CN111413507A - 检测s蛋白rbd抗体评价恢复期血浆抗病毒能力的方法 - Google Patents
检测s蛋白rbd抗体评价恢复期血浆抗病毒能力的方法 Download PDFInfo
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Abstract
本发明公开了一种检测S蛋白RBD抗体评价恢复期血浆抗病毒能力的方法,包括如下步骤:S1.制备恢复期血浆;S2.利用抗原抗体特异性结合的原理检测S蛋白的RBD抗体;S3.以S2的RBD抗体的含量评价恢复期血浆抗病毒能力。本发明采用针对RBD的表达产物检测,建立新的方法,操作简单、成本低,实验室要求低,安全性高,普通临床实验室即可检测。
Description
技术领域
本发明涉及生物化学技术领域,具体涉及一种检测S蛋白RBD抗体评价恢复期血浆抗病毒能力的方法。
背景技术
恢复期血浆疗法是一种基于血浆或血浆衍生物的治疗方式,即采用严重感染患者恢复健康后的血浆或血浆衍生物治疗相应病原体感染的患者。这些恢复期患者的血浆中含有高浓度特异性的抗病原体抗体,输入患者体内后,可以中和病原体,也会激活补体,介导有效的免疫反应,从而达到治疗疾病、清除病原体的目的。恢复期血浆疗法可以追溯到20世纪初,被成功应用于很多传染性疾病,包括炭疽病、瘟疫、猩红热、麻疹、白喉、痢疾、流行性脑脊髓膜炎、狂犬病、肺炎球菌性肺炎等。2003年SARS流行期间、H1N1流行期间,血浆疗法也对感染患者尤其是对部分药物治疗效果不佳的患者或重症患者显示出良好的效果。
目前针对新发病原体COVID-19尚无特效药物针对性治疗,疫苗研发尚需时日,特异性抗体的生产和检定也需要一定的周期。近期治愈出院的患者血浆中含有高效价的抗病原体抗体。有研究也指出,危重症患者血浆中分离出的部分新病毒可以被多个感染患者的血清中和,说明患者的血清中存在针对新病毒的特异性中和抗体。因此,利用恢复期血浆进行治疗,有望为救治感染新病原体患者提供有效的治疗手段,降低死亡率,保障患者生命安全。
目前评价恢复期血浆或免疫球蛋白抗病毒能力的方法只有中和试验,中和试验成本高、检测周期长,条件要求高,需要在P3实验室里操作,安全风险大,需要使用活病毒。
发明内容
有鉴于此,为解决上述技术问题,本发明的目的在于一种检测S蛋白RBD抗体评价恢复期血浆抗病毒能力的方法,该方法操作简单、成本低,实验室要求低,安全性高,普通临床实验室即可检测。
所采用的技术方案为:
一种检测S蛋白RBD抗体评价恢复期血浆抗病毒能力的方法,包括如下步骤:
S1.制备恢复期血浆;
S2.利用抗原抗体特异性结合的原理检测S蛋白的RBD抗体;
S3.以S2的RBD抗体的含量评价恢复期血浆抗病毒能力。
进一步地,S3中,当RBD抗体浓度大于50稀释度时,则恢复期血浆具有良好的临床抗病毒能力。
进一步地,S2中,利用ELISA法或化学发光法来检测S蛋白的RBD抗体。
进一步地,S1中,所述恢复期血浆为COVID-19恢复期血浆或SARS-CoV恢复期血浆。当然恢复期血浆包括于此,但不局限于此,也可以包括其他病原体的恢复期血浆。
作为一种替代方案,将恢复期血浆用免疫球蛋白替代。即该替代方案为:
一种检测S蛋白RBD抗体评价免疫球蛋白抗病毒能力的方法,包括如下步骤:
S1.制备免疫球蛋白;
S2.利用抗原抗体特异性结合的原理检测S蛋白的RBD抗体;
S3.以S2的RBD抗体的含量评价免疫球蛋白抗病毒能力。
制备免疫球蛋白的步骤,可以从被替代方案中的制备恢复期血浆中进一步浓缩纯化得到。
还可以有将恢复期血浆替换为该恢复期血浆的其他衍生物的替代方案。
本发明的有益效果在于:
目前评价恢复期血浆或免疫球蛋白抗病毒能力的方法只有中和试验,中和试验成本高、检测周期长,条件要求高,需要在P3实验室里操作,安全风险大,需要使用活病毒。本发明采用针对RBD的表达产物检测,建立新的方法,操作简单、成本低,实验室要求低,安全性高,普通临床实验室即可检测。
附图说明
图1为本发明方法与传统的活病毒中和试验有较好的相关性的实验结果图。
图2为比较实验组与对照组患者症状无改善的累积率的实验结果图。
具体实施方式
下面通过具体的实施例对本发明进行详细说明,但这些例举性实施方式的用途和目的仅用来例举本发明,并非对本发明的实际保护范围构成任何形式的任何限定,并非将本发明的保护范围局限于此。
实施例1
检测S蛋白RBD抗体评价恢复期血浆抗COVID-19新冠肺炎病毒能力的方法,包括以下步骤:
S11.恢复期血浆的制备(IVIG的制备方式同恢复期血浆的制备,增加浓缩纯化步骤):
主要采用血细胞分离机和密闭式专用管道,选择单采血浆专用程序,采集血浆量为200-600mL,采集后的血浆用无菌接驳器进行接驳和分装成100mL的小包装,迅速放置-40℃以下速冻,-20℃以下保存;
然后留样进行血浆质量检测。两次采浆间隔期不少于14天,按照GB18469进行血浆质量检测。
此外,还进行COVID-19新冠肺炎病毒血清学定性检测:研究其反应性,并进行定量检测(滴度检测):滴度不少于50。
S2.RBD抗体检测:(目前的方法有ELISA法和化学发光法,本实施例为ELISA法)
S21.以包被液稀释COVID-19新冠肺炎病毒血清(终浓度为1ug/ml-5ug/ml),以每孔100ul加入96孔酶标板;4℃过夜包被;
S22.甩掉包被液,加封闭液200ul/孔封闭过夜(或者37℃封闭2小时);
S23.洗涤液洗涤3次,用稀释液稀释恢复期血浆,稀释后按100ul/孔加入恢复期血浆,37℃孵育1小时;
S24.洗涤液洗涤3次,加酶标第二抗体(抗抗体)(按照试剂说明以稀释液稀释),100ul/孔。37℃孵育1小时;
S25.洗涤液洗涤3次,加AB显色液,100ul/孔,室温避光呈色4分钟;
S26.加入终止液50ul/孔终止反应;
S27.以酶标仪测量450nm OD值;
S3.计算RBD抗体浓度,当RBD抗体浓度大于50稀释度时,则评价:恢复期血浆具有良好的临床抗病毒能力。
其中,包被液、封闭液、稀释液、洗涤液、终止液等均是本领域ELISA法中常规的溶液。例如(1)包被液(pH9.6 0.05M碳酸盐缓冲液):
NaCO3 1.59克,
NaHCO3 2.93克,
加蒸馏水至 1000ml。
(2)洗涤液(pH7.4 PBS):0.15M
(3)稀释液:
牛血清白蛋白(BSA) 0.1克
加洗涤缓冲液至 100ml
或以羊血清、兔血清等血清与洗涤液配成5~10wt%使用。
(4)终止液(2M H2SO4):
蒸馏水178.3ml,逐滴加入浓硫酸(98vol%)21.7ml。
(5)封闭液;1%的BSA。每100mL的PBST(PBS溶液加上Tween-20即为PBST)中加入1g的牛血清白蛋白(BSA)。
实施例2
本实施例具体操作步骤同实施例1。不同的是,实施例1的恢复期血浆为COVID-19恢复期血浆,本实施例是SARS-CoV恢复期血浆。
本实施例相应是一种检测S蛋白RBD抗体评价恢复期血浆抗SARS-CoV能力的方法。
实施例3
参照实施例1,与实施例一不同的是,本实施例用免疫球蛋白(IVIG)来替代实施例1的恢复期血浆。其中S1.制备免疫球蛋白,包括如下步骤:
以恢复期血浆为原料,通过两步离子交换层析实现蛋白分离纯化,结合纳米膜除病毒过滤,制备出以甘氨酸为稳定剂的静注人免疫球蛋白制剂。
一、实施例1的效果测试(实验室评价验证):
测试方法:
1.检测COVID-19恢复期血浆抗体浓度;
2.在感染COVID-19活病毒前24小时将宿主细胞(Vero细胞104)接种在96孔板中;
3.接种活病毒,并在37℃5vol%CO2细胞培养箱中孵育2小时;
4.将上述恢复期血浆在56℃孵育30分钟,稀释1-10倍,加入上述细胞培养板实验组中,在37℃含有5vol%CO%细胞培养箱中5天,在显微镜下检查细胞病变效应+;
5.分析RBD抗体浓度与活病毒中和效应的相关性,试验结果参见图1所示。
试验结果表明,该方法与传统的活病毒中和试验有较好的相关性,R值为0.69,P值为0.0139。因此推测检测RBD抗体浓度即可检测恢复期血浆的抗病毒能力。
二、实施例1的的效果测试(临床评价):
检测献血者恢复期血浆中RBD抗体浓度,不同献血者具有不同的抗体浓度,当RBD抗体浓度大于50稀释度时,推测恢复期血浆具有良好的临床治疗效果。
测试方法:
1.招募受试者,用随机方法分为实验组和对照组;
2.将RBD抗体浓度大于50稀释度的恢复期血浆或IVIG输入实验组,其他处理方法和对照组完全相同。
恢复期血浆输入方法如下:
(1).除常规治疗外,联合静脉注入RBD抗体滴度高于50稀释度的恢复期血浆,宜尽早输注,第一天输注一次。输注开始和结束时,均应记录血浆输注的日期和时间(24小时制),以及输注量。
(2).恢复期血浆输注原则:按次侧相容性原则交叉配血输注,献血者不规则抗体筛查阴性的血浆可直接进行ABO相容性输注,优先使用ABO同型血浆。
(3).恢复期血浆输注剂量:由临床医生根据临床状况、患者体重及新型冠状病毒抗体滴度决定,治疗组患者经静脉输注抗体滴度高于50稀释度的血浆,输注量100-400mL。
(4).恢复期血浆输注速度:缓慢输注,推荐速度100mL/小时,不超过200mL/小时,严密监测是否发生输血不良反应。如有不良反应发生,首先可以通过减慢输注速度来缓解不良反应,必要时,可以暂停或终止血浆输注,并详细记录血浆输注后的不良反应情况以及血浆中断输注的原因。
3.记录实验组和对照组患者病程。
4.分析输入RBD抗体浓度大于50稀释度的恢复期血浆与实验组生存状况差异,发现实验组与对照相比,患者症状无改善的累积率降低,即具有良好的症状改善累积率,试验结果参见图2所示。
试验结果表明:输入恢复期血浆后患者症状无改善的累积率与对照组相比降低,即患者症状改善累积率提高。
综上,本发明采用针对病原体S蛋白与宿主细胞受体结合的区域(RBD)的表达产物检测恢复期血浆或免疫球蛋白抗病毒能力,建立新的方法,操作简单、成本低,实验室要求低,安全性高,普通临床实验室即可检测。
上文所列出的一系列的详细说明仅仅是针对本发明的可行性实施例的具体说明,它们并非用以限制本发明的保护范围,凡未脱离本发明技艺精神所作的等效实施例或变更均应包含在本发明的保护范围之内。
Claims (5)
1.一种检测S蛋白RBD抗体评价恢复期血浆抗病毒能力的方法,其特征在于,包括如下步骤:
S1.制备恢复期血浆;
S2.利用抗原抗体特异性结合的原理检测S蛋白的RBD抗体;
S3.以S2的RBD抗体的含量评价恢复期血浆抗病毒能力。
2.根据权利要求1所述的检测S蛋白RBD抗体评价恢复期血浆抗病毒能力的方法,其特征在于,S3中,当RBD抗体浓度大于50稀释度时,则恢复期血浆具有良好的临床抗病毒能力。
3.根据权利要求1所述的检测S蛋白RBD抗体评价恢复期血浆抗病毒能力的方法,其特征在于,S2中,利用ELISA法或化学发光法来检测S蛋白的RBD抗体。
4.根据权利要求1所述的检测S蛋白RBD抗体评价恢复期血浆抗病毒能力的方法,其特征在于,S1中,所述恢复期血浆为COVID-19恢复期血浆或SARS-CoV恢复期血浆。
5.根据权利要求1所述的检测S蛋白RBD抗体评价恢复期血浆抗病毒能力的方法,其特征在于,将所有的恢复期血浆均用免疫球蛋白替代。
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