Detection of one milliattomole of ferritin by novel and ultrasensitive enzyme immunoassay.
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'-beta-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 epsilon N-dinitrophenyl-L-lysine and transferred to streptavidin-coated polystyrene balls. The beta-D-galactosidase activity bound to streptavidin-coated polystyrene balls was assayed by fluorometry. Nonspecifically bound beta-D-galactosidase activity was remarkably lowered but there was much less decrease in specifically bound beta-D-galactosidase activity. As a result, the detection limit of ferritin was lowered to 1 milliattomole (1 x 10(-21) mol, 600 molecules as calculated from Avogadro's number). This technique will be useful for measuring, for example, antigens in single cells.
Hashida
S
S
Department of Biochemistry, Medical College of Miyazaki.
Ishikawa
E
E
D000936:Antigen-Antibody Complex; D004306:Dose-Response Relationship, Immunologic; D005293:Ferritins / Q000032:analysis* / Q000276:immunology; D006801:Humans; D007124:Immunoenzyme Techniques; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D008099:Liver / Q000737:chemistry; D012680:Sensitivity and Specificity; D001616:beta-Galactosidase / Q000378:metabolism
D000936:Antigen-Antibody Complex; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D005293:Ferritins; D001616:beta-Galactosidase