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

将抗神经丝蛋白（NF）抗体标记的胶体金应用于人工膜上神经近端残端的位置，可以判断其活力，神经近端残端的制备是神经缝合成功的重要因素之一。对树桩的评价主要是从肉眼观察的角度进行的。它是如此主观，有时甚至不准确。然后，我们利用胶体金标记的NF抗原和抗NF抗体在人工膜上的免疫反应，建立了一种新的检测方法。1）将近端残端在醋酸纤维素膜上压印成斑点2）将膜置于专用装置上3）用磷酸盐缓冲液（PBS）洗涤膜4）将抗NF-抗体标记的胶体金涂于斑点上，等待数分钟后通过膜吸出，再次用PBS洗涤，如果残端正常且斑点含有适当的神经丝，胶体金固定在斑点上，呈粉红色，方法简便、快速。然而，用同样的方法来标记神经残端并不容易重复，而用另一种方法来检测NF抗原的数量也很难进行，因此我们尝试了另一种方法。即：1）取神经残端切取薄片，制成提取液; 2）将提取液点样; 3）与胶体金反应，若点样中含有NF抗原，则点样呈红色，颜色根据抗原量的多少而分级。我们进行了一项实验，粉碎和切断兔的神经，并在八周后切除神经，用这种方法进行检查。斑点的颜色与残端的组织学和ELISA检测的NF含量有很好的相关性，虽然我们的方法只适用于近端残端，不能区分再生神经和完整神经，但这种简单快速的方法可能对周围神经缝合有很大的帮助。

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.