Quantification of lipoprotein(a) particles containing various apolipoprotein(a) isoforms by a monoclonal anti-apo(a) capture antibody and a polyclonal anti-apolipoprotein B detection antibody sandwich enzyme immunoassay

Abstract

A quantitative sandwich ELISA for lipoprotein(a) [Lp(a)], utilizing a monoclonal capture antibody that recognizes human and rhesus monkey apolipoprotein(a) [apo(a)] isoforms in combination with a polyclonal anti-apolipoprotein B-peroxidase conjugate was developed. This assay generates a linear calibration curve from 31.2 to 1000 mg/L, is highly reproducible (intra- and interassay CV of < 5% and < or = 12%, respectively), and shows no interference from plasminogen (1 g/L), low-density lipoprotein (6.00 g/L), triglycerides (27.00 g/L from chylomicrons and 10.00 g/L from very-low-density lipoprotein), hemoglobin (5 g/L), or bilirubin (30 mg/L). This assay format quantifies the concentration of Lp(a) on an equal molar basis regardless of apo(a) isoform. In contrast, a commercially available ELISA [Macra Lp(a)] method with a monoclonal anti-apo(a) capture antibody and a polyclonal anti-apo(a) conjugate was found to underestimate the Lp(a) concentrations of individuals with lower-M(r) apo(a) isoforms--whether quantifying the Lp(a) in plasma or the purified lipoprotein. This demonstrates the importance of assay format selection in quantifying Lp(a).

Serum lipids, lipoprotein(a) level, and apolipoprotein(a) isoforms as prognostic markers in patients with coronary heart disease

“…Western blot analysis was performed with monoclonal antibody 2‐D1 (PerImmune, Rockville, MD, USA) as described [29, 30]. The apo(a) isoforms were separated into seven categories according to their electrophoretic mobility, as described by Utermann et al .…”

Lipoprotein(a): what's in a measure?

“…In`sandwich' ELISAs, a popular antibody combination that has been used is a capture apo(a) antibody bound to a solid phase to isolate Lp(a) particles and subsequent detection by (enzyme-linked) anti-LDL or anti-apo B antibody since each Lp(a) particle includes only one such moiety. 20 The Mercodia IRMA (Mercodia AB) method, however, uses two different anti-apo(a) antibodies (the detector antibody is 125 I-linked).…”

Effect of sample storage on quantitation of lipoprotein (a) by an enzyme‐linked immunosorbent assay

“…Again our data for loss of Lp(a) are closer to those reported by Kronenberg et al (39) for plasma (EDTA) samples: 7% after two years at -80~ as measured by ELISA (Boehringer). Fless et al (31) and Taddei-Peters et al (30) both studying filter sterilized EDTA plasma and using ELISA employing apo(a) capture and apoB detection antibodies reported that Lp(a) was stable for at least one month and one year, respectively.…”

“…Additional documentation of the response of these two isoforms to the antibody is unavailable. Standardization of the assay is further complicated by the occurrence of over 30 isoforms (5,6), and Taddei-Peters et al (30) report that this assay underestimates the concentration of the smaller isoforms. These limitations, however, do not affect the comparison of Lp(a) concentrations in the same groups of samples stored for different periods of time and at different temperatures, although it complicates attempts to interpret the influence of isoform size.…”