Human FIGF ELISA Matched Antibody Pair

A method to measure free form of insulin-like growth factor I (IGF-I) in human plasma using octadecylsilyl silica (Sep-Pak C-18) cartridge has been developed. IGF-I was adsorbed by Sep-Pak C18 cartridge and eluted with 75% ethanol-0.01 M HCl. Labeled and non-labeled IGF-I were recovered in yields 92.5 +/- 2.1% (Mean +/- SEM) and 94.4 +/- 6.3% after adsorption to and elution from the Sep-Pak, respectively. When EDTA plasma was applied to the Sep-Pak, less than 5% of total IGF-I was recovered in the eluate. However, when acid-ethanol extracted plasma was applied to the Sep-Pak, IGF-I was recovered in yields greater than 75% of total IGF-I. When the Sep-Pak eluate was gel filtered, 88.4 +/- 4.0% of immunoreactive IGF-I eluted in the same fraction as synthetic IGF-I did, but the fraction passed through the Sep-Pak was observed as a high molecular weight form (bound form) of IGF-I. These data indicate that this Sep-Pak method does not extract all of the IGF-I in plasma, but extracts mainly the free form IGF-I. Using this method, IGF-I values of free form (fIGF-I) in EDTA plasma were measured. The fIGF-I values in normal adults, patients with acromegaly, and patients with growth hormone (GH)-deficiency were 2.4 +/- 0.1, 13.8 +/- 1.6, and 1.1 +/- 0.1 ng/ml, respectively. Plasma fIGF-I and fIGF-I% of pregnant women were elevated during the second and the third trimester of pregnancy, and total IGF-I levels in cord blood were low, but flGF-I% was elevated in comparison with normal adults. These data suggest that measurement of free form of IGF-I in plasma would be useful in order to study the pathophysiological role of IGF-I in various clinical conditions.

Satellite stem cell activation is the process by which quiescent precursor cells resident on muscle fibers are recruited to cycle and move. Two processes are reported to affect satellite cell activation. In vivo, nitric oxide (NO) produced by NO synthase in fibers (NOS-Imu) promotes activation. In cell cultures, hepatocyte growth factor (HGF) is the major activating factor isolated from crushed muscle extract (CME). In this study we hypothesized that distinct and possibly related events were mediated by NO and HGF during activation. Intact fibers were cultured in the presence of bromodeoxyuridine (BrdU) to label DNA synthesis over 48 h. Experiments were designed to test the effects of CME, HGF, a NOS substrate L-arginine, and the NOS inhibitor L-NAME on activation, determined as the number of BrdU-positive satellite cells per fiber. Activation was increased significantly by CME, HGF, and L-arginine. L-Arginine increased activation in a dose-response manner. CME-induced activation was reduced significantly by NOS inhibition. Exposure to marcaine (10 min) caused reversible membrane damage without hypercontraction, as shown by characterizing the sarcolemmal integrity. The resulting decrease in satellite cell activation could be overcome by exogenous FIGF. Results support the hypothesis that NO is involved in recruiting to cycle those satellite cells resident on fibers. Separate assessments of resident and free muscle cells showed that FIGF and NO also participate in mobilizing satellite cells. Since HGF counteracted NOS inhibition and marcaine-induced membrane damage, data suggest that NO may mediate early steps in activation and precede HGF-mediated events. (C) 2002 Elsevier Science (USA). All rights reserved.