Venembre P, Seta N, Boutten A, Dehoux M, Aubier M, Durand G
Laboratoire de Biochimie A, Hôpital Bichat, Paris, France.
Clin Chim Acta. 1994 Jun;227(1-2):175-84. doi: 10.1016/0009-8981(94)90145-7.
In order to detect, characterize and quantify blotted proteins, such as human alpha 1-antitrypsin (AAT), there is a need for a specific, extremely sensitive, non-radioactive and uniform revelation system applicable to diluted biological fluids and to culture supernatants of cells isolated from such fluids. We compared two immunochemical revelation systems, enhanced chemiluminescence (ECL) and colorimetric procedures, applied to human ATT, after determining their optimal conditions of performance. ECL was the most sensitive method (down to 50 pg blotted AAT), but could not be used to quantify AAT. In contrast, the colorimetric method enables quantification of blotted AAT, either simply dotted or transferred after SDS-polyacrylamide gel electrophoresis, but is not as sensitive as ECL. Using these two complementary procedures, we have been able to detect AAT in the culture supernatant of a monocytic cell line (THP-1), to characterize the different forms of AAT present in the culture supernatant of blood monocytes and to quantify both.
为了检测、表征和定量印迹蛋白，如人α1-抗胰蛋白酶（AAT），需要一种适用于稀释生物体液以及由此类体液分离的细胞培养上清液的特异性、极其灵敏、非放射性且均匀的显色系统。在确定了两种免疫化学显色系统（增强化学发光法（ECL）和比色法）的最佳性能条件后，我们将它们应用于人AAT进行了比较。ECL是最灵敏的方法（可检测低至50 pg的印迹AAT），但无法用于定量AAT。相比之下，比色法能够对简单点样或经SDS-聚丙烯酰胺凝胶电泳转移后的印迹AAT进行定量，但不如ECL灵敏。通过使用这两种互补方法，我们得以在单核细胞系（THP-1）的培养上清液中检测到AAT，表征血液单核细胞培养上清液中存在的不同形式的AAT，并对两者进行定量。
