Isolation of a Somatomedin-Binding Protein from Preterm Amniotic Fluid. Development of a Radioimmunoassay*

Publisher Website
Google Scholar
Abstract
Amniotic fluid binding protein (AFBP) is a heat and acid stable somatomedin (Sm)-binding protein with a mol wt of 35–40,000 and an isoelectric point of ±4.7. It is reactive in RRAs for Sm and inhibits Sm activity in Sm bioassays. AFBP was purified from midgestational human amniotic fluid (AF) using acid-ethanol extraction, Sephadex G-150 chromatography, high speed gel filtration chromatography, and disc gel-electrophoresis. Specific binding activity (microgram equivalents per mg protein) was quantitated by incubation with 125I-insulin-like growth factor II and dextran-coated charcoal separation. Protein recovery was less than 1%. AFBP antiserum was produced by immunizing rabbits with purified AFBP. The antiserum was cleared of human serum albumin antibodies by affinity chromatography. Immunoelectrophoresis of 20× concentrated preterm AF and fetal serum resulted in one precipitin line. AFBP was labeled by the chloramine-T method. The AFBP antiserum specifically bound ±35% of added 126I-AFBP at a final dilution of 1:5000. A double antibody RIA was developed. The AFBP level measured by RIA in midgestation AF (n = 30) was 148 ± 18 (SEM) and in term AF (n = 12) 72 ± 36 μgeq/ml. Insuluvlike growth factor I/Sm-C values (determined by RIA) in the same samples were uniformly very low (<0.10 U/ml). When serum was chromatographed on Sephadex G-200 at pH 2.2, AFBP-RIA activity eluted in one peak corresponding to a mol wt of 35–40,000. Highest activity was found in fetal serum (gestational age ± 20 weeks) and lowest in serum from adults. The development of the AFBP-RIA may contribute to further elucidation of the physiological importance of Sm and the Sm-binding proteins in pre-and postnatal growth.

This publication has 16 references indexed in Scilit: