碱性磷酸酶标A蛋白加强的PAP技术

本文介绍一种碱性磷酸酶标Ａ蛋白加强的ＰＡＰ技术。采用ＰＡＰ技术、碱性磷酸酶标Ａ蛋白（ＰＡＡＰ）技术ＰＡＡＰ和加强的ＰＡＰ（ＰＡＰ－ＰＡＡＰ）技术显示下丘脑室旁核催产素（ＯＴ）能神经元。结果发现，其中使用ＰＡＰ－ＰＡＡＰ技术免疫反应产物的显色最深。此技术的原理可能是，由于Ａ蛋白分子至少有四个位点能与ＩｇＧ分子的Ｆｃ段高亲合性地结合，故在该技术中，先经过ＰＡＰ程序的三步免疫反应并显色后，每个与一抗结合的二抗分子上和每个与二抗结合的ＰＡＰ复合物分子上各暴露一个能与Ａ蛋白分子结合的Ｆｃ段，在随后经过ＰＡＡＰ技术处理时，部分ＰＡＡＰ复合物分子就结合在这些Ｆｃ段上，经显色后，ＰＡＡＰ技术显示的浅紫兰色与ＰＡＰ技术显示的浅棕褐色重叠，变成更深的反差明显的深棕褐色。

A PAP TECHNIQUE REINFORCED BY THE PROTEIN A LABELED WITH ALKALINE PHOSPHATASE

In the present study a PAP technique reinforced by alkaline phosphatase-labeled protein A is reported. Oxytocin(OT)-ergic neurons in the paraventricular nucleus(PVN) were immunohistochemically demonstrated by using the PAP technique, the technique of the protein A labeled with alkaline phosphatase(protein A-alkaline phosphatase, PAAP) and the PAP technique reinforced by the PAAP(PAP-PAAP). It was found that of three kinds of techniques the staining of the reaction products from the PAP-PAAP technique was darkest and brightest. The principle of this reinforced technique may be summarized as follows: there are at least four Fc-binding regions which can bind in high affinity with Fc-fragment at the IgG molecule in the protein A molecule. In the process of the PAAP-reinforced technique, after the sections are first processed with the PAP procedure and developed, on each second antibody molecule bound with the first antibody one Fc-segment is exposed; and on each molecule of the PAP complex bound with the second antibody another Fe-segment is exposed. When these sections are then treated with the PAAP procedure, a part of the PAAP complex is bound in the protein A at this complex with these Fc-segments exposed. After development the blue colour exerted by the PAAP procedure and the brown colour produced by the PAP procedure overlap, thereby assuming a very dark brown-black colour.