Preparation Of Specific Immunoadsorbent In Myasthenia Gravis And Its Property

Objective:A desired specific immunoadsorbent for anti-AChR antibodies in myasthenia gravis patients was prepared. The adsorption of the prepared specific immunoadsorbent to anti-AChR antibodies in myasthenia gravis patients was studied by static and dynamic absorption tests on the patients’serum. The blood compatibility character of specific immunoadsorbent was studied by whole blood perfusion extracorporeal circulation experiment.Methods:10%cellulose beads activated by epichlorohydrin were prepared as carriers by suspension regeneration. The content of epoxy could achieve to92μmol/g. As ligands, L-tryptophan and synthesized polypeptide of AChR at amino acid residues67-76were immobilized on cellulose beads to make immunoadsorbent, respectively. The affection of reactive condition to fixative dose was observed simultaneously. In the static adsorption test, the adsorbents were reacted with patients’ serum which contained anti-AChR antibodies at37℃for3hours with shaking. Before and after the reaction, the optical densities of the antibodies were determined by ELISA respectively and adsorption capacities were calculated. The differences of adsorption rate in immunoadsorbents were compared. In the dynamic absorption test, the adsorbents were reacted with patients’serum which contained anti-AChR antibodies at37℃. The whole blood perfusion was cycled for4hours at a speed of2ml/min. Before and after the reaction, the optical densities of the antibodies were determined by ELISA respectively and adsorption capacities were calculated. The blood compatibility character of specific immunoadsorbent was studied by whole blood perfusion extracorporeal circulation experiment. In this experiment the normal rabbits were randomly divided into three groups, including control group (only cellulose beads), specific group and L-tryptophan group. Blood cells, ions and plasma proteins were examined before and after experiment.t test was applied in analysis of the data. The rates of loss were compared by t test, too.Results:The optimal conditions of polypeptide immobilization were37℃, pH9.7and20hours. The highest adsorption capacity of polypeptide adsorbent for anti-AChR antibody was40.33%±2.29%, followed by L-tryptophan adsorbent with22.35%±1.47%. The statistical difference was significant (p<0.05). The adsorbent with cellulose as carrier and polypeptide as ligands, was of high affinity with antibody in dynamic absorption test on the patients’serum. The adsorption capacity rose continuously as the time prolonged, and there was a peak after2hours of the adsorption. The adsorption capacity was26.59%. In the blood compatibility, there was no significant difference in control group in blood cells, ions and proteins before and after experiment (P>0.05). There were non-significant differences between specific group and L-tryptophan group in blood cells and in ions, but plasma proteins were significant decreased. In plasma total proteins (TP) were57.50±2.52and46.50±3.32(P<0.05), Plasma globulins (Glo) were23.75±1.89and16.75±2.36(P<0.05) and plasma albumins (Alb) were33.75±2.63and29.50±1.91(P<0.05) respectively in specific group. TP were60.50±7.68and51.00±9.42(P<0.05), Glo were24.25+4.57and19.50±6.40(P<0.05) and Alb were36.25±3.30and31.50±3.70(P<0.05) respectively in L-tryptophan group. The plasma proteins were not significant in specific group and L-tryptophan group (P>0.05).Conclusion:Fixed dose is influenced immediately by temperature, pH and time. The statistical difference is significant in L-tryptophan and polypeptide group of the static absorption test. The result of polypeptide in dynamic absorption tests is better than others. The blood compatibility character of specific immunoadsorbent is favourable. The adsorbent with cellulose as carrier and polypeptide fragment as ligands, which is of high affinity with antibody and favorable in blood compatibility character, is an ideal immunoadsorbent in specific immunotherapy of myasthenia gravis.