A novel enzyme immunoassay specific for ABCA1 protein quantification in human tissues and cells.
ATP-binding cassette transporter A1 (ABCA1) mediates the transport of cholesterol and phospholipids from cells to lipid-poor HDL and maintains cellular lipid homeostasis. Impaired ABCA1 function plays a role in lipid disorders, cardiovascular disease, atherosclerosis, and metabolic disorders. Despite the clinical importance of ABCA1, no method is available for quantifying ABCA1 protein. We developed a sensitive indirect competitive ELISA for measuring ABCA1 protein in human tissues using a commercial ABCA1 peptide and a polyclonal anti-ABCA1 antibody. The ELISA has a detection limit of 8 ng/well (0.08 mg/l) with a working range of 9-1000 ng/well (0.09-10 mg/l). Intra- and interassay coefficient of variations (CVs) were 6.4% and 9.6%, respectively. Good linearity (r = 0.97-0.99) was recorded in serial dilutions of human arterial and placental crude membrane preparations, and fibroblast lysates. The ELISA measurements for ABCA1 quantification in reference arterial tissues corresponded well with immunoblot analysis. The assay performance and clinical utility was evaluated with arterial tissues obtained from 15 controls and 44 patients with atherosclerotic plaques. ABCA1 protein concentrations in tissue lysates were significantly lower in patients (n = 24) as compared with controls (n = 5; 9.37 +/- 0.82 vs. 17.03 +/- 4.25 microg/g tissue; P < 0.01). The novel ELISA enables the quantification of ABCA1 protein in human tissues and confirms previous semiquantitative immunoblot results.
Physiology Weihenstephan, Technical University Munich, Weihenstephaner Berg 3, 85350 Freising, Germany.
Vijay
V
Paul
Heinrich H D
HH
Meyer
Katharina
K
Leidl
Soni
S
Soumian
Christiane
C
Albrecht
D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000276:immunology; D000906:Antibodies / Q000276:immunology; D001158:Arteries / Q000166:cytology / Q000276:immunology / Q000473:pathology; D050197:Atherosclerosis / Q000276:immunology / Q000473:pathology; D001667:Binding, Competitive; D016022:Case-Control Studies; D002477:Cells / Q000737:chemistry* / Q000276:immunology / Q000473:pathology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D015151:Immunoblotting; D016014:Linear Models; D010920:Placenta / Q000166:cytology / Q000276:immunology / Q000473:pathology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors
C577836:ABCA1 protein, human; D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters; D000906:Antibodies