DEVELOPMENT OF A HETEROGENEOUS AMPEROMETRIC IMMUNOSENSOR FOR THE DETERMINATION OF APOLIPOPROTEIN-E IN SERUM

Apolipoprotein (ape) E is a constituent of serum lipoproteins and can serve as a diagnostic parameter for the assessment of disorders of lip id metabolism. For the determination of apo E in serum a sandwich-type amperometric immunosensor using disposable membranes was developed. T he best results were obtained by using a site-directed attachment of a monoclonal capture antibody to a hydrazide-functionalized membrane su rface. Bound antigen was then determined with the aid of a polyclonal antibody labelled with alkaline phosphatase in conjunction with p-amin ophenyl phosphate as substrate. In this approach a carbon working elec trode (vs. Hg/HgCl2) was used, and enzymatically generated p-aminophen ol could be monitored with a detection limit of 40 pmol and a linear r ange 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 a ssay configuration. The approach with site-directed immobilization how ever, not only allowed the determination of apo E in serum, but also t he determination of antibody cross-reactivity against the apolipoprote ins AI, AII and B.