CN110616247A - 脾氨肽口服冻干粉活力快速测定方法 - Google Patents
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Abstract
本发明公开了一种脾氨肽口服冻干粉活力快速测定方法,脾氨肽口服冻干粉与健康小鼠脾淋巴细胞共孵时,可将其免疫活性过继给鼠脾淋巴细胞,使之成为致敏淋巴细胞。该致敏淋巴细胞释放出白细胞粘附抑制因子,使白细胞粘附抑制指数增高。因此,可通过测定白细胞粘附抑制指数来检定成品的活力。本法引入MTT(噻唑蓝),是因为MTT可被活细胞线粒体中琥珀酸脱氢酶还原成蓝紫色化合物(Formazan),此还原产物的多少与细胞数在一定范围内成正比,用二甲基亚砜将沉积在细胞中的Fromazan溶解,用酶联检测仪测定490nm波长的A值,将数值代入公式,即可求出粘附抑制指数。该方法简单,检测数据更准确。
Description
技术领域
本发明涉及一种测定方法,具体是一种脾氨肽口服冻干粉活力快速测定方法。
背景技术
脾氨肽溶液的活力测定方法,是采用兔心脏取血40ml,用淋巴细胞分离液分离出白色淋巴细胞为实验用的白细胞,再采用琼脂糖打孔法利用白细胞移动的生物活性,在5%CO2 37℃温箱中培养24小时观察,白细胞在琼脂糖移动的距离来测定其免疫活性。本方法一次实验需要的兔心脏血40ml,需采3-4只兔子,采血技术要求很高,需要专职老师负责采血,且本单位实验需求量大,240ml-320ml/周;实验结果需要在显微镜下用测微尺测量每一孔细胞移动的长短直径均值。本方法耗时,费力,主观性强。
发明内容
为了克服上述缺陷,本发明提供了一种脾氨肽口服冻干粉活力快速测定方法。
本发明为实现上述目的所采用的技术方案是:一种脾氨肽口服冻干粉活力快速测定方法,步骤如下:
(1)取体重22-25g小鼠3-5只,雌雄均可,雌鼠无孕,体表采用75%的酒精消毒后放净化工作台内,无菌操作取出脾脏,在灭菌平皿内置于灭菌尼龙网袋中,加适量RPMI-1640液,用灭菌针芯研磨后吸集于灭菌小试管中,将脾细胞悬液缓缓重层于淋巴细胞分离液上面,在水平离心机上于20秒内使离心力达2000r/min,离心20min,吸取淋巴细胞层,用适量1640培养液悬浮,1500r/min,离心5min,如此洗2次,计数淋巴细胞,并用1640培养液调整浓度为3×106/ml-5×106/ml备用;
(2)样品2mg/ml :脾氨肽口服冻干粉1支,加1640培养液制成含多肽2mg/ml 溶液;
(3)取灭菌的96孔细胞培养板,每份样品设至少三个实验孔和三个对照孔,按下表操作:
(4)将培养板置CO2培养箱中,在37℃、5%CO2及饱和湿度条件下培养2.5小时;
(5)弃去培养板内液体,每孔加MTT应用液200ul,在上述CO2培养箱中培养4小时,将培养板在微量板离心机上离心,离心机转速1000r/min,离心15min,然后弃去孔内培养液;
(6)每孔加入二甲基亚砜150ul,用微量混振器振荡3min,使其充分溶解,室温放置5min;
(7)选择490nm波长,在酶联检测仪上测定各孔A值,记录结果,按下式计算粘附抑制指数:
。
本发明的有益效果为:脾氨肽口服冻干粉与健康小鼠脾淋巴细胞共孵时,可将其免疫活性过继给鼠脾淋巴细胞,使之成为致敏淋巴细胞。该致敏淋巴细胞释放出白细胞粘附抑制因子,使白细胞粘附抑制指数增高。因此,可通过测定白细胞粘附抑制指数来检定成品的活力。本法引入MTT(噻唑蓝),是因为MTT可被活细胞线粒体中琥珀酸脱氢酶还原成蓝紫色化合物(Formazan),此还原产物的多少与细胞数在一定范围内成正比,用二甲基亚砜将沉积在细胞中的Fromazan 溶解,用酶联检测仪测定490nm波长的A值,将数值代入公式,即可求出粘附抑制指数。该方法简单,检测数据更准确。
具体实施方式
下面结合具体实施例对本发明作进一步解释说明。
(1)取体重22-25g小鼠3-5只,雌雄均可(雌者无孕),脱颈处死,浸入75%酒精中,体表消毒后放净化工作台内,无菌操作取出脾脏,在灭菌平皿内置于灭菌尼龙网袋中。加适量RPMI-1640液,用灭菌针芯研磨后吸集于灭菌小试管中。将脾细胞悬液缓缓重层于淋巴细胞分离液上面,在水平离心机上于20秒内使离心力达2000r/min,离心20min,吸取淋巴细胞层,用适量1640培养液悬浮,1500r/min,离心5min,如此洗2次。计数淋巴细胞,并用1640培养液调整浓度为3×106/ml-5×106/ml备用。
(2)样品2mg/ml :脾氨肽口服冻干粉1支,加1640培养液 制成含多肽2mg/ml 溶液。
(3)取灭菌的96孔细胞培养板,每份样品设三个实验孔和三个对照孔,按下表操作:
(4)将培养板置CO2培养箱中,在37℃、5%CO2及饱和湿度条件下培养2.5小时。
(5)弃去培养板内液体,每孔加MTT应用液200ul,在上述CO2培养箱中培养4小时。将培养板在微量板离心机上离心(1000r/min,15min),然后弃去孔内培养液。
(6)每孔加入二甲基亚砜150ul,用微量混振器振荡3min,使其充分溶解。室温放置5min。
(7)选择490nm波长,在酶联检测仪上测定各孔A值,记录结果。按下式计算粘附抑制指数:
。
一般情况, LAI指数(%)应不低于30%。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明披露的技术范围内,根据本发明的技术方案及其构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
Claims (2)
1.脾氨肽口服冻干粉活力快速测定方法,其特征在于,
(1)取小鼠3-5只,雌雄均可,雌鼠无孕,体表消毒后放净化工作台内,无菌操作取出脾脏,在灭菌平皿内置于灭菌尼龙网袋中,加适量RPMI-1640液,用灭菌针芯研磨后吸集于灭菌小试管中,将脾细胞悬液缓缓重层于淋巴细胞分离液上面,在水平离心机上于20秒内使离心力达2000r/min,离心20min,吸取淋巴细胞层,用适量1640培养液悬浮,1500r/min,离心5min,如此洗2次,计数淋巴细胞,并用1640培养液调整浓度为3×106/ml-5×106/ml备用;
(2)样品2mg/ml :脾氨肽口服冻干粉1支,加1640培养液制成含多肽2mg/ml 溶液;
(3)取灭菌的96孔细胞培养板,每份样品设至少三个实验孔和三个对照孔,按下表操作:
(4)将培养板置CO2培养箱中,在37℃、5%CO2及饱和湿度条件下培养2.5小时;
(5)弃去培养板内液体,每孔加MTT应用液200ul,在上述CO2培养箱中培养4小时,将培养板在微量板离心机上离心,离心机转速1000r/min,离心15min,然后弃去孔内培养液;
(6)每孔加入二甲基亚砜150ul,用微量混振器振荡3min,使其充分溶解,室温放置5min;
(7)选择490nm波长,在酶联检测仪上测定各孔A值,记录结果,按下式计算粘附抑制指数:
。
2.根据权利要求1所述的脾氨肽口服冻干粉活力快速测定方法,其特征在于,取体重22-25g的小鼠3-5只,小鼠体表采用75%的酒精消毒后放净化工作台内。
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