CN117054647A - 一种检测HPV IgG抗体试剂盒及其制备方法 - Google Patents
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Abstract
本发明涉及检测技术领域,具体公开了一种检测HPV IgG抗体试剂盒及其制备方法。该试剂盒包括鼠源人IgG抗体‑吖啶酯发光试剂、HPV L1‑VLP抗原‑磁微粒偶联物;所述鼠源人IgG抗体‑吖啶酯发光试剂包括鼠源人IgG抗体和吖啶酯;所述HPV L1‑VLP抗原‑磁微粒偶联物包括HPV L1‑VLP抗原和磁微粒;所述HPV L1‑VLP抗原通过将HPV病毒的L1基因删除N末端序列同时采用点突变改造L1基因并进行蛋白质表达、蛋白质纯化和颗粒组装制得。本发明检测HPV IgG抗体试剂盒灵敏度高、特异性强,并且具有良好的重复性和准确性。
Description
技术领域
本发明涉及检测技术领域,具体涉及一种检测HPV IgG抗体试剂盒及其制备方法。
背景技术
人乳头瘤病毒(HPV)是一种嗜人体皮肤和黏膜组织细胞的DNA病毒,可广泛感染人类皮肤及生殖道、呼吸道上皮组织,它与人类多种良恶性肿瘤密切相关。目前已知HPV有近100个基因型,与人类常见疾病相关的有10多个基因型。人们根据其致癌性把其分为高危型和低危型,高危型HPV主要有16、18、31、33、39、45、51、52、56、58、59、68型;低危型HPV主要有6、11、42、43、44型。高危型HPV与宫颈癌关系密切,低危型HPV常在湿疣病人中发现。WHO在《关于保证重组人乳头瘤病毒类颗粒(VLP)疫苗安全性、有效性及质量的建议(WHO/BS/2015.2252)》中指出,中和抗体是评价HPV疫苗诱导的免疫反应是否有保护作用的金标准。《柳叶刀-感染病学》最新发表的一项研究发明,HPV疫苗诱导的中和抗体可用作疫苗效力的替代指标,来评估疫苗对高危型HPV感染和相关癌前病变的预防能力。建立稳健和标准化的血清学抗体检测方法,对当前和未来的HPV疫苗评估至关重要。
其中IgG抗体(immunoglobulin G)在免疫应答中起着激活补体,中和多种毒素的作用。IgG抗体持续时间长,是唯一能在母亲妊娠期穿过胎盘保护胎儿的抗体。它们还从乳腺分泌进入初乳,使新生儿第一时间得到抗体保护。IgG是四链单体,占血清Ig总量的75%,是血清和细胞外液中最主要的抗体成分。
化学发光免疫分析(Chemiluminescence Immunoassay,CLIA)是近十年来在世界范围内发展非常迅速的非放射性免疫分析。它具有高灵敏度、检测范围宽、操作简便快速、标记物稳定性好、无污染、仪器简单经济等优点。它是放射性免疫分析与普通酶免疫分析的取代者,是免疫分析重要的发展方向。
目前,接种HPV疫苗仍是预防宫颈癌主要方式之一,但检测HPV抗体的相关技术存在灵敏度低、特异性差的缺点,且对进行检测的环境条件(例如实验室安全等级)与样品质量要求较高,检测步骤繁复且容易造成受试者不适等。
因此,亟需提供一种检测HPV IgG抗体试剂盒及其制备方法,提高检测的灵敏度和特异性,可以在常规或低生物安全等级的实验室环境内进行操作,操作简便,结果判断客观,从而达到快速评估IgG抗体的水平。
发明内容
本发明旨在至少解决上述现有技术中存在的技术问题之一,为此本发明提出一种检测HPV IgG抗体试剂盒及其制备方法,提高检测的灵敏度和特异性,可以在常规或低生物安全等级的实验室环境内进行操作,操作简便,结果判断客观,从而达到快速评估中和抗体的水平。
本发明的第一方面提供一种检测HPV IgG抗体试剂盒。
具体的,包括鼠源人IgG抗体-吖啶酯发光试剂、HPV L1-VLP抗原-磁微粒偶联物;
所述鼠源人IgG抗体-吖啶酯发光试剂包括鼠源人IgG抗体和吖啶酯;
所述HPV L1-VLP抗原-磁微粒偶联物包括HPV L1-VLP抗原和磁微粒;
所述HPV L1-VLP抗原通过将HPV病毒的L1基因删除N末端序列同时采用点突变改造L1基因并进行蛋白质表达、蛋白质纯化和颗粒组装制得。
优选的,所述鼠源人IgG抗体与吖啶酯的质量比为1-3:1。
进一步优选的,所述鼠源人IgG抗体与吖啶酯的质量比为2:1。
优选的,所述HPV L1-VLP抗原与磁微粒的质量比为1:15-25。
进一步优选的,所述HPV L1-VLP抗原与磁微粒的质量比为1:20。
优选的,所述HPV病毒的L1基因为HPV6 L1基因、HPV11 L1基因、HPV16L1基因、HPV18 L1基因、HPV31 L1基因、HPV33 L1基因、HPV35 L1基因、HPV45 L1基因、HPV52 L1基因、HPV58 L1基因、HPV59 L1基因中的至少一种。
进一步优选的,所述HPV病毒的L1基因为HPV16 L1基因、HPV18 L1基因中的至少一种。
优选的,所述磁微粒为羧基磁微粒。
优选的,所述的检测HPV IgG抗体试剂盒还包括激发液和预激发液。
优选的,所述激发液包括无机碱和增强剂。
优选的,所述预激发液包括无机酸和氧化剂。
本发明的第二方面提供一种检测HPV IgG抗体试剂盒的制备方法。
具体的,包括以下步骤:
使用洗涤缓冲液洗涤磁微粒,加入等量的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基丁二酰亚胺振荡活化;将磁微粒与HPV L1-VLP抗原反应4-6h。加入封闭缓冲液封闭1-3h,洗涤缓冲液洗涤后制得HPV L1-VLP抗原-磁微粒偶联物;
将鼠源人IgG抗体与吖啶酯混合,偶联0.1-1.5h,透析法去除游离的吖啶酯,过凝胶层析柱纯化,根据发光强度收集流出液,制得鼠源人IgG抗体-吖啶酯发光试剂。
本发明的第三方面提供一种检测HPV IgG抗体试剂盒的检测方法。
具体的,包括以下步骤:
将待测样品与HPV L1-VLP抗原-磁微粒偶联物、鼠源人IgG抗体-吖啶酯发光试剂混合,外加磁场制得沉淀复合物,弃上清液,沉淀复合物清洗后使用化学发光免疫分析法测量。
相对于现有技术,本发明的有益效果如下:
本发明通过HPV L1-VLP抗原-磁微粒偶联物与IgG抗体的特异性结合,在外置磁场的作用下能够检测出血清中IgG抗体水平。本发明检测HPV IgG抗体试剂盒灵敏度高、特异性强,并且具有良好的重复性和准确性。
具体实施方式
为了让本领域技术人员更加清楚明白本发明所述技术方案,现列举以下实施例进行说明。需要指出的是,以下实施例对本发明要求的保护范围不构成限制作用。
以下实施例中所用的原料、试剂或装置如无特殊说明,均可从常规商业途径得到,或者可以通过现有已知方法得到。
实施例1
一种检测HPV IgG抗体试剂盒。
检测HPV IgG抗体试剂盒包括鼠源人IgG抗体-吖啶酯发光试剂、HPV L1-VLP抗原-磁微粒偶联物、激发液、预激发液。
实施例2
一种检测HPV IgG抗体试剂盒的制备方法。
1.HPV L1-VLP抗原的制备方法:
(1)HPV16(ANA05496)L1基因(其核苷酸序列如SEQ ID NO.1所示),HPV18(AAQ92369)L1基因(其核苷酸序列如SEQ ID NO.2所示)用于L1表达和假病毒制备。将HPV16L1基因、HPV18 L1基因的N末端序列删除并克隆到pTO-T7载体中。使用快速突变试剂盒(Vazyme,中国南京)在HPV16 L1基因和HPV18 L1基因的C175位点和C428位点进行定点突变。将突变的基因克隆到pTO-T7表达载体中,并使用大肠杆菌ER2566菌株进行蛋白质表达。
(2)蛋白质纯化和颗粒组装。在ER2566大肠杆菌菌株中产生野生型HPV(HPV WT)和突变L1蛋白。菌株在37℃的LB培养基中生长,直到OD600达到0.6。通过添加异丙基-β-D-硫代半乳糖苷(IPTG,终浓度为10μM)诱导L1蛋白表达,菌株在25℃下进一步孵育8h。通过离心收集菌株,并用裂解溶液(20mM Tris,pH 7.4,300mM NaCl和5mM EDTA)重悬。从细胞中释放HPV16L1蛋白和HPV18 L1蛋白并与20mM DTT结合,然后通过SP琼脂糖(GE Healthcare,美国)和CHT-II树脂(Bio-Rad,美国)进一步纯化。纯化后,使用SDS-PAGE分析蛋白质,并以1mg/mL的最终浓度储存在含有20mM磷酸盐缓冲液(pH 8.0)、500mM NaCl和20mM DTT的溶液中。然后将单个(WT)或混合的L1蛋白改变为组装缓冲液(10mM磷酸盐缓冲液,pH 6.5,500mMNaCl),衣壳体杂交VLPs。
2.HPV L1-VLP抗原-磁微粒偶联物的制备方法:
0.05mol/L吗啉磺酸(pH6.0)洗涤羧基磁微粒数次后,加入等量的EDC和NHS,(EDC:1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐,NHS:N-羟基丁二酰亚胺)常温下振荡活化40min;洗涤缓冲液洗涤4次后,按照HPV L1-VLP抗原与磁微粒1:20的质量比混合,室温振荡反应5h。经封闭缓冲液(0.05mol/L三羟甲基氨基甲烷(Tris)+2%牛血清清蛋白+0.05%Triton X100+0.5% Proclin300)封闭2h后,洗涤缓冲液洗涤后得到HPV L1-VLP抗原-磁微粒偶联物,用微球稀释液(0.05mol/LTris+3%牛血清清蛋白+0.05% TritonX-100+5%海藻糖+1%氯化钠+0.2% Proclin300)溶解,放置于4℃冰箱中备用。
3.鼠源人IgG抗体-吖啶酯发光试剂的制备方法:
将鼠源人IgG抗体(购于北京博尔西科技有限公司)与吖啶酯按2:1质量比进行混合,常温下振荡偶联0.5h,透析法去除游离的吖啶酯,过凝胶层析柱(SephadexG50)纯化,收集发光强度高的流出液,制得鼠源人IgG抗体-吖啶酯发光试剂,用发光稀释液(0.01mol/L磷酸盐缓冲液(PBS)+0.05%聚乙二醇辛基苯基醚(TritonX-100)+5%海藻糖+0.2%酪蛋白+0.5% Proclin300)稀释,避光放置于4℃下备用。
实施例3
一种检测HPV IgG抗体试剂盒的检测方法。
将待测样品与HPV L1-VLP抗原-磁微粒偶联物、鼠源人IgG抗体-吖啶酯发光试剂混合,37℃下温育反应20min,外加磁场促使磁微粒及其磁微粒偶联物聚集到试管底部,使用洗涤缓冲液(PBS+0.05% Tween 20)轻轻洗涤试管去除游离物质,用洗液清洗沉淀复合物4次,分别加入100μL激发液和100μL预激发液后,进入标本测定室,仪器自动泵入底物液,自动测量化学发光反应的结果。
样本检测效果测定:
HPV阳性血清样本为未感染接种HPV二价疫苗人群血清样本(完成全程接种后28-60天采集);HPV阴性血清样本为未感染未接种HPV疫苗的健康人群血清样本,使用HPV阳性血清样品50份,HPV阴性血清样品50份,通过该试剂盒进行测试,检测步骤如下:
阳性血清样本试验:取HPV L1-VLP抗原-磁微粒偶联物,加入60μL的HPV阳性血清和100μL鼠源人IgG抗体-吖啶酯发光剂混合,37℃下温育反应20min,外加磁场促使磁微粒及其磁微粒偶联物聚集到试管底部,使用洗涤缓冲液(PBS+0.05% Tween 20)轻轻洗涤试管去除游离物质;分别加入100μL激发液和100μL预激发液后,应用化学发光仪自动检测结果。
阴性血清样本试验:取HPV L1-VLP抗原-磁微粒偶联物,加入60μL的HPV阴性血清和100μL鼠源人IgG抗体-吖啶酯发光剂,37℃下温育反应20min,外加磁场促使磁微粒及其磁微粒偶联物聚集到试管底部,使用洗涤缓冲液(PBS+0.05% Tween 20)轻轻洗涤试管去除游离物质;分别加入100μL激发液和100μL预激发液后,应用化学发光仪自动检测结果。
阴性结果判定:统计阴性样品RLU值的平均值和标准差,即当样品RLU值>平均值+3×标准差时,判定为阳性;样品RLU值<平均值+2×标准差,判定为阴性;平均值+2×标准差≤样品RLU值≤平均值+3×标准差,判定为疑似。
表1 50组HPV阴性血清样本检测结果
表2 50组HPV阳性血清样本检测结果
表3 100组血清样本检测结果统计
由表1和表2可知,检测结果的阳性符合率为96%,阴性符合率为100%,总符合率为98%。试剂灵敏度为100%,特异度为100,准确度为98%。说明本发明检测HPV IgG抗体试剂盒灵敏度高、特异性好。
以上详细描述了本发明的较佳具体实施例。应当理解,本领域的普通技术人员无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此,凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验所作的任何修改、等同替换、改进等得到的技术方案,皆应在由权利要求书所确定的保护范围内。
Claims (10)
1.一种检测HPV IgG抗体试剂盒,其特征在于,包括鼠源人IgG抗体-吖啶酯发光试剂、HPV L1-VLP抗原-磁微粒偶联物;
所述鼠源人IgG抗体-吖啶酯发光试剂包括鼠源人IgG抗体和吖啶酯;
所述HPV L1-VLP抗原-磁微粒偶联物包括HPV L1-VLP抗原和磁微粒;
所述HPV L1-VLP抗原通过将HPV病毒的L1基因删除N末端序列同时采用点突变改造L1基因并进行蛋白质表达、蛋白质纯化和颗粒组装制得。
2.根据权利要求1所述的检测HPV IgG抗体试剂盒,其特征在于,所述鼠源人IgG抗体与吖啶酯的质量比为1-3:1。
3.根据权利要求1所述的检测HPV IgG抗体试剂盒,其特征在于,所述HPV L1-VLP抗原与磁微粒的质量比为1:15-25。
4.根据权利要求1所述的检测HPV IgG抗体试剂盒,其特征在于,所述HPV病毒的L1基因为HPV6 L1基因、HPV11 L1基因、HPV16 L1基因、HPV18 L1基因、HPV31 L1基因、HPV33 L1基因、HPV35 L1基因、HPV45 L1基因、HPV52L1基因、HPV58 L1基因、HPV59 L1基因中的至少一种。
5.根据权利要求1所述的检测HPV IgG抗体试剂盒,其特征在于,所述磁微粒为羧基磁微粒。
6.根据权利要求1所述的检测HPV IgG抗体试剂盒,其特征在于,还包括激发液和预激发液。
7.根据权利要求6所述的检测HPV IgG抗体试剂盒,其特征在于,所述激发液包括无机碱和增强剂。
8.根据权利要求6所述的检测HPV IgG抗体试剂盒,其特征在于,所述预激发液包括无机酸和氧化剂。
9.权利要求1至8中任一项所述的检测HPV IgG抗体试剂盒的制备方法,其特征在于,包括以下步骤:
使用洗涤缓冲液洗涤磁微粒,加入等量的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基丁二酰亚胺振荡活化;将磁微粒与HPV L1-VLP抗原反应4-6h。加入封闭缓冲液封闭1-3h,洗涤缓冲液洗涤后制得HPV L1-VLP抗原-磁微粒偶联物;
将鼠源人IgG抗体与吖啶酯混合,偶联0.1-1.5h,透析法去除游离的吖啶酯,过凝胶层析柱纯化,根据发光强度收集流出液,制得鼠源人IgG抗体-吖啶酯发光试剂。
10.权利要求1至8中任一项所述的检测HPV IgG抗体试剂盒的检测方法,其特征在于,包括以下步骤:
将待测样品与HPV L1-VLP抗原-磁微粒偶联物、鼠源人IgG抗体-吖啶酯发光试剂混合,外加磁场制得沉淀复合物,弃上清液,沉淀复合物清洗后使用化学发光免疫分析法测量。
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