Schreyer D J, Andersen P L, Williams K, Kosatka I, Truong T N
Department of Physiology, Queen's University, Kingston, Canada.
J Neurosci Methods. 1997 Apr 4;72(2):137-45. doi: 10.1016/s0165-0270(96)02171-1.
A cell-ELISA technique is described which allows the quantification of GAP-43 protein in a large number of microcultures of adult dorsal root ganglion neurons. GAP-43 is measured in the 1-10 ng range, corresponding to the amount of GAP-43 present in fewer than 500 DRG neurons. Specificity of the assay is confirmed using Western blotting and immunocytochemistry. The GAP-43 content of adult DRG microcultures rises during 2 weeks in culture, although the number of surviving neurons decreases. The GAP-43 content of cultured adult DRG neurons is not increased by chronic exposure to added nerve growth factor after 7 days in vitro. However, GAP-43 is increased in DRG taken from animals with prior peripheral nerve injury, and is decreased by chronic exposure to dibutyryl cyclic AMP after 7 days in vitro. The method affords the sensitivity and statistical power to document modest changes in GAP-43 protein abundance in complex cultures.
本文描述了一种细胞酶联免疫吸附测定（cell-ELISA）技术，该技术可对大量成年背根神经节神经元的微培养物中的GAP-43蛋白进行定量。所测定的GAP-43含量在1-10 ng范围内，这相当于少于500个背根神经节（DRG）神经元中所含的GAP-43量。通过蛋白质印迹法和免疫细胞化学法确认了该测定方法的特异性。尽管存活神经元的数量减少，但成年DRG微培养物中的GAP-43含量在培养2周期间有所增加。体外培养7天后，长期暴露于添加的神经生长因子并不会增加成年DRG培养神经元的GAP-43含量。然而，取自先前有周围神经损伤动物的DRG中GAP-43含量增加，并且体外培养7天后长期暴露于二丁酰环磷腺苷会使其含量降低。该方法具有足够的灵敏度和统计学效力，能够记录复杂培养物中GAP-43蛋白丰度的适度变化。
