Marcel Y L, Jewer D, Vézina C, Milthorp P, Weech P K
J Lipid Res. 1987 Jul;28(7):768-77.
The expression and immunoreactivity of apolipoprotein (apo) A-I epitopes in high density lipoproteins (HDL) and serum has been investigated using two series of monoclonal antibodies (Mabs) which have been described elsewhere. Series 1 Mabs, identified as 3D4, 6B8, and 5G6, were obtained by immunization and screening with apoA-I, and series 2 Mabs, identified as 2F1, 4H1, 3G10, 4F7, and 5F6, were obtained by immunization and screening with HDL. These Mabs were characterized with respect to their binding to HDL particles in solution. In series 2 Mabs, 2F1, 3G10, and 4F7, which react with apoA-I CNBr-fragments 1 and 2, could precipitate 100% of 125I-labeled HDL, while 4H1 and 5F6, which react with CNBr fragments 1 and 3, precipitated 90 and 60% of 125I-labeled HDL, respectively. Therefore, three distinct epitopes mapped to CNBr fragments 1 and 2 have been identified which are expressed on all HDL particles, indicating that several antigenic do mains exist on apoA-I which have the same conformation on all apoA-I-containing lipoproteins. The Mabs reacting at these sites have significantly higher affinity constants for 125I-labeled HDL than those that failed to precipitate 100% of HDL. This suggests that the high affinity Mabs react with apoA-I epitopes that are both expressed on all lipoproteins and located in thermo-dynamically stable regions of the molecules. All Mabs from series 1 precipitated 35% or less of 125I-labeled HDL prepared from freshly collected serum, but the proportion of HDL particles expressing the epitopes for these Mabs doubled or more upon serum storage at 4 degrees C. The time course of the alteration of apoA-I antigen in vitro was measured in three normolipemic donors. Upon storage of serum at 4 degrees C, the immunoreactivity of series 2 Mabs (4H1, 3G10) remained unchanged. However, the immunoreactivity of series 1 Mab 3D4 increased linearly at 38%/day for 4 weeks and by 12 weeks had plateaued at about 280-fold compared to day 1. The immunoreactivity of other series 1 Mabs also increased significantly with time in vitro. This process was partially inhibited in the presence of EDTA and by addition of antioxidants, however, the exact molecular nature of this in vitro alteration of apoA-I antigen was not identified.
使用此前已描述的两组单克隆抗体（Mab）研究了高密度脂蛋白（HDL）和血清中载脂蛋白（apo）A-I表位的表达及免疫反应性。第1组Mab，即3D4、6B8和5G6，是通过用apoA-I免疫和筛选获得的；第2组Mab，即2F1、4H1、3G10、4F7和5F6，是通过用HDL免疫和筛选获得的。对这些Mab与溶液中HDL颗粒的结合特性进行了表征。在第2组Mab中，与apoA-I CNBr片段1和2反应的2F1、3G10和4F7能够沉淀100%的125I标记的HDL，而与CNBr片段1和3反应的4H1和5F6分别沉淀了90%和60%的125I标记的HDL。因此，已鉴定出三个定位于CNBr片段1和2的不同表位，它们在所有HDL颗粒上均有表达，这表明apoA-I上存在几个抗原结构域，在所有含apoA-I的脂蛋白上具有相同的构象。在这些位点反应的Mab对125I标记的HDL的亲和常数明显高于那些未能沉淀100% HDL的Mab。这表明高亲和力Mab与在所有脂蛋白上均有表达且位于分子热力学稳定区域的apoA-I表位发生反应。第1组的所有Mab沉淀的新鲜采集血清制备的125I标记的HDL均不超过35%，但在4℃储存血清后，表达这些Mab表位的HDL颗粒比例增加了一倍或更多。在三名血脂正常的供体中测量了体外apoA-I抗原变化的时间进程。在血清于4℃储存时，第2组Mab（4H1、3G10）的免疫反应性保持不变。然而，第1组Mab 3D4的免疫反应性在4周内以38%/天的速度线性增加，到12周时与第1天相比达到约280倍的平台期。其他第1组Mab的免疫反应性在体外也随时间显著增加。在EDTA存在下和添加抗氧化剂后，这一过程受到部分抑制，然而，apoA-I抗原体外变化的确切分子性质尚未确定。
