Development of a novel sandwich ELISA for measuring cell lysate ABCA1 protein levels.
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.
Troutt
Jason S
JS
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA.
Alborn
William E
WE
Bellinger
Melissa A
MA
Cox
Karen L
KL
Ehsani
Mariam E
ME
Wang
Xiliang
X
Qian
Yue-wei
YW
Sloan
John H
JH
Willey
Mark B
MB
Cao
Guoqing
G
Konrad
Robert J
RJ
D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters / Q000032:analysis* / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D047468:Immunoprecipitation; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism
C577836:ABCA1 protein, human; D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters