Abelev G I, Karamova E R
Laboratory of Immunochemistry, Cancer Research Center, Moscow, U.S.S.R.
Mol Immunol. 1989 Jan;26(1):41-7. doi: 10.1016/0161-5890(89)90018-7.
Urine with trace amounts of different proteins from healthy people or B-lymphoma patients was concentrated and separated simultaneously by counterflow isotachophoresis on cellulose acetate membranes (CAM). The protein zones were blotted onto nitrocellulose membrane (NCM) by direct contact of CAM and NCM. NCM-blots were exposed to second isotachophoresis with the leading electrolyte 0.06 M Tris-HCl and the terminating one, 0.012 M Tris-beta-alanine. Under these conditions the moving boundary formed by Cl-/beta-alanine- migrated towards the anode with decreasing velocity. At a certain point the rate of migration of the moving boundary became completely compensated by the electroendosmotic counterflow. In this steady state position the boundary stopped on the NCM support, while the electroendosmotic rate in the area before the boundary was much higher than the rate of the opposite migration of any protein to the anode. Under these conditions electroendosmosis served as a "conveyer belt" which transferred consecutively the immunoreagents, antibodies, immunoconjugates, or antiperoxidase-peroxidase system through the protein blots "printed" on NCM. The immunoblots obtained in this way were developed by the substrate for the immunoenzyme complex used in the experiment. The technique could be used to characterize light chains present in the urine of normal donors and monoclonal light chains in the urine of patients with B-cell malignancies.
来自健康人或B淋巴瘤患者的含有微量不同蛋白质的尿液，通过在醋酸纤维素膜（CAM）上进行逆流等速电泳同时进行浓缩和分离。通过CAM与硝酸纤维素膜（NCM）直接接触，将蛋白质区带印迹到硝酸纤维素膜上。将NCM印迹膜置于以0.06 M Tris-HCl为前导电解质、0.012 M Tris-β-丙氨酸为终止电解质的第二次等速电泳中。在这些条件下，由Cl⁻/β-丙氨酸形成的移动边界以逐渐降低的速度向阳极迁移。在某一点上，移动边界的迁移速率完全被电渗逆流所补偿。在这个稳定状态位置，边界停留在NCM载体上，而边界之前区域的电渗速率远高于任何蛋白质向阳极反向迁移的速率。在这些条件下，电渗起到了“传送带”的作用，它将免疫试剂、抗体、免疫结合物或抗过氧化物酶-过氧化物酶系统依次通过“印”在NCM上的蛋白质印迹。通过这种方式获得的免疫印迹用实验中使用的免疫酶复合物的底物进行显色。该技术可用于鉴定正常供体尿液中存在的轻链以及B细胞恶性肿瘤患者尿液中的单克隆轻链。
