An enzyme-linked immunosorbent assay for measuring GPIHBP1 levels in human plasma or serum

Abstract

BACKGROUND GPIHBP1, a glycosylphosphatidylinositol (GPI)-anchored protein of capillary endothelial cells, transports lipoprotein lipase to the capillary lumen and is essential for the lipolytic processing of triglyceride-rich lipoproteins. OBJECTIVE Because some GPI-anchored proteins have been detected in plasma, we tested whether GPIHBP1 is present in human blood, and whether GPIHBP1 deficiency or a history of cardiovascular disease affected GPIHBP1 circulating levels. METHODS We developed two monoclonal antibodies against GPIHBP1and used the antibodies to established a sandwich ELISA to measure GPIHBP1 levels in human blood. RESULTS The GPIHBP1 ELISA was linear in the 8–500 pg/ml range and allowed the quantification of GPIHBP1 in serum and in pre- and post-heparin plasma (including lipemic samples). GPIHBP1 was undetectable in the plasma of subjects with null mutations in GPIHBP1. Serum GPIHBP1 median levels were 849 pg/ml (range: 740–1014) in healthy volunteers (n = 28) and 1087 pg/ml (range: 877–1371) in patients with a history of cardiovascular or metabolic disease (n = 415). There was an extremely small inverse correlation between GPIHBP1 and triglyceride levels (r = 0.109; P <0.0275). GPIHBP1 levels tended to be slightly higher in patients who had a major cardiovascular event after revascularization. CONCLUSION We developed an ELISA for quantifying GPIHBP1 in human blood. This assay will be useful to identify patients with GPIHBP1 deficiency and patients with GPIHBP1 autoantibodies. The potential of plasma GPIHBP1 as a biomarker for metabolic or cardiovascular disease is yet questionable but needs additional testing.

Unfolding of monomeric lipoprotein lipase by ANGPTL4: Insight into the regulation of plasma triglyceride metabolism

“…Having shown that the "hotspot residues" in the 5D2 epitope participate directly in the binding interface of LPL homodimers, we next investigated the impact of 5D2 on homodimer formation. For these studies, we generated Fab fragments of both mAb 5D2 (37,40) and the human GPIHBP1-specific mAbs RF4 and RE3 (41,42). The epitope for RF4 is centered on Arg 53 −Leu 54 downstream from GPIHBP1's disordered acidic domain (43), whereas RE3 binds GPIHBP1's Ly6/uPAR domain and prevents GPIHBP1 from binding LPL (41).…”

An ELISA for quantifying GPIHBP1 autoantibodies and making a diagnosis of the GPIHBP1 autoantibody syndrome

“…While we had no difficulty in documenting multiple cases of GPIHBP1 autoantibodies with our ELISA [9, 12], we have yet to identify a single case of LPL autoantibodies (even in samples that were thought to have LPL autoantibodies). In the past, the antigens and antibodies used to detect LPL autoantibodies have not been optimal and have resulted in misleading results [15, 16].…”

“…Plasma triglyceride levels were determined with enzymatic assay (Quick Neo TG II, Shino-Test Corporation, Tokyo). Plasma GPIHBP1 levels were measured with a solid-phase monoclonal antibody–based sandwich ELISA (Immuno-Biological Laboratories, Fujioka, Japan) [12]. For the GPIHBP1 ELISA, serum and plasma were diluted 10-fold in PBS; 100 μL of the diluted samples were added to wells of the 96-well ELISA plate.…”

Chylomicronemia from GPIHBP1 autoantibodies

“…Seventeen of 22 GPIHBP1 autoantibody patients (patients 1-9, 13-15, 17-21) had very low plasma levels of GPIHBP1, as judged by a monoclonal antibody-based ELISA (35,38,55).…”

“…Monoclonal antibody-based ELISAs to measure plasma GPIHBP1 levels have been described in detail (35,38,55), and an ELISA kit to measure GPIHBP1 is available from Immuno-Biological Laboratories. Measuring GPIHBP1 levels is not essential.…”