Determination of the concentration of a-fetoprotein using an Alexa fluor-based fluorescence immunoassay

Alpha fetoprotein (AFP) has been identified as a specific marker of hepatocellular carcinoma (HCC). Our perpose is to develop a new fluorescence immunoassay based on Alexa fluor-647 to determine the concentration of AFP in serum. Alexa fluor-647 and magnetic beads were applied in labeling two different anti-AFP monoclonal antibodies. Both labeled antibodies and AFP antigen formed a sandwiched immunocomplex. After washing in a magnetic field, the fluorescent intensity of Alexa fluor-647 in the immunocomplex was measured and the value was directly in proportion to the levels of AFP present in the samples. The influence of two physicochemical parameters involved in the assay signals were optimized and the parameters of the proposed method were assessed. The consequence showed the detection limit of the proposed method was 1.1 ng/mL. The coefficient of variation (CV) was less than 6% for intra-assay precision. An entire assay could be finished in 25 min. This assay provided a new way to quantitatively measure AFP in serum for the diagnosis of HCC.