Hoff H F, Dusek D M, Lynn M P
Lab Invest. 1986 Sep;55(3):377-86.
An immunotransfer procedure has been developed which can determine both the spatial distribution of low density lipoproteins (LDL) along the intima-media of large blood vessels such as the aorta, and can quantify LDL accumulation along its length. Aortas which were opened longitudinally along their ventral aspect were positioned so that their intimal side abutted against a gel containing glyoxyl agarose to which anti-LDL had been covalently coupled. LDL was electrophoresed out of the agarose gel where it was immunofixed. This distribution was then visualized first by incubating the gel with 125I-anti-LDL which bound to free epitopes on the immunofixed LDL, and second by subjecting the washed and dried gel to autoradiography. Plasma LDL was applied to wells of different shapes and sizes in an agarose gel substituting for aortic tissue, and the transfer procedure was performed as described. The resultant patterns matched those of the original wells, suggesting that the spatial distribution of LDL in the autoradiogram probably mimicked that in the aortic tissue. The transfer procedure appeared to be specific for the antigen under study since minimal silver grains were observed in autoradiograms when an IgG fraction of nonimmune serum was used in place of anti-LDL. Application of increasing concentrations of LDL to wells in a gel substituting for tissue, resulted in a dose-dependent increase in autoradiographic grain density. If such standards were applied to gels adjacent to tissue samples, the amounts of LDL in the tissue could be quantified from the standard curve of grain density versus LDL concentration. The distribution of LDL along the abdominal aortas of 10- and 31-week-old swine was determined by converting autoradiographic grain densities to isopleths of LDL concentrations by computer assisted image analysis. These distributions were focal and were found to range between 10 and 225 ng of apoB/mm2 of intimal surface area. This procedure lends itself not only to studies relating lipoprotein accumulation to atherogenesis, but also to any studies dealing with tissue accumulation of macromolecules.
已开发出一种免疫转移程序，该程序既能确定低密度脂蛋白（LDL）沿大动脉（如主动脉）内膜中层的空间分布，又能对其沿长度方向的LDL积累进行定量。将沿腹侧纵向切开的主动脉放置好，使其内膜面与含有已共价偶联抗LDL的乙二醛琼脂糖的凝胶接触。LDL从琼脂糖凝胶中电泳出来并在其中进行免疫固定。然后首先通过用与免疫固定的LDL上的游离表位结合的125I - 抗LDL孵育凝胶来可视化这种分布，其次通过对洗涤并干燥的凝胶进行放射自显影来实现。将血浆LDL施加到替代主动脉组织的琼脂糖凝胶中不同形状和大小的孔中，并按所述进行转移程序。所得图案与原始孔的图案匹配，表明放射自显影图中LDL的空间分布可能模拟了主动脉组织中的分布。该转移程序似乎对所研究的抗原具有特异性，因为当使用非免疫血清的IgG组分代替抗LDL时，在放射自显影图中观察到的银粒极少。将越来越高浓度的LDL施加到替代组织的凝胶中的孔中，导致放射自显影颗粒密度呈剂量依赖性增加。如果将这些标准应用于与组织样品相邻的凝胶，则可以根据颗粒密度与LDL浓度的标准曲线对组织中的LDL量进行定量。通过计算机辅助图像分析将放射自显影颗粒密度转换为LDL浓度等值线，确定了10周龄和31周龄猪腹主动脉中LDL的分布。这些分布是局灶性的，发现其范围在内膜表面积每平方毫米10至225 ng载脂蛋白B之间。该程序不仅适用于将脂蛋白积累与动脉粥样硬化发生相关的研究，也适用于任何涉及大分子组织积累的研究。
