Characterization of antibodies to synthetic nerve growth factor (NGF) and ProNGF peptides

Abstract

Sequence data for the mature nerve growth factor (NGF) protein and its precursor are available from molecular cloning of the NGF gene in several species, including mice, humans, rats, and chickens. Hydrophilicity analysis of the predicted rat and chicken prepro‐NGF was carried out to locate putative antigenic determinants. Eight peptides were selected and synthesized based on hydrophilicity profiles. Two peptides represent sequences in the rat (and mouse) pro‐NGF, one peptide (our peptide P3) represents a highly conserved region of the mature NGF protein (identical in humans, mice, rats, and chickens), two peptides are specific for the mature chicken NGF, and the remaining three peptides are specific for the mature rat NGF (each with only one amino acid substitution compared with corresponding segments of the mouse NGF). For immunization, the peptides were conjugated to keyhold limpet hemocyanin and used to produce antisera in rabbits. After bleeding, peptidespecific antibodies were purified on affinity columns prepared by coupling each of the synthetic peptides. The different peptide antisera and affinity‐purified antibodies then were characterized by enzyme‐linked immunoassay (ELISA) and immunohistochemistry of the male mouse submandibular gland, a rich exocrine source of NGF. ELISA analysis showed that all peptide antisera bound two to four orders of magnitude better than normal rabbit serum to a coat of their proper peptide. The higher binding was retained by the purified peptide antibodies compared with normal rabbit immunoglobulin. Specific tests, in which one peptide antiserum was checked against different peptide coats in the ELISA, also showed two to four orders of magnitude higher binding of antibodies to the proper synthetic peptide. The peptide antibodies also were tested for their ability to bind to native mouse βNGF coated to the immunoplates. Only antibodies raised to the conserved P3 peptide recognized native NGF to an extent similar to that obtained with polyclonal anti‐NGF antibodies. Conversely, P3 was well recognized by several different NGF antisera. Immunohistochemically, both peptide antisera against the pro‐NGF stained the perinuclear cytoplasm in the basal part of the cells of the granulated convoluted tubules in the mouse submandibular gland. Several of the peptide antisera against the mature rat NGF strongly stained the secretory granules in the same cells. It was concluded that the conserved P3 epitope of the mature βNGF is a region useful to raise peptide antibodies for recognition of the native NGF, possibly from a number of species, and that several of the antisera raised in this study have potential use for immunohistochemical localization of NGF and prepro‐NGF.