Biochimica et Biophysica A cta, 1176 (1993) 299-304 299 © 1993 Elsevier Science Publishers B.V, All rights reserved 0167-4889/93/$06.00 BBAMCR 13354 Establishment of an enzyme immunoassay system for gliostatin/platelet-derived endothelial cell growth factor (PD-ECGF) Takayoshi Hirano, Kiyofumi Asai, Kohei Matsukawa, Taiji Kato, Masanori Takeuchi, Masato Yonezawa, Takanobu Otsuka and Nobuo Matsui Departments of Bioregulation Research and Orthopedic Surgery, Nagoya City University Medical School, Mizuho-ku, Nagoya (Japan) (Received 12 August 1992) Key words: Enzyme immunoassay; Gliostatin; Platelet-derived endothelial cell growth factor PD-ECGF A two-site enzyme immunoassay for gliostatin (GLS)/platelet-derived endothelial cell growth factor (PD-ECGF) has been developed. The detection limit of gliostatin/PD-ECGF was 30 pg/well, and the optimal assay range was 0.1 to 10 ng/well. This assay system enabled us to confirm the immunochemical identity of both factors and to detect immunoreactive gliostatin/PD- ECGF (IR-GLS/PD-ECGF) in human biological body fluids. The age-related analysis from newborn to 69 years revealed that the serum IR-GLS/PD-ECGF level was high in infants younger than 1 year old (1.8 ng/ml) and in the 20-year-old age group (1.8 ng/ml), and highest in the umbilical cord blood (2.1 ng/ml). Curiously high concentrations were detected in saliva with a significant sex difference (11.3 ng/ml for males and 48.7 ng/ml for females), and in synovial fluids (3.7 ng/ml). A number of human tumor cells, gastric cancer cells, MKN-74, neuroblastoma cells, GOTO, as well as epidermoid carcinoma cells, A431, were found to produce a significant amount of IR-GLS/PD-ECGF (0.2 to 21.8 ng/mg protein), and some of them secreted the IR-GLS/PD-ECGF in the conditioned medium (~ 0.5 ng/ml). The enzyme immunoassay system is sufficiently sensitive for the basic and clinical study of gliostatin/PD-ECGF in human body fluids, tissues and organs. Introduction Gliostatin (GLS) is a protein factor, which was ini- tially identified in neurofibroma type 1 (NF1) extracts as a growth inhibitory factor against glial cells such as astrocytes and glial tumor cells, with a relative molecu- lar mass of 100 kDa comprised of two 50-kDa subunits [1,2]. This factor has lately been found to play an essential role on the neuron-glial interaction during development or regeneration of the nervous system [3]. More recently, we have demonstrated the chemical and biological identity of gliostatin and platelet-derived en- dothelial cell growth factor (PD-ECGF) [4], which had Correspondence to: T. Kato, Department of Bioregulation Research, Nagoya City University Medical School, Mizuho-ku, Nagoya 467, Japan. Abbreviations: GLS, gliostatin; PD-ECGF, platelet-derived endothe- lial cell growth factor; IR-, immunoreactive-; CM, conditioned medium; CMF-Tyrode, Ca2+-, Mg2+-free Tyrode's solution; PVDF, polyvinylidene difluoride; NF1, neurofibromatosis type 1; SDS, sodium dodecyl sulfate; aFGF, acidic fibroblast growth factor; bFGF, basic fibroblast growth factor; GMF, glia maturation factor; CNTF, ciliary neurotrophic factor and PMSF, phenylmethanesulfonyl fluo- ride. been purified from platelets and placenta as a growth factor promoting proliferation and chemotactic migra- tion of endothelial cells resulting in angiogenesis [5,6]. In this article, we establish a sensitive enzyme im- munoassay system for gliostatin/PD-ECGF, enabling the measurement of a minute amount of immuno- reactive-GLS/PD-ECGF in human biological body flu- ids. Materials and Methods Anti-gliostatin / PD-ECGF antiserum 40 ~g of gliostatin/PD-ECGF, which were purified from human placenta in accordance with the method previously described [2,4], were emulsified with 0.2 ml of complete Freund's adjuvant (Difco, UK) and in- jected subcutaneously into a New Zealand albino rab- bit. Booster immunizations of 20 ~g were given after 2 and 5 weeks. Anti-gliostatin/PD-ECGF antibody was raised 6 weeks after the initial immunization. The y-globulin fraction of anti gliostatin/PG-ECGF serum (10 ml) was prepared by two successive steps of ammo- nium sulfate precipitation (40% and 33% saturation). The precipitates were collected by centrifugation