Immunosensing system for alpha-fetoprotein through boronate immunoaffinity column in combination with flow injection chemiluminescence

A novel immunosensing system for determination of human a-fetoprotein (AFP) was proposed by using a boronate immunoaffinity column as the glycated antigen collector in combination with flow injection chemiluminescence. The column was fabricated by filling boronic acid-modified sepharose gel into a glass tube. With a sugar-boronic acid interaction, the AFP antigen could be effectively immobilized on the sepharose gel matrix. After an off-line incubation, the mixture of the analyte AFP and horseradish peroxidase-labeled AFP antibody (HRP-anti-AFP) was injected into the column. The free HRP-anti-AFP was trapped by the immobilized antigen in the column and detected via chemiluminescence due to its sensitive effect on the reaction of luminol and hydrogen peroxide. A calibration curve with two linear ranges of 5-120 and 300-1000 ng mL(-1) was obtained under the optimized conditions. The whole assay process including regeneration of the reactor can be completed in 36 min. The presented immunoassay exhibited a high sensitivity, a wide linear range, a low interference with other antigens and a good reproducibility. It is potentially used to detect the serum AFP level in clinical diagnosis.