Development of a heterogeneous amperometric immunosensor for the determination of apolipoprotein E in serum.

Apolipoprotein (apo) E is a constituent of serum lipoproteins and can serve as a diagnostic parameter for the assessment of disorders of lipid metabolism. For the determination of apo E in serum a sandwich-type amperometric immunosensor using disposable membranes was developed. The best results were obtained by using a site-directed attachment of a monoclonal capture antibody to a hydrazide-functionalized membrane surface. Bound antigen was then determined with the aid of a polyclonal antibody labelled with alkaline phosphatase in conjunction with p-aminophenyl phosphate as substrate. In this approach a carbon working electrode (vs. Hg/HgCl2) was used, and enzymatically generated p-aminophenol could be monitored with a detection limit of 40 pmol and a linear range of 26-20000 nM. The sensor displayed a linear response from 50 to 1000 ng ml-1 apo E. In contrast, antibody coupling through primary amino groups led to a total loss of antigen binding capacity in this assay configuration. The approach with site-directed immobilization however, not only allowed the determination of apo E in serum, but also the determination of antibody cross-reactivity against the apolipoproteins AI, AII and B.