Noncompetitive enzyme immunoassay for alpha-fetoprotein using flow injection chemiluminescence

A novel, direct noncompetitive flow injection enzyme immunoassay for alpha-fetoprotein (AFP) was developed by enhanced chemiluminescence detection. The method was based on off-line incubation of AFP and horseradish peroxidase (HRP)-labeled anti-AFP, and then trapping of the unbound enzyme conjugate by an immunoaffinity column filled with AFP-modified Sepharose. The immunocomplex formed in incubation passed through the column and then was directly detected by a postcolumn chemiluminescence technique. The optimal conditions for the immunoassay procedure and chemiluminescence detection were established. At a 1:10 dilution of enzyme conjugate solution, the linear range for chemiluminescence detection of AFP was from 2.0 to 75 ng/mL with a correlation coefficient of 0.993 and a coefficient of variation of 2.67% at 30 ng/mL. The detection limit was 0.5 ng/mL. This method was flexible, sensitive, and rapid. The immunoaffinity column of 200 microL could be repeatedly used 100 times without a single decrease. The whole assay time including the preincubation step was only 30 min for one sample.