A novel enzyme immunoassay specific for ABCA1 protein quantification in human tissues and cells

Abstract

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 5 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 5 24) as compared with controls (n 5 5; 9.37 6 0.82 vs. 17.03 6 4.25 mg/g tissue; P , 0.01). The novel ELISA enables the quantification of ABCA1 protein in human tissues and confirms previous semiquantitative immunoblot results.-Paul, V., H. H. D. Meyer, K. Leidl, S. Soumian, and C. Albrecht. A novel enzyme immunoassay specific for ABCA1 protein quantification in human tissues and cells.

An In-Silico Model of Lipoprotein Metabolism and Kinetics for the Evaluation of Targets and Biomarkers in the Reverse Cholesterol Transport Pathway

“…In particular, ABCA1 gene expression and protein concentration on leukocytes has been measured in patients with type 2 diabetes, where the data suggested a negative correlation between ABCA1 expression and HbA1c levels [59]. While there are assays that can quantify ABCA1 protein levels in specific cell types [60], an experimental technique for the assessment of ABCA1 activity in-vivo at the whole body level has yet to be developed. Given the current experimental limitations, there is an interest to evaluate the potential effectiveness of plasma-based biomarkers for quantitatively assessing ABCA1 activity.…”

Increased Placental Phospholipid Levels in Pre-Eclamptic Pregnancies

“…Cholesterol and phospholipid efflux from various tissues is mediated via ATP-binding cassette (ABC) transporters, in particular ABCA1 [44–48]. ABCA1 is highly expressed in the placenta [46–48] and was suggested to play a role in cholesterol transfer from the maternal to the fetal circulation [49].…”

Type 2 Diabetes Is Associated with Reduced ATP-Binding Cassette Transporter A1 Gene Expression, Protein and Function

“…The clear leukocyte cell lysates were stored at −80°C until ABCA1 protein measurement was performed. Frozen leukocyte lysates were thawed and ABCA1 protein concentration was measured using an ELISA method as previously described [18]. Briefly, either 50 µl ABCA1 peptide (ab14148, Abcam, UK) calibrators (range 8–1000 ng) or 50 µl of unknown leukocyte lysates were incubated in a ABCA1 peptide pre-coated and BSA blocked micro titre plate along with 100 µl (2 µg/l diluted in PBST) anti-ABCA1 rabbit antibody (ab7360, Abcam, UK).…”