Drop S L, Kortleve D J, Guyda H J
J Clin Endocrinol Metab. 1984 Nov;59(5):899-907. doi: 10.1210/jcem-59-5-899.
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 20x 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 125I-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 mu geq/ml. Insulin-like growth factor I/Sm-C values (determined by RIA) in the same samples were uniformly very low (less than 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.
羊水结合蛋白（AFBP）是一种对热和酸稳定的生长调节素（Sm）结合蛋白，分子量为35000 - 40000，等电点为±4.7。它在生长调节素的放射受体分析（RRAs）中有反应，并在Sm生物测定中抑制Sm活性。使用酸 - 乙醇提取、葡聚糖凝胶G - 150层析、高速凝胶过滤层析和圆盘凝胶电泳从妊娠中期人羊水（AF）中纯化AFBP。通过与125I - 胰岛素样生长因子II孵育并进行葡聚糖包被活性炭分离来定量特异性结合活性（每毫克蛋白质的微克当量）。蛋白质回收率小于1%。用纯化的AFBP免疫兔子制备AFBP抗血清。通过亲和层析清除抗血清中的人血清白蛋白抗体。对20倍浓缩的早产羊水和胎儿血清进行免疫电泳产生一条沉淀线。用氯胺 - T法标记AFBP。在最终稀释度为1:5000时，AFBP抗血清特异性结合约35%添加的125I - AFBP。开发了一种双抗体放射免疫分析法（RIA）。通过RIA测定，妊娠中期羊水（n = 30）中AFBP水平为148±18（标准误），足月羊水（n = 12）中为72±36微克当量/毫升。相同样本中胰岛素样生长因子I/Sm - C值（通过RIA测定）始终非常低（小于0.10 U/ml）。当血清在pH 2.2条件下在葡聚糖凝胶G - 200上进行层析时，AFBP - RIA活性在对应分子量为35000 - 40000的一个峰中洗脱。在胎儿血清（胎龄约20周）中发现活性最高，在成人血清中最低。AFBP - RIA的开发可能有助于进一步阐明生长调节素和Sm结合蛋白在产前和产后生长中的生理重要性。
