A sensitive two-site (sandwich) enzyme immunoassay system for measuring nerve growth factor (NGF)

Abstract

Nerve growth factor (NGF) is a prototype of neurotrophin family neurotrophic factors that have nearly 50% amino acid sequence homology with each other. NGF is known to support survival and stimulate differentiation of sympathetic and neural crest-derived some sensory neurons in the peripheral nervous system and the basal forebrain cholinergic neurons in the central nervous system. Recent investigations have expanded NGF action to much wider cell populations than previously known. Namely, NGF stimulates differentiation and/or proliferation of immune cells including lymphocytes, leucocytes and mast cells. These findings suggest important roles of NGF in the crosstalk between the nervous system and immune system. Therefore, a practical and sensitive method to quantify physiological NGF is in great demand. We developed a sensitive twosite enzyme immunoassay (EIA) system for mouse NGF, based on the sandwiching of the antigen between anti-mouse NGF antibody IgG coated to a plastic plate and biotinylated-anti-NGF antibody. Avidin-β-D-galactosidase was then bound to biotines, and galactosidase activity was determined fluorometrically. This method is simple, rapid and sensitive. Purified NGF is detectable at a concentration as low as a few pg/ml, which is enough to detect physiological NGF in various tissues and NGF secreted from cultured non-neuronal cells such as fibroblasts, astrocytes and Schwann cells.