Highly sensitive enzyme immunoassays for β-nerve growth factor

Abstract

A comparison was made between three different strategies for measuring β-nerve growth factor (NGF) by fluorometric enzyme immunoassay. The substrate used was 4-methylumbelliferyl-β-galactoside and the enzyme reaction was followed in a Microfluor plate reader (Dynatech). After optimizing incubation times, concentrations, buffers, pH, and washings, a primary anti-NGF antibody directly conjugated to β-galactosidase gave the best detection limit (2 × 10 −17 M) of purified mouse NGF ( M r 26 000) in a two-site sandwich assay. Biotinylated secondary antibodies followed by streptavidin conjugated β-galactosidase proved to be 200-fold less sensitive in a similar assay. Finally, blotting NGF onto nitrocellulose membranes for detection with the same biotin-streptavidin steps after incubation with unlabelled primary antibodies resulted in a detection limit of 3 × 10 −12 M. All three methods indicated the same level (4 × 10 −11 M) of endogenous NGF in the rat brain hippocampus.