Interaction of monoclonal antibodies with a neurite outgrowth factor from chicken gizzard extract

A neurite outgrowth factor (NOF), which promotes neurite outgrowth of cultured peripheral neurons, was purified to homogeneity from chicken gizzard extract and used as an immunogen for generating monoclonal antibodies. From 603 hybridoma clones, 55 positive hybridoma cell lines were detected. Four of them (IgG1 (kappa-chain) producers designated as 5-10A, M1-2G, 4-2C and 1-4D) were selected for further characterization. These antibodies immunoprecipitated 125I-labelled gizzard NOF molecules, whereas they did not inhibit the biological activity of NOF (or NOF-like molecules) derived from the tissues tested. The subcellular distribution of NOF in chick embryonal tissues was investigated using monoclonal antibody 4-2C. Both ciliary ganglionic (CG) neurons and gizzard muscle tissues were stained. The NOF content of embryonic CG, measured by an immunoblot assay, increased about 20-fold during embryogenesis from 6 to 15 days, whereas the sensitivity of neurons to NOF reciprocally decreased. At least two different sizes of NOFs (700 and 800 kDa) were found in CG of various ages, with the 800/700 kDa ratio gradually increasing with age. A mixture of these 4 monoclonal antibodies was used for immunoaffinity purification of NOF from chicken gizzard muscles.