Highly sensitive enzyme immunoassays for beta-nerve growth factor.
A comparison was made between three different strategies for measuring beta-nerve growth factor (NGF) by fluorometric enzyme immunoassay. The substrate used was 4-methylumbelliferyl-beta-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 beta-galactosidase gave the best detection limit (2 X 10(-17) M) of purified mouse NGF (Mr 26,000) in a two-site sandwich assay. Biotinylated secondary antibodies followed by streptavidin conjugated beta-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 X 10(-12) M. All three methods indicated the same level (4 X 10(-11) M) of endogenous NGF in the rat brain hippocampus.
D000818:Animals; D000906:Antibodies; D007124:Immunoenzyme Techniques; D007202:Indicators and Reagents; D051379:Mice; D008970:Molecular Weight; D009414:Nerve Growth Factors / Q000032:analysis*; D013363:Submandibular Gland / Q000032:analysis; D001616:beta-Galactosidase
D000906:Antibodies; D007202:Indicators and Reagents; D009414:Nerve Growth Factors; D001616:beta-Galactosidase