoB) and apolipoprotein AI (apoAI) are strong risk predictors for atherosclerosis. Non-human primates (NHP), including rhesus monkeys, cynomolgus monkeys, and African green monkeys, are important preclinical species for studying dyslipidemia and atherosclerosis as they more closely resemble humans in lipid metabolism and disease physiology compared to lower species such as rodents. However, no commercial assays are currently available for measuring apoB and apoAI in NHP. We therefore evaluated analytical methods for routinely measuring apoB and apoAI in our NHP dyslipidemia and atherosclerosis research. Since NHP apoB and apoAI sequences are likely highly similar to human, we focused on the clinically validated and widely utilized human apoB and apoAI immunoturbidity assays. We carried out technical validation of these assays with NHP samples and leveraged orthogonal technical platforms including mass spectrometry, independent ELISA assay, and absolute quantitation via SDS–PAGE for further characterization. Analysis of purified lipoproteins demonstrated that the immunoturbidity assays detect NHP apoAI and apoB, with good dilution linearity and spike recovery from NHP plasma. Orthogonal studies showed apoAI correlated with protein concentration and apoB levels correlated with LC/MS and an independent ELISA. NHP samples from a drug treatment study were analyzed with the immunoturbidity assays and levels of apoB and apoAI fit our understanding of biology and expectations from literature. These studies serve as important technical and biological validation of the immunoturbidity assays for NHP samples, and demonstrate that these assays provide a high-throughput, fully automated analytical platform for NHP samples. Our studies pave the way for future translational research in NHP for developing therapies for treating dyslipidemia and atherosclerosis.

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Acknowledgments

We thank Sang Ho Lee for assistance with FPLC analysis on lipoprotein particles and Doug Johns for discussions. We thank Wei Wang for bioinformatics support on NHP sequence assembly and comparison to human. For declaration of conflicting interests, all authors are employees and may hold stocks or stock options of Merck & Co.

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Authors and Affiliations

  1. Atherosclerosis, Cardiovascular Diseases, Merck Research Laboratories, RY80T-A100, 126 E. Lincoln Ave., Rahway, NJ, 07065, USA

    Zhu Chen, Alison M. Strack, Alice C. Stefanni, Ying Chen, Weizhen Wu, Theresa McLaughlin, Neil Geoghagen, Michael E. Lassman, Thomas P. Roddy, Kenny K. Wong & Brian K. Hubbard

  2. Bioanalytics & Pathology, In Vivo Sciences, Merck Research Laboratories, RY80B-100, 126 E. Lincoln Ave., Rahway, NJ, 07065, USA

    Yi Pan, Olga Urosevic-Price, Li Wang & Amy M. Flattery

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  1. Zhu Chen
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Supplementary Fig. 1

FPLC analysis of the LDL preparations showing their size difference. The LDL preparation from the ultracentrifugation separation method from African green monkeys, rhesus monkeys, and cynomolgus monkeys was further fractionated by FPLC, in the amount of 207 mg, 128 mg, and 113 mg protein input, respectively. x-axis is retention time, and y-axis is absorbance at 600 nm resulted from the in-line colorimetric detection of total cholesterol. Retention time of the LDL peak in each sample is noted on the graph. A small amount of VLDL in the preparation is also noted (PPT 79 kb)

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Chen, Z., Strack, A.M., Stefanni, A.C. et al. Validation of Human ApoB and ApoAI Immunoturbidity Assays for Non-human Primate Dyslipidemia and Atherosclerosis Research. J. of Cardiovasc. Trans. Res. 4, 373–383 (2011). https://doi.org/10.1007/s12265-011-9264-4

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