Performance characteristics of agar gel based
adsorbent in removing immunoglobulin E**★

Abstract

AIM： To study the effect of agar gel based adsorbent in removing excessive immunoglobulin E (IgE) in plasma.

METHODS： The experiment was accomplished from April 2002 to September 2004 in Nankai University. ① The serum of high level IgE was supplied by Immunity Room of Tianjin Changzheng Hospital. Agar powder was purchased from Beijing Yili Fine Chemicals Co., Ltd. with the batch number of 20000810. Epichlorohydrin was purchased from Tianjin Jinda Fine Chemicals Co., Ltd. with the batch number of 20010502. ② The IgE immunoadsorbent was prepared by using agar gel beads of 20-40 meshes, activated by epichlorohydrin, immobilized spacer and tryptophan as ligand. The in vitro static adsorption was tested, and the adsorption result was examined by using IgE test kit. ③ The blood compatibility of the agar-tryptophan adsorbent was examined, and the content of IgG, IgM, IgA, C3 and C4 in serum were tested. The changes of leucocyte, erythrocyte and platelet were examined by using an auto hemocytomater.

RESULT:① Effects of different ligands on adsorption capacity: The adsorbent had the best adsorption capacity when tryptophan was immobilized as ligand; there exists nonpolar reaction between tryptophan ligand and IgE. ② Effects of different buffer solutions and pH values on adsorption capability: The immobilization content was the lowest at pH=5-6 because the saturated solubility of tryptophan was the lowest at this pH value. The phosphate radical ion took part in the ring-opening process of epoxy group in the phosphate buffer system (pH=6-8), and the activation efficiency of the beads fell and the amino acid ligand which can be immobilized on the beads decreased correspondingly. The saturated solubility of tryptophan was not so high in Tris-HCl and sodium carbonate-sodium bicarbonate buffer solutions. The effect was much better in boric acid-borax buffer solution (pH=7-8). ③ Effects of different spacers on adsorption capacity: The adsorption capability to IgE was enhanced when succinic anhydride and phthalic anhydride was added as spacers compared to adsorbent without spacer （56.02%，68.00%，60.87%）. ④ Adsorption dynamics study of adsorbent containing succinic anhydride spacer and tryptophan ligand: The adsorption efficiency to IgE was 70.73% at 37 ℃. ⑤ The blood compatibility of adsorbent: The adsorption efficiency to 5 components was IgA> IgG> C4> C3> IgM after 2 hours of whole blood static adsorption in vitro, whereas the adsorption efficiency to IgA was only 26.17%, which was far below the adsorption efficiency to IgE (70.73%). There was certain destructive effect on whole blood cells, and the contents of leucocyte, erythrocyte and platelet decreased by 27.3%, 2.4% and 6.9% respectively after 2-hour static adsorption.

CONCLUSION:IgE immunoadsorbent is successfully prepared through cross-linking, activation of agar gel beads and immobilization of tryptophan ligand. The adsorbent has high adsorption efficiency to IgE and better blood compatibility, which can be used to cure the bronchial asthma through clinical blood perfusion.

摘要
目的：观察以琼脂凝胶球为载体的吸附剂对血浆中多余免疫球蛋白Ｅ的清除效果。
方法：实验于2002－04/2004－09在南开大学完成。①高免疫球蛋白E患者血清由天津市长征医院免疫室提供。琼脂粉（北京益利精细化学品有限公司，批号20000810）。环氧氯丙烷（天津津达精细化学品厂，批号20010502）。②以20～40目的琼脂凝胶微球为载体，经环氧氯丙烷活化，键联配基色氨酸及手臂制得免疫球蛋白E免疫吸附剂，进行体外静态吸附测试。吸附结果采用酶标法免疫球蛋白E试剂盒检测。③连接手臂的琼脂－色氨酸吸附剂进行血液相容性测试，检测吸附前后血清中免疫球蛋白G、免疫球蛋白M、免疫球蛋白A以及C3，C4含量的变化。全自动血球计数分析仪检测白细胞、红细胞、血小板数目的变化。
结果：①不同配基对吸附剂吸附效果的影响：琼脂凝胶载体连接色氨酸配基后对免疫球蛋白E的吸附性能最好，对其吸附主要是一种非极性作用力。②不同缓冲体系及pH值对吸附效果的影响：pH=5～6的范围内色氨酸的饱和溶解度最低，键联量也很低。pH=6～8的磷酸盐缓冲体系，磷酸根离子参与环氧基的开环反应，降低载体的活化效率，同时可键联的活性氨基酸配基的量也相应减少。Tris-HCl和碳酸钠－碳酸氢钠缓冲体系，对色氨酸的饱和溶解度也不是很高。相对来说，pH=7～8的硼酸－硼砂缓冲体系效果较好。③不同手臂对吸附剂吸附效果的影响：与连接前比较，连接丁二酸酐、邻苯二甲酸酐手臂后的吸附剂对免疫球蛋白E的吸附能力均有所提高（56.02%，68.00%，60.87%）。④以色氨酸为配基、丁二酸酐为手臂的琼脂凝胶吸附剂吸附动力学实验：吸附剂在37 ℃对免疫球蛋白E的吸附率为70.73%。⑤吸附剂的血液相容性实验结果：经过2 h的体外全血静态吸附后，吸附剂对5种组分的吸附率为免疫球蛋白A > 免疫球蛋白G > C4 > C3 > 免疫球蛋白M，而对最大的免疫球蛋白A的吸附率也仅为26.17%，远低于对免疫球蛋白E的吸附率70.73%。吸附剂对全血细胞有一定的破坏性，静态吸附2 h后，白细胞下降27.3%，红细胞下降2.4%，血小板下降6.9%。
结论：以琼脂凝胶微球载体，通过交联、活化和固定配基色氨酸成功制备免疫球蛋白E免疫吸附剂。此吸附剂对免疫球蛋白E有较高的清除率，同时具备较好的血液相容性，适用于临床血液灌流治疗支气管哮喘疾
病。
关键词：哮喘；免疫吸附剂；免疫球蛋白Ｅ；琼脂凝胶

王为超，董瑞玲，张红梅，张玉环，俞耀庭，王深琪.琼脂凝胶球载体吸附剂清除免疫球蛋白Ｅ的性能特征[J].中国组织工程研究与临床康复，2007，11(1):90-94 [www.zglckf.com/zglckf/ejournal/upfiles/07-1/1k-90(ps)pdf]