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

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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.