重症肌无力特异性免疫吸附剂的制备及其性能研究
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摘要
目的：制备一种对重症肌无力(myasthenia gravis, MG)致病性抗乙酰胆碱受体(acetylcholine receptor, AChR)抗体具有特异性免疫吸附作用的特异性免疫吸附剂。通过对MG患者血清的体外静态吸附实验及体外动态吸附实验研究所制备的特异性免疫吸附剂对致病性抗AChR抗体的吸附性能。通过对家兔的全血灌流体外循环实验研究特异性免疫吸附剂的血液相容性。
方法：利用悬浮再生法制备10%的球型纤维素载体,并以环氧氯丙烷对载体进行活化,环氧基含量可达92μmol/g。分别制备以球型纤维素为载体,以人工合成的人AChRa亚单位67-76位10氨基酸多肽为配基的MG特异性免疫吸附剂和以L-色氨酸为配基的免疫吸附剂,并研究了反应条件对多肽固定量的影响。MG患者血清体外静态吸附实验将以1份免疫吸附剂与3倍体积的抗AChR抗体阳性的MG患者血清于37℃振荡反应3h,应用酶联免疫吸附法检测吸附前后血清中抗体含量计算吸附率。同时比较相同载体不同配基的两种免疫吸附剂之间的吸附率差异。MG患者血清体外动态吸附实验以1份特异性免疫吸附剂与10倍体积的抗AChR抗体阳性的MG患者血清在37℃条件下以2ml/min的速度循环反应4h,应用酶联免疫吸附法检测吸附前后血清中抗体含量计算吸附率。通过对家兔的全血灌流体外循环实验研究所制备的MG特异性免疫吸附剂的血液相容性。血液相容性实验将实验动物分为球形纤维素载体对照组和以相同载体不同配基的实验组。对3组实验动物进行全血灌流体外循环实验,于灌流前后采血进行血常规、血浆离子和血浆蛋白浓度测定。所得数据进行灌流前后差数t检验,比较实验本身及两种免疫吸附剂对血液各项指标的影响。计算各项指标灌流后损失率,对两种免疫吸附剂同一指标损失率进行t检验。
结果：多肽的最佳固定化条件为：最佳温度37℃,最佳pH值9.7,最佳反应时间20h。多肽为配基的特异性免疫吸附剂对血清中的抗AChR抗体的吸附率最高,为40.33%±2.29%。L-色氨酸为配基的免疫吸附剂对血清中的抗AChR抗体的吸附率也较高,为22.35%±1.47%。二者比较显示差别有统计学意义(P<0.05)。以球形纤维素为载体,多肽为配基所制备的特异性免疫吸附剂,在血清体外动态吸附实验中对血清中的抗AChR抗体的吸附率随吸附时间的延长而上升,并在吸附开始后2h达到最高峰,吸附率可达26.59%。血液相容性实验对照组实验前后各项指标均无明显变化(P>0.05)。实验组灌流2h后血常规及血浆离子均无明显变化(P>0.05),血浆蛋白明显下降。多肽为配基的特异性免疫吸附剂组实验前后血浆总蛋白分别为57.50±2.52和46.50±3.32(g/L),血浆球蛋白分别为23.75±1.89和16.75±2.36(g/L),血浆白蛋白分别为33.75±2.63和29.50±1.91(g/L)(P<0.05)；L-色氨酸为配基的免疫吸附剂组实验前后血浆总蛋白分别为60.50±7.68和51.00±9.42(g/L),血浆球蛋白分别为24.25±4.57和19.50±6.40(g/L),血浆白蛋白分别为36.25±3.30和31.50±3.70(g/L)(P<0.05)。对两种免疫吸附剂同一指标损失率进行t检验,结果显示两种免疫吸附剂对血浆蛋白的吸附率没有差异(P>0.05)。
结论：温度、pH值、时间对多肽的固定量有直接影响。两种免疫吸附剂对血清中的抗AChR抗体的静态吸附率有显著性差异,多肽为配基的特异性免疫吸附剂动态吸附效果明显,并具有良好的血液相容性。所以,以球型纤维素为载体、多肽为配基的特异性免疫吸附剂是一种对致病性抗AChR抗体吸附率较高且血液相容性较好的适用于全血灌流免疫吸附治疗MG的特异性免疫吸附剂。
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.
方法：利用悬浮再生法制备10%的球型纤维素载体,并以环氧氯丙烷对载体进行活化,环氧基含量可达92μmol/g。分别制备以球型纤维素为载体,以人工合成的人AChRa亚单位67-76位10氨基酸多肽为配基的MG特异性免疫吸附剂和以L-色氨酸为配基的免疫吸附剂,并研究了反应条件对多肽固定量的影响。MG患者血清体外静态吸附实验将以1份免疫吸附剂与3倍体积的抗AChR抗体阳性的MG患者血清于37℃振荡反应3h,应用酶联免疫吸附法检测吸附前后血清中抗体含量计算吸附率。同时比较相同载体不同配基的两种免疫吸附剂之间的吸附率差异。MG患者血清体外动态吸附实验以1份特异性免疫吸附剂与10倍体积的抗AChR抗体阳性的MG患者血清在37℃条件下以2ml/min的速度循环反应4h,应用酶联免疫吸附法检测吸附前后血清中抗体含量计算吸附率。通过对家兔的全血灌流体外循环实验研究所制备的MG特异性免疫吸附剂的血液相容性。血液相容性实验将实验动物分为球形纤维素载体对照组和以相同载体不同配基的实验组。对3组实验动物进行全血灌流体外循环实验,于灌流前后采血进行血常规、血浆离子和血浆蛋白浓度测定。所得数据进行灌流前后差数t检验,比较实验本身及两种免疫吸附剂对血液各项指标的影响。计算各项指标灌流后损失率,对两种免疫吸附剂同一指标损失率进行t检验。
结果：多肽的最佳固定化条件为：最佳温度37℃,最佳pH值9.7,最佳反应时间20h。多肽为配基的特异性免疫吸附剂对血清中的抗AChR抗体的吸附率最高,为40.33%±2.29%。L-色氨酸为配基的免疫吸附剂对血清中的抗AChR抗体的吸附率也较高,为22.35%±1.47%。二者比较显示差别有统计学意义(P<0.05)。以球形纤维素为载体,多肽为配基所制备的特异性免疫吸附剂,在血清体外动态吸附实验中对血清中的抗AChR抗体的吸附率随吸附时间的延长而上升,并在吸附开始后2h达到最高峰,吸附率可达26.59%。血液相容性实验对照组实验前后各项指标均无明显变化(P>0.05)。实验组灌流2h后血常规及血浆离子均无明显变化(P>0.05),血浆蛋白明显下降。多肽为配基的特异性免疫吸附剂组实验前后血浆总蛋白分别为57.50±2.52和46.50±3.32(g/L),血浆球蛋白分别为23.75±1.89和16.75±2.36(g/L),血浆白蛋白分别为33.75±2.63和29.50±1.91(g/L)(P<0.05)；L-色氨酸为配基的免疫吸附剂组实验前后血浆总蛋白分别为60.50±7.68和51.00±9.42(g/L),血浆球蛋白分别为24.25±4.57和19.50±6.40(g/L),血浆白蛋白分别为36.25±3.30和31.50±3.70(g/L)(P<0.05)。对两种免疫吸附剂同一指标损失率进行t检验,结果显示两种免疫吸附剂对血浆蛋白的吸附率没有差异(P>0.05)。
结论：温度、pH值、时间对多肽的固定量有直接影响。两种免疫吸附剂对血清中的抗AChR抗体的静态吸附率有显著性差异,多肽为配基的特异性免疫吸附剂动态吸附效果明显,并具有良好的血液相容性。所以,以球型纤维素为载体、多肽为配基的特异性免疫吸附剂是一种对致病性抗AChR抗体吸附率较高且血液相容性较好的适用于全血灌流免疫吸附治疗MG的特异性免疫吸附剂。
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.
引文
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