Two-site column enzyme immunoassay for neuron-specific enolase (NSE) in human serum using monoclonal antibodies

A new method for the rapid determination of neuron-specific gamma-enolase (NSE), gamma-subunit of alpha gamma- and gamma gamma-enolase in human serum was developed by employing monoclonal antibodies for the separation method. The assay system consists of 0.1 ml Sepharose 4B column with immobilized rabbit anti-mouse IgG antibodies for the separation of bound label, Fab' fragments of rabbit anti-bovine gamma gamma-enolase IgG labeled with beta-D-galactosidase from Escherichia coli, and F(ab')2 fragments of two mouse monoclonal antibodies to gamma gamma-enolase. Serum samples or standard NSE solutions were incubated at 30 degrees C with the monoclonal antibody fragments. 10 min later, the galactosidase-labeled antibody fragments were added to the mixture, and incubated at 30 degrees C for 30 min. Then the reaction mixture was applied to a micro-column of Sepharose 4B with immobilized anti-mouse IgG antibodies. From the galactosidase activity bound in the column, NSE concentration in the samples could be estimated within 2 h. The minimum detection limit of the assay system was 30 pg/tube, being sufficiently sensitive for the assay of serum NSE with a satisfactory precision. Serum concentrations of NSE determined by the present method correlated well with that by the colorimetric solid-phase immunoassay method.