Use of microwell plates carrying hydrazide groups to enhance antibody immobilization in enzyme immunoassays.

Using microwell plates functionalized with surface hydrazide groups, we site-specifically immobilized antibodies to the surface of the wells. The procedure for site-specific, oriented immobilization of IgG to hydrazide surfaces is simple. It requires a brief, mild oxidation of the carbohydrate side chains of IgG with NaIO4 at a pH of approximately 5 and incubation of the oxidized IgG with the hydrazide surface. These oxidized immunoglobulins bind to the hydrazide groups of these plates preferentially by the Fc region of the molecule, resulting in improved specific activity. Enzyme immunoassays for horseradish peroxidase and human IgG were tested on both standard (hydrophobic) and hydrazide group-carrying surfaces. The improved antigen binding capacity of the hydrazide-modified plates results in greatly increased sensitivity (approximately 4 x) and improved linearity in both assays when compared to native, nonoxidized antibody used with standard microwell plates. Furthermore, the hydrazide functionalized plates exhibited lower non specific binding.

Spectrophotometric assay for ornithine decarboxylase.

A rapid and sensitive spectrophotometric assay for ornithine decarboxylase is described. It is based on the observation that the product of ornithine decarboxylase, putrescine, reacts with 2,4,6-trinitrobenzenesulfonic acid to give a colored product soluble in 1-pentanol whereas ornithine does not. The amount of putrescine produced by the enzyme was determined by measuring the absorbance of the 1-pentanol extract of the reaction mixture at 420 nm, and by comparing the results to those obtained by the trapping of 14CO2 and by HPLC assays. The three assays were found to be equivalent in sensitivity, with the spectrophotometric assay having the advantages of being relatively rapid, requiring only common laboratory equipment, and not requiring the use of radioactive isotopes.