Two-site enzyme immunoassay for alpha-fetoprotein in dried-blood samples collected on filter paper

This is a method for measuring alpha-fetoprotein (AFP) in eluates of dried-blood samples on filter paper by use of a simple, sensitive two-site enzyme immunoassay. The spot, 6 mm in diameter (equivalent to about 12 microL of whole blood), is incubated overnight with alkaline phosphatase conjugated to rabbit anti-AFP antibody in a tube containing a polystyrene bead coated with mouse monoclonal antibody to AFP. After the beads are washed the enzyme activities associated with them are determined colorimetrically, with p-nitrophenyl phosphate as substrate. The measurable range of AFP is from 9 to 900 micrograms per liter of plasma. AFP in the dried-blood spot as determined by this method correlated well with the AFP value for serum from the same blood sample as determined by radioimmunoassay (r = 0.957, p less than 0.001). Preliminary studies in which we used this method with 242 healthy blood donors and 60 patients with hepatocellular carcinoma indicate that it may be suitable for use in mass screening for hepatocellular carcinoma in high-risk populations.