Chang W S, Friedman C H, Iqbal M A, Neff N T
Cephalon, Inc., West Chester, PA 19380.
J Neurosci Methods. 1991 Jan;36(1):71-6. doi: 10.1016/0165-0270(91)90139-q.
An ELISA assay for the growth associated protein GAP-43 was developed to determine rapidly its relative abundance in neuronal tissue. The assay was performed with affinity-purified anti-GAP-43 antibody that detected a single band of Mr = 42,000-45,000 on Western blots of rat brain homogenates but no bands on blots of liver homogenates. GAP-43 was determined by ELISA assay in as little as 0.6 microgram protein of brain homogenate. The assay was highly reproducible; the standard error of the mean of sample to sample variation was less than 5%. When ELISA development time was held constant, the standard error of the mean of inter-assay variation was between 2 and 7%. Using this method, GAP-43 immunoreactivity was examined in developing rat brain. At post-natal day 1, GAP-43 immunoreactivity was 3-4 times greater than that observed in the adult, remained elevated for several weeks, and decreased by the end of the first month of life. These results are in accord with previous studies on the expression or synthesis of GAP-43 during neuronal development.
开发了一种用于生长相关蛋白GAP-43的酶联免疫吸附测定（ELISA），以快速测定其在神经组织中的相对丰度。该测定使用亲和纯化的抗GAP-43抗体进行，该抗体在大鼠脑匀浆的蛋白质免疫印迹上检测到一条Mr = 42,000 - 45,000的条带，但在肝匀浆的印迹上未检测到条带。通过ELISA测定，在低至0.6微克脑匀浆蛋白中即可测定GAP-43。该测定具有高度可重复性；样品间变异均值的标准误差小于5%。当ELISA显色时间保持恒定时，批间变异均值的标准误差在2%至7%之间。使用该方法，对发育中的大鼠脑进行了GAP-43免疫反应性检测。在出生后第1天，GAP-43免疫反应性比成年大鼠高3 - 4倍，持续升高数周，并在出生后第一个月末下降。这些结果与先前关于神经发育过程中GAP-43表达或合成的研究一致。
