RESUMO
An immunoadsorbent that removes anti-acetylcholine receptor antibodies (AChRAb) in abnormal serum of myasthenia gravis (MG) patient was efficiently prepared by an expression product, the functional fragment of AChR(alpha205) fused with maltose binding protein (MBP). The ligand can then covalently bind to amylose resin through MBP fusion protein. It was shown from the result of this study with anti-AChR mice sera that the removal rate of AChRAb on this immunoadsorbent reached 87+/-10% (mean value of 10 mice) and the maximally binding capacity of AChRAb was approximately 260 microg/g immunoadsorbent (wet weight). Moreover, the immunoadsorption test of sera in two MG patients indicated that about 90% and 96% of abnormal AChRAb could be eliminated, while other serum components such as albumin, IgG, IgM and IgA only dropped 18%, 35%, 22%, 15% and 24%, 27%, 15%, 12%, respectively, for two MG patient sera. It is anticipated from this study that the immunoadsorbent reported here could, with further development, find its clinical application for removal of AChRAb from patient serum.
Assuntos
Autoanticorpos/isolamento & purificação , Autoantígenos/genética , Técnicas de Imunoadsorção , Imunoadsorventes/síntese química , Imunoadsorventes/metabolismo , Receptores Colinérgicos/imunologia , Proteínas Recombinantes de Fusão/síntese química , Animais , Autoanticorpos/sangue , Autoanticorpos/metabolismo , Autoantígenos/imunologia , Sítios de Ligação de Anticorpos , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Eletroforese em Gel de Poliacrilamida/métodos , Humanos , Proteínas Ligantes de Maltose , Camundongos , Camundongos Endogâmicos C57BL , Miastenia Gravis/sangue , Miastenia Gravis/imunologia , Plasmídeos , Proteínas Recombinantes de Fusão/metabolismoRESUMO
An open reading frame of the alpha-subunit 1-205 residues (alpha205) of human acetylcholine receptor (AchR) was amplified by PCR with pUC-AChR alpha205 as the template and inserted into vector pMAL-c2X. The constructed pMAR alpha205 was transferred into E. coli BL21 which were then grown in LB medium. The amount of soluble MBP-AChR alpha205 protein reached about 25% of total soluble proteins from the cell lysate. Using amylose-affinity chromatography, about 35 mg MBP-AChR alpha205 could be obtained from 1 l culture. Western blot analysis and ELISA showed that immunoreactivities of both MBP-AChR alpha205 and AChR alpha205 were similar to that of AChR alpha-subunit from Torpedo.