HCC Biomarkers

AFP-L3 and DCP

AFP-L3% Methodology

The AFP-L3% test is FDA cleared.

The LiBASys® (liquid phase binding assay system) is an automated analyzer that measures the total AFP concentration and the AFP-L3% in serum simultaneously using a liquid phase binding assay method. With this method, the antigen-antibody reaction is performed in the liquid phase, and B/F separation is performed in a short time using column chromatography and without using a solid phase.

AFP-L3% is a ratio of LCA-reactive AFP to total AFP. The LiBASys® assay involves LCA; 3 anti-AFP monoclonal antibodies, each detecting a different epitope; and a substrate for fluorophotometric measurement. When a serum sample is mixed with LCA and anion 1 conjugated anti-AFP monoclonal antibody, LCA binds to the sugar chain of LCA-reactive AFP, which has an additional fucose residue, whereas anion 1-conjugated anti-AFP monoclonal antibody binds to all the present AFP molecules, forming immune complexes.

These immune complexes then react with peroxidase-labeled anti-AFP monoclonal antibody and anion 2 conjugated anti-AFP monoclonal antibody. Due to steric hindrance, previously reacted LCA prohibits the binding of anion 2-conjugated anti-AFP on AFP-L3 (LCA-reactive AFP), however it is able to bind with all LCA-nonreactive AFP. The peroxidase-labeled anti-AFP monoclonal antibody binds to all the present AFP molecules.

Two immune complexes with different anionic charges are formed. The reaction mixture, which includes the 2 complexes, is introduced to an anion-exchange column, and by means of a stepwise gradient with buffers containing different salt concentrations, immune complex fractions are eluted into separate reaction cups.

The peroxidase activity of each complex is measured after the addition of a substrate and is determined as the increase of fluorescence intensity of 5,5'-diacetoamide-2,2'-bisphenol formed by the reaction of hydrogen peroxide and 4-acetamidophenol. The total fluorescence intensity of complex 1 and complex 2 represents the AFP concentration, and the intensity of complex 1/(complex 1 + complex 2) represents the AFP-L3% result. The AFP concentration and AFP-L3% are calculated by comparing the increasing rate in fluorescence intensity obtained with those of the standards with known AFP concentrations (ng/mL) and AFP-L3%.

AFP-L3% Flow of Measurement