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DEVELOPMENT OF THE INTRAOPERATIVE ESTIMATION OF THE PROXIMAL NERVE STUMP USING ANTIGEN-ANTIBODY REACTION ON THE ARTIFICIAL MEMBRANE

利用人工膜上抗原抗体反应进行近端神经残端术中估计的研究进展

基本信息

批准号：
02670648
负责人：
OCHIAI Naoyuki
金额：
$ 1.22万
依托单位：
UNIVERSITY OF TOKYO
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

Applying colloidal gold labelled by anti-neurofilament (NF) antibody to the spot of proximal nerve stumps on the artificial membrane made it possible to estimate their vitality.One of the important factor of the successful nerve suture is to prepare the good proximal stumps. The assessment of the stump has been done mainly from the view with the naked eyes. It was so subjective and sometimes inaccurate. Then we developed a new method using the immunological reaction of NF antigen and anti-NF antibody labelled with colloidal gold on the artificial membrane.First we developed the method :1) Stamp the proximal stump on the cellulose acetate membrane to make a spot.2) Set the membrane on the special device.3) Wash the membrane with the phosphate buffer (PBS).4) Apply the antiNF-antibody labelled colloidal gold to the spot and aspirated through the membrane after several minutes waiting and wash with PBS again.If the stump is normal and the spot contain appropriate neurofilament, the colloidal gold is fixed to the spot and shows pinkish color.It was easy and quick method. However, to stamp a nerve stump was not so easy to repeat in the same manner, and to check the quantity of the NF antigen by the other method was hard to perform.So we tried an alternative method. That is :1) Make extract of the resected thin slice of the nerve stump2) Dot the extract3) React the colloidal gold.If the spot includes NF antigen, the spot shows red color and the color graded according to the quantity of the antigen. We performed an experiment to crush and cut the nerve of a rabbit and after eight weeks the nerve was resected to examine with this method. The color of the spots well correlated to the histology of the stumps and the quantity of NF detected by ELISA.Though our method is applicable only to the proximal stumps and distinction of regenerated nerve from intact nerve is impossible, this simple and quick method may be a great help to the peripheral nerve suture.

将抗神经丝抗体标记的胶体金应用于人工膜上近端神经残端部位，可以估计其活力。神经缝合成功的重要因素之一是准备好近端残端。对树桩的评估主要是通过肉眼观察来完成的。它是如此主观，有时是不准确的。在此基础上，建立了一种利用胶体金标记的NF抗原和抗NF抗体在人工膜上的免疫反应的新方法。首先，我们开发了一种方法：1)将近端残端压在醋酸纤维素膜上形成一个点。2)在专用设备上设置膜。3)用磷酸盐缓冲液（PBS）清洗膜。4)将抗f抗体标记的胶体金涂抹在斑点上，等待几分钟后通过膜抽吸，再次用PBS洗涤。如果残端正常且斑点含有适当的神经丝，则胶体金固定在斑点上并显示粉红色。这是一种简单快捷的方法。然而，用相同的方法对神经残端进行踩踏并不容易重复，用其他方法检测NF抗原的数量也很困难。所以我们尝试了另一种方法。即：1)取切除的神经残端薄片提取液2)将提取液点上3)与胶体金反应。如果斑点含有NF抗原，斑点呈红色，颜色根据抗原的数量分级。我们做了一个实验，粉碎和切断兔子的神经，8周后切除神经，用这种方法检查。斑点的颜色与树桩的组织学和酶联免疫吸附法检测到的NF的数量有很好的相关性。虽然我们的方法只适用于近端残端，无法区分再生神经和完整神经，但这种简单快捷的方法可能对周围神经的缝合有很大的帮助。