CN1151174C - 抗人淋巴细胞球蛋白制备方法 - Google Patents
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Abstract
本发明涉及抗人淋巴细胞球蛋白制备方法,其特征在于它采用人胸腺或胸导管引流液或人血中的T淋巴细胞为抗原,免疫家兔或马,分离免疫血清,用缓冲剂或生理盐水稀释加入醛化人血小板、红细胞和胎盘组织,除杂抗体,再用乙醇沉淀和离子交换或用正辛酸和硫酸铵提纯,得到95%以上纯度的抗人淋巴细胞球蛋白的产品,该方法有效的除去杂抗,降低毒副作用,纯度高,治疗效果好。
Description
本发明涉及一种抗人淋巴细胞球蛋白制备方法,属于生物制剂的制备方法。
抗人淋巴细胞球蛋白是一种生物制剂,临床上用于治疗急性再生障碍性贫血和组织器官移植排异反应,由于人体免疫机制和免疫功能失调,T淋巴细胞的参与有很大作用,它不仅释放有毒物质,同时还刺激其它细胞的分泌一些细胞因子,而器官移植,由于配型不当等原因,引起免疫功能失调,现有的抗人淋巴细胞球蛋白,由于制备方法不完善,提纯不好,杂抗体多,造成治疗效果不好,毒副作用大。
本发明的目的正是为了克服上述已有技术的缺点与不足,而提供一种简便有效的抗人淋巴细胞球蛋白的制备方法,从而提高了纯度,降低了毒副作用,更好的发挥药效作用,提高治疗效果。
本发明的目的是通过下述技术方案实现的;
抗人淋巴细胞球蛋白制备方法,其特征在于它按下述步骤进行:
a)采用人胸腺或胸导管引流液或人血中的T淋巴细胞液为抗原与福氏完全佐剂充分混合进行一次基础免疫家兔或马,再与不完全佐剂充分混合加强免疫家兔或马2-4次,采集血液测定血清的免疫效价达到1∶400时,停止免疫,并分离合格免疫血清;
b)在(a)项的合格免疫血清中用磷酸盐缓冲液或生理盐水稀释1-3倍后,加入醛化的其终浓度分别为105-108细胞/ml的人血小板和红细胞与胎盘组织混合3-4小时,离心除去血浆中的抗血小板、抗红细胞抗体及其它杂抗体,收集上清液,即得到初级抗人淋巴细胞球蛋白血清;
c)然后在(b)项的初级抗人淋巴细胞球蛋白血清中加入-5℃乙醇进行沉淀,使其最终浓度为19%,搅拌后静置1-2小时,离心分离,收集沉淀,尾弃上清液,再将沉淀用DEAE平衡缓冲液溶解制成悬液,然后把悬液加入到EDAE离子交换柱进行层析,再用缓冲液洗涤,直至无球蛋白为止后用0.1-1.0M的氯化钠溶液梯度洗脱,收集球蛋白峰。经超滤脱盐浓缩,即得到提纯的抗人淋巴细胞球蛋白液体,再加入赋型剂后除菌过滤,收集滤液;
d)最后将滤液分装或冻干即为产品。
抗人淋巴细胞球蛋白血清的制备方法,其特征在于在初级抗人淋巴细胞球蛋白血清中用0.06M、pH=4.0醋酸缓冲液稀释一倍后加入正辛酸,使其最终浓度为0.2-1.0%,搅拌1小时后离心取上清,尾弃沉淀,再在上清液中加入硫酸铵,最终饱和度为35-45%,搅拌1-2小时,离心分离,收集沉淀,尾弃上清液,再将沉淀用蒸馏水溶解,经超滤脱盐、浓缩得到提纯淋巴细胞球白液体。
抗人淋巴细胞球蛋白血清的制备方法,其特征在于在初级抗人淋巴细胞球蛋白血清中加入-5℃乙醇进行沉淀,使其最终浓度为19%,搅拌静置1-2小时,离心分离,收集沉淀,再将沉淀用磷酸盐缓冲液溶解制成悬液,再加入硫酸铵至饱和度达35-45%,搅拌1-2小时离心分离,收集沉淀,尾弃上清液,再用蒸馏水溶解沉淀,经超滤脱盐,浓缩得到提纯的抗人淋巴细胞球蛋白液体。
免疫血清与醛化人血小板、免疫血清与红细胞的用量按体积比分别为10∶1-10∶2,免疫血清与胎盘组织用量按体积(ml)与重量(g)比为10∶0.2、10∶0.3和10∶0.5;赋型剂为甘氨酸。
本发明的制备方法,采用免疫纯化方法,使免疫血清中通过醛化的人血小板、红细胞和胎盘组织可更好的吸收血清中的相应杂抗体,得到初级抗人淋巴细胞球蛋白血清,采用多种方法提纯,使本发明的制备方法更加灵活选择,而因地制宜,用乙醇沉淀,通过离子交换提纯,也可用正辛酸、硫酸铵两步沉淀提纯,也可用乙醇沉淀,再用硫酸铵沉淀提纯,均取得较好的效果。
按着本发明的制备方法,制备的抗人淋巴细胞球蛋白产品经过质量检测和临床观察,均达到了较好的指标,其结果如下:
(a)质量检测标准为:
1)红细胞抗体测定:凝集滴度小于1∶32。
2)抗人血小板抗体测定:凝集滴度小于1∶4。
3)抗人血浆抗体测定:免疫双扩无法沉淀线。
4)效力测定:抑制花环25%以上的高稀释度的倒数表示,大于1∶512。
5)球蛋白纯度大于95%。
(b)临床观察:
经三家医院对急性再生障碍性贫血、组织器官移植排异反应治疗观察,治疗急性再生障碍性贫血,治疗量200-300mg/日,有效率达55%-70%。治疗量100mg/日,能预防和治疗组织器官移植的排异反应,在整个治疗过程中,未见明显的毒副反应。
由于采取上述技术方案,使本发明技术与已有技术相比具有如下优点及效果;
a)本发明的制备方法,可有效的除去杂抗体,最大限度地降低毒副作用,因此提高了治疗效果,经过临床观察已取得明显的效果;
b)产品纯度大于95%以上,效价高,质量达到国家标准;
c)制备方法简便、有效、适用性强、选择性高。
实施例1
采用人胸腺中T淋巴细胞液为抗原与福氏完全佐剂充分混合进行一次基础免疫家兔,再与福氏不完全佐剂充分混合加强免疫家兔两次,当效价达到1∶400时,停止免疫,采集血清,并分离免疫血清,取免疫血清1000ml,然后加入一倍体积的磷酸盐缓冲液,浓度为0.02M,PH=7.2,再加入浓度为105细胞/ml醛化人血小板100ml,红细胞100ml和胎盘组织20g混合3小时,离心除去血浆中的抗血小板、抗红细胞抗体及其它杂抗体,收集上清液,得到初级抗人淋巴细胞球蛋白血清,再在上述血清中逐渐加入-5℃乙醇搅拌,使其最终浓度为19%,静置1小时,离心收集沉淀,再用DEAE缓冲液溶解沉淀制成悬液100ml加入到DEAE离子交换柱进行层析,用缓冲液洗涤,直至无球蛋白为止,最后用0.5M的氯化纳梯度洗脱,收集球蛋白峰值,经超滤器超滤、脱盐、浓缩后加入甘氨酸赋型剂用0.22u膜除菌滤器过滤除菌,收集滤液即为抗人淋巴细胞球蛋白成品液,经分装或冻干即为合格产品,其纯度为96%。
实施例2
采用胸导管引流中T淋巴细胞液为抗原与福氏完全佐剂充分混合进行一次基础免疫家兔,再与福氏不完全佐剂混合加强免疫家兔三次,当效价达到1∶400时,停止免疫,采集血清并分离免疫血清,取免疫血清1000ml,然后加入一倍的生理盐水,再加入浓度为108细胞/ml醛化人血小板200ml,红细胞200ml和胎盘组织30g混合4小时,离心分离,收集上清液制成初级抗人淋巴细胞球蛋白血清,然后用0.06M,PH=4.0醋酸缓冲液稀释一倍后,加入正辛酸,最终浓度为0.7%,搅拌1小时后离心取上清,再逐渐加入硫酸胺其饱和度为35%,搅拌1.5小时,离心分离收集沉淀,尾弃上清液,再将沉淀用蒸溜水溶解,用超滤器脱盐,加入甘氨酸赋型剂用0.22u膜除菌器过滤除菌,收集滤液即为抗人淋巴细胞球蛋白成品液,经分装冻干即为合格产品,其纯度为95%。
实施例3
采用人血T淋巴细胞为抗原,然后按实施例1的佐剂基础免疫马1次,加强免疫马4次,采集血液提取免疫血清,取免疫血清1000ml,然后加入一倍生理盐水稀释后,再加入浓度为1010/ml醛化人血小板、红细胞各150ml和胎盘组织50g混合3.5小时,离心分离,收集上清液,并加入-5℃乙醇,使其最终浓度为19%,静置1.5小时,离心分离,收集上清液,即为初级抗人淋巴细胞球蛋白血清,再将沉淀用磷酸盐缓冲液溶解,再加入硫酸铵,其饱和度为45%,搅拌1.5小时,离心分离,收集沉淀用蒸溜水溶解,经超滤器脱盐、浓缩、加入甘氨酸赋型剂,用0.22u膜除菌滤器过滤除菌,收集滤液即为抗人淋巴细胞球蛋白液,经分装或冻干即为合格产品,其纯度为95.5%。
Claims (5)
1、抗人淋巴细胞球蛋白制备方法,其特征在于它按下述步骤进行:
a)采用人胸腺或胸导管引流液或人血中的T淋巴细胞液为抗原与福氐完全佐剂充分混合进行一次基础免疫家兔或马,再与不完全佐剂充分混合加强免疫家兔或马2-4次,采集血液测定血清的免疫效价达到1∶400时,停止免疫,并分离合格免疫血清。
b)在(a)项的合格免疫血清中用磷酸盐缓冲液或生理盐水释1-3倍后,加入醛化的其终浓度分别为105-108细胞/ml的人血小板和红细胞与胎盘组织混合3-4小时,离心除去血浆中的抗血小板、抗红细胞抗体及其它抗体,收集上清液,即得到初级抗人淋巴细胞球蛋白血清。
c)然后在(b)项的初级抗人淋巴细胞球蛋白血清中加入-5℃乙醇进行沉淀,使其最终浓度为19%,搅拌后静置1-2小时,离心分离,收集沉淀,尾弃上清液,再将沉淀用EDAE平衡缓冲液溶解制成悬液,然后把悬液加入到EDAE离子换柱进行层析,再用缓冲液洗涤,直至无球蛋白为止后用0.1-1.0M的氯化钠溶液梯度洗脱,收集球蛋白峰。经超滤脱盐浓缩,即得到提纯的抗人淋巴细胞球蛋白液体,再加入赋型剂后除菌过滤,收集滤液。
d)最后将滤液分装或冻干即为产品。
2、抗人淋巴细胞球蛋白血清的制备方法,其特征在于在初级抗人淋巴细胞球蛋白血清中用0.06M、pH=4.0醋酸缓冲液稀释一倍后加入正辛酸,使其最终浓度为0.2-1.0%,搅拌1小时后离心取上清,尾弃沉淀,再在上清液中加入硫酸铵,最终饱和度为35-45%,搅拌1-2小时,离心分离,收集沉淀,尾弃上清液,再将沉淀用蒸馏水溶解,经超滤脱盐、浓缩得到提纯的抗人淋巴细胞球蛋白血清液体。
3、抗人淋巴细胞球蛋白血清的制备方法,其特征在于在初级抗人淋巴细胞球蛋白血清中加入-5℃乙醇进行沉淀,使其最终浓度为19%,搅拌静置1-2小时,离心分离,收集沉淀,再将沉淀用磷酸盐缓冲液溶解制成悬浮液,再加入硫酸铵至饱和度达35-45%,搅拌1-2小时离心分离,收集沉淀,尾弃上清液,再用蒸馏水溶解沉淀,经超滤脱盐,浓缩得到提纯的抗人淋巴细胞球蛋白血清液体。
4、根据权利要求1所述的方法,其特征在于所述的免疫血清与醛化人血小板、免疫血清与红细胞用量按体积比为分别为10∶1-10∶2,免疫血清与胎盘组织用量按体积(ml)与重量(g)比为10∶0.2、10∶0.3和10∶0.5。
5、根据权得要求1所述的方法,其特征在于赋型剂为甘氨酸。
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| CN104530227A (zh) * | 2014-11-24 | 2015-04-22 | 广西大学 | 一种抗神经炎细胞抗体的制取工艺 |
| CN115232783B (zh) * | 2022-08-03 | 2023-03-10 | 武汉中生毓晋生物医药有限责任公司 | 抗人t细胞猪免疫球蛋白的制备方法及醛化胎盘 |
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