Eto M, Watanabe K, Moriyama T, Makino I
Second Department of Internal Medicine, Asahikawa Medical College.
Tohoku J Exp Med. 1990 Apr;160(4):301-9. doi: 10.1620/tjem.160.301.
A method for apolipoprotein (apo) E phenotyping directly from plasma by isoelectric focusing (IEF) and immunoblotting was confirmed. Ten microliters plasma were delipidated. IEF in 5% polyacrylamide flat gel with 6.4 mol/l urea and 2.8% pharmalyte (PH 4-6.5) was carried out at 3,000 V for 1 hr. Seventeen samples were applied per one flat gel, and IEF of two flat gels was made. Then, Western blotting on nitrocellulose membrane was done at 75 V for 3 hr. Immunostaining was performed using goat-anti-human apo E as first antibody and biotinylated anti-goat IgG as second antibody, and 4-chlorodel-1-naphthol as a substrate. In approximately 5% of the samples, we had difficulty in discriminating between homozygotes and heterozygotes (i.e., apo E3/3 and apo E3/2, or apo E4/4 and apo E4/3) because of equally strong sialated band, but this problem was solved by sialidase treatment of plasma before delipidation. As a result, six apo E phenotypes were clearly demonstrated. Apo E phenotyping of 34 samples could be made simultaneously in 2 days. It is concluded that the polyacrylamide gel IEF and immunoblotting method is useful for apo E phenotyping if it is made up for by sialidase treatment.
一种通过等电聚焦（IEF）和免疫印迹直接从血浆中进行载脂蛋白（apo）E表型分析的方法得到了验证。取10微升血浆进行脱脂处理。在含有6.4摩尔/升尿素和2.8%两性电解质（pH 4 - 6.5）的5%聚丙烯酰胺平板凝胶中进行IEF，在3000伏电压下进行1小时。每块平板凝胶上点样17个样本，制作两块平板凝胶的IEF。然后，在硝酸纤维素膜上进行75伏电压、3小时的蛋白质印迹。使用山羊抗人apo E作为一抗、生物素化抗山羊IgG作为二抗以及4 - 氯 - 1 - 萘酚作为底物进行免疫染色。在大约5%的样本中，由于唾液酸化条带同样强烈，我们难以区分纯合子和杂合子（即apo E3/3和apo E3/2，或apo E4/4和apo E4/3），但通过在脱脂前对血浆进行唾液酸酶处理解决了这个问题。结果，清晰地显示出六种apo E表型。34个样本的apo E表型分析可在2天内同时完成。结论是，如果通过唾液酸酶处理进行补充，聚丙烯酰胺凝胶IEF和免疫印迹方法对于apo E表型分析是有用的。
