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Keywords:

  • Nerve growth factor;
  • Immunoassay;
  • Sex difference;
  • Mice

Abstract: Levels of the β-subunit of nerve growth factor (β-NGF) were measured in the central nervous and peripheral tissues of mice using a highly sensitive, sandwich-type enzyme immunoassay system. Antiserum was raised in rabbits against the 7S form of NGF, which was purified from mouse submandibular glands. β-NGF-specific antibody isolated on a column of Sepharose CL-4B coupled with purified β-NGF reacted only with β-NGF. The assay for β-NGF was performed by incubation of F(ab′)2 fragments of the antibody immobilized on a polystyrene ball with tissue extract and then with the same antibody Fab’ fragments labeled with β-D-galactosidase, followed by measurement of galactosidase activity. Our assay system was found to be highly sensitive (minimal detection limit, 0.3 pg/0.3 ml of assay mixture). Furthermore, the presence of gelatin hydrolysates and protease inhibitors during preparation of tissue extracts enabled us to determine the precise levels of β-NGF in almost all organs of mice. The amount of β-NGF in submandibular glands was extremely high, and its level increased rapidly until mice were 2 months of age; then, the level continued to increase slowly until mice were 1 year old (3–5 mg/g of tissue). In serum, some of the 2-month-old males, but none of the females, exhibited a fairly high level of β-NGF (> 100 pg/ml). The level of β-NGF at 4 months of age was relatively high (2–5 ng/g) in the pancreas, spleen, and ovary among the organs examined, excluding the submandibular glands. An unexpected finding was that levels of β-NGF not only in submandibular glands, but also in the brain, adrenal glands, and spinal cord, of males were three to seven times as high as levels in the analogous organs of females. Possible mechanisms regulating the level of β-NGF in neural and paraneural tissues are discussed.