Detection of One Milliattomole of Ferritin by Novel and Ultrasensitive Enzyme Immunoassay

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Abstract
A novel and ultrasensitive enzyme immunoassay (immune complex transfer two-site enzyme immunoassay) for ferritin is described. Ferritin was reacted simultaneously with affinity-purified dinitrophenyl biotinyl anti-ferritin IgG and affinity-purified anti-ferritin Fab′-β-D-galactosidase conjugate. The complex formed of the three components was trapped onto affinity-purified (anti-dinitrophenyl group) IgG-coated polystyrene balls. After eliminating excess conjugate by washing, the complex was eluted from the polystyrene balls with an excess of εN-dinitrophenyl-L-lysine and transferred to streptavidincoated polystyrene balls. The β-D-galactosidase activity bound to streptavidin-coated polystyrene balls was assayed by fluorometry. Nonspecifically bound β-D-galactosidase activity was remarkably lowered but there was much less decrease in specifically bound β-D-galactosidase activity. As a result, the detection limit of ferritin was lowered to 1 milliattomole (1×1O−21 mol, 600 molecules as calculated from Avogadro's number). This technique will be useful for measuring, for example, antigens in single cells.