Résumé :
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Communication n° 489 INTRODUCTION : The pathology of the autoimmune disease myasthenia gravis (MG) is largely due to the produced autoantibodies against the human muscle acetylcholine receptor (AChR), a ligand-gated ion channel composed of five subunits (?2??? or ?2??? - embryonic or adult AChR). Plasmapheresis is a therapeutic option, but in addition to anti-AChR antibodies it also removes indispensable plasma components. OBJECTIVE : To develop a specific therapy for MG and replace the non-specific plasmapheresis, through the construction of "immunoadsorbent" columns carrying recombinant AChR fragments: passage of MG plasmas through the column will specifically withhold the anti-AChR antibodies. METHODS : We expressed the extracellular domains (ECDs, amino acids 1~210) of the ?, ?, ?, ? and ? subunits of the human AChR in Pichia pastoris and purified them by Ni2+NTA-agarose. Each ECD was immobilized on CNBr-Sepharose and tested for its immunoadsorbing capacity. RESULTS : The ECDs adopted a near-native structure. After immobilization on CNBr-Sepharose and use in immunoadsorption of autoantibodies from 64 MG sera, we found that, on the average, ?1-210, ?1-221, ?1-218, ?1-224 and ?1-219 removed 35%, 22%, 21%, 0% and 18% of the anti-AChR antibodies, respectively. Combination of ?1-210 and ?1-218 additively removed antibodies from 10 tested sera. The immobilized ECDs remained stable and did not dissociate from their matrix after incubation with human plasma. Immunoadsorption was complete in a few minutes, while 1?g of polypeptide sufficed to remove 2-4 pmoles of anti-AChR antibodies. CONCLUSIONS : The application of immunoadsorbent columns with all subunit ECDs could be applied as a novel, antigen-specific therapy for MG. Future work will focus on the use of all five ECDs on large-scale, real-time experiments and the use of the ? subunit variant which carries the p3A exon
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