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Development of a Novel Sandwich ELISA for Measuring Cell Lysate ABCA1 Protein Levels

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Lipids

Abstract

ATP-binding cassette transporter A-1 (ABCA1) mediates the transfer of cellular cholesterol to lipid-poor apolipoproteins. Liver X receptors (LXRs) are regulators of cholesterol homeostasis that increase transcription of ABCA1. Synthetic LXR agonists developed to date have been shown to induce ABCA1 mRNA expression and increase reverse cholesterol transport. Unfortunately, there have been few options for quantitatively measuring ABCA1 protein levels, including a previously described competitive ELISA standardized to an ABCA1 peptide with a sensitivity of 80 ng/ml. To address this unmet need, we developed a novel sandwich ELISA standardized to full-length human recombinant ABCA1 protein with sensitivity of approximately 0.5 ng/ml. To determine if the sandwich ELISA had adequate sensitivity to detect LXR-induced increases in ABCA1, we utilized it to measure ABCA1 levels in untreated and LXR agonist-treated human (THP-1) macrophage cells and human peripheral blood mononuclear cells (PBMC). Data obtained from the ELISA demonstrated an approximately eightfold increase in ABCA1 levels in both macrophages as well as PBMC in response to LXR agonist treatment, and results were highly correlated with those obtained by immunoprecipitation and western blotting. Together, these results suggest that the sandwich ELISA may be a sensitive and effective method for quantitating ABCA1 protein levels.

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Abbreviations

ABCA1:

ATP-binding cassette transporter A-1

ApoA1:

Apolipoprotein A1

CPT:

Cell preparation tube

DMSO:

Dimethyl sulfoxide

ELISA:

Enzyme-linked immunosorbent assay

HDL:

High density lipoprotein

HDL-C:

High density lipoprotein cholesterol

LXR:

Liver X receptor

LDL:

Low density lipoprotein

LDL-C:

Low density lipoprotein cholesterol

PBMC:

Peripheral blood mononuclear cells

PMA:

Phorbol 12-myristate 13-acetate

PPAR:

Peroxisome proliferator-activated receptor

RCT:

Reverse cholesterol transport

TD:

Tangier disease

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Authors and Affiliations

  1. Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, 46285, USA

    Jason S. Troutt, William E. Alborn, Melissa A. Bellinger, Karen L. Cox, Mariam E. Ehsani, Xiliang Wang, Yue-wei Qian, John H. Sloan, Mark B. Willey, Guoqing Cao & Robert J. Konrad

Authors
  1. Jason S. Troutt
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  2. William E. Alborn
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  3. Melissa A. Bellinger
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  4. Karen L. Cox
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  5. Mariam E. Ehsani
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  6. Xiliang Wang
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  7. Yue-wei Qian
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  8. John H. Sloan
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  11. Robert J. Konrad
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Correspondence to Robert J. Konrad.

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Troutt, J.S., Alborn, W.E., Bellinger, M.A. et al. Development of a Novel Sandwich ELISA for Measuring Cell Lysate ABCA1 Protein Levels. Lipids 45, 757–764 (2010). https://doi.org/10.1007/s11745-010-3448-6

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  • DOI: https://doi.org/10.1007/s11745-010-3448-6

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