Enzyme-linked Sandwich Immunoassay of Ornithine <5-Aminotransferase from Rat Liver Using Antibody-coupled Glass Rods as Solid Phase

A macromolecular antigen, ornithine 5-aminotransferase [EC 2.6.1.13] from rat liver (OAT) was assayed by the sandwich procedure using a rabbit (anti-OAT) Fab'-£-r> galactosidase complex and rabbit (anti-OAT) IgG-coupled glass rods as a solid phase. The Fab ' fragments of the rabbit (anti-OAT) IgG were conjugated with ^-D-galacto-sidase [EC 3.2.1.23] from Escherichia coli using N, N'-o-phenylenedimaleimide. The rabbit (anti-OAT) IgG was coupled to the aminoalkylsilyl glass rods using glutaral-dehyde. The rabbit (anti-OAT) IgG-coupled glass rods were incubated with OAT and then with the rabbit (anti-OAT) Fab'-/3-D-galactosidase complex. The amount of OAT was determined from the activity of ^9-r>galactosidase bound to the glass rods. A minimum of 0.03 fmoles of OAT could be determined by this method and use of the glass rods gave greater reproducibility, and was more sensitive and simpler than use of Sepharose 4B. Several procedures have been developed for the enzyme-linked solid phase sandwich im-munoassay of macromolecular antigens (1—7). The enzymes that have been used for labelling antibodies are alkaline phosphatase [orthophos-phoricmonoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] (1), peroxidase [donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7] (2), glucose oxidase [/9-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4] (3), and /3-D-ga-lactosidase [£-r>galactoside galactohydrolase, EC 3.2.1.23] (4-7). Antibodies have been labelled with enzymes using glutaraldehyde (1—3) or N, N'-o-phenylenedimaleimide (4—7)