The Development and Evaluation of a Time-Resolved Fluoroimmunoassay for Lipoprotein (a) in Human Serum

Lipoprotein (a) (Lp(a)) has a complex structure which includes two apolipoproteins, apolipoprotein B (which is also present in other serum lipoproteins) and apolipoprotein (a) (which is unique to Lp(a)). Raised serum concentrations of Lp(a) have been associated with an increased risk of coronary heart disease. Commercially-available Lp(a) assay systems suffer from poor analytical performance. In this study, cell lines secreting anti-Lp(a) antibodies are identified using a series of immunoassay-based screening tests. Monoclonal antibodies developed from these cells lines are used (in conjunction with commercially-available polyclonal anti-Lp(a) antibodies) to develop a two-site time-resolved fluoroimmunoassay for Lp(a) in human serum. This assay format requires one antibody to be immobilised onto the surface of a microtitre plate and a second antibody to be labelled with an element from the lanthanide series (europium) which, under certain reaction conditions, is fluorescent. Protocols for the labelling of polyclonal and monoclonal antibodies with europium are presented in this thesis. Various combinations of anti-Lp(a) antibodies are used in assay development. The combination found to give the best assay performance with the minimum of interference from other biomolecules was:- a monoclonal anti-apo (a) capture antibody with a europium-labelled polyclonal anti-apo B detection antibody. Data obtained during the optimisation of antibody concentrations, sample dilution and reaction times is also presented. In the optimised assay, 200μl of a 1:1000 dilution of serum is incubated with the capture antibody for three hours. Following a wash step, immune complexes are detected fluorimetrically after incubation with a europium-labelled second antibody for two hours. The performance of the assay is compared to that of commercially-available kit assays. The time-resolved fluorescence assay is shown to be more precise and to have a much wider working range (1-1500 mg/L) than the commercial assays. However, data from analysis of samples distributed as part of an external quality control scheme is used to demonstrate the wide inter-assay variability of Lp(a) measurements. Measured Lp(a) concentrations in a series of human serum samples demonstrate the highly skewed distribution previously reported in adult Caucasian populations. No significant differences are observed in the mean serum Lp(a) concentrations of males and females and no correlation could be made between age and serum Lp(a) concentration in the adult population studied. In a limited study, a wide day-to-day percentage variation in serum Lp(a) concentration is demonstrated in one subject