CN117074668A - Hpv病毒分型检测试剂盒及其制备方法 - Google Patents
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Abstract
本发明属于生物免疫检测分析技术领域,具体涉及一种HPV病毒分型检测试剂盒及其制备方法。本发明的HPV病毒分型检测试剂盒,包括试纸条和标本提取液,所述试纸条包括底板、样品垫、金标垫、硝酸纤维素膜和吸水垫,所述硝酸纤维素膜上包被有检测线和质控线,所述质控线上包被有胶体金标记的鼠抗人HPV16蛋白单克隆抗体和鼠抗人HPV18蛋白单克隆抗体,所述质控线上包被有抗鼠HPV16IgG抗体和HPV18IgG抗体。
Description
技术领域
本发明属于生物免疫检测分析技术领域,具体涉及一种HPV病毒分型检测试剂盒及其制备方法。
背景技术
人***瘤病毒(HPV)属于乳多空病毒科***瘤空泡病毒A属,是球形DNA 病毒,能引起人体皮肤黏膜的鳞状上皮增殖。目前已分离出130多种,不同的型别引起不同的临床表现,根据侵犯的组织部位不同可分为:(1)皮肤低危型:包括HPV1、2、3、4、7、10、12、15等与寻常疣、扁平疣、跖疣等相关;(2) 皮肤高危型:包括HPV5、8、14、17、20、36、38等与疣状表皮发育不良有关,其他还与可能HPV感染有关的恶性肿瘤包括:外阴癌、***癌、***癌、***癌、膀胱癌;(3)黏膜低危型如HPV6、11、13、32、34、40、42、43、44、 54等与感染生殖器、***、口咽部、食道黏膜;(4)黏膜高危型HPV16、18、 30、31、33、35、53、39与***、直肠癌、口腔癌、扁桃体癌等。
目前关于HPV的检测方法有很多种,包括杂交捕获法、荧光原位杂交法、芯片技术杂交法、实时荧光PCR法和斑点印记法等,每种检测方法的敏感性、特异性均有所不同。但是按检测结果来说可以分为两类,即定量检测和分型检测。
无论是哪种检测方法均对设备要求较高,而且在条件相对完善的实验室内方可进行,平均检测时间需要4个小时以上,操作复杂,依赖于实时定量PCR 仪和高水平技术人员。
发明内容
本发明的目的在于提供一种HPV病毒分型检测试剂盒及其制备方法。
根据本发明具体实施方式的HPV病毒分型检测试剂盒,所述试剂盒包括试纸条和标本提取液,所述试纸条包括底板、样品垫、金标垫、硝酸纤维素膜和吸水垫,所述硝酸纤维素膜上包被有检测线和质控线,所述质控线上包被有胶体金标记的鼠抗人HPV16蛋白单克隆抗体和鼠抗人HPV18蛋白单克隆抗体,所述质控线上包被有抗鼠HPV16 IgG抗体和HPV18IgG抗体。
优选的,所述标本提取液包括100mmol Tris-Hcl缓冲液、25mmol EDTA溶液、500mmol NaCl溶液以及1wt%SDS,其中,Tris-Hcl缓冲液的pH为8.0, EDTA溶液的pH为8.0。
根据本发明具体实施方式的HPV病毒分型检测试剂盒,将抗鼠HPV16 IgG 抗体和HPV18 IgG抗体与包被缓冲液混合,配制成为浓度为0.5mg/mL质控混合溶液,以涂布量为1uL/cm的速度喷涂在硝酸纤维素膜上,得到质控线。
根据本发明具体实施方式的HPV病毒分型检测试剂盒,将鼠抗人HPV16单克隆抗体与包被缓冲溶液混合,配制成为浓度为0.4mg/mL混合溶液,喷在硝酸纤维素膜上,得到检测线T16。
根据本发明具体实施方式的HPV病毒分型检测试剂盒,将鼠抗人HPV18单克隆抗体与包被缓冲溶液混合,配制成为浓度为0.4mg/mL混合溶液,喷在硝酸纤维素膜上,得到检测线T18。
根据本发明具体实施方式的HPV病毒分型检测试剂盒,所述包被缓冲液为硼酸盐、碳酸盐、磷酸盐、Tris-HCl或Tris-磷酸盐缓冲液,缓冲液的pH值为7.0-7.5。
根据本发明具体实施方式的HPV病毒分型检测试剂盒的制备方法,所述制备方法包括以下步骤:
(1)将HPV IgG抗体加入到金标缓冲液中,加入BSA,混匀,静置,得到沉淀,将沉淀复溶得到胶体金标记的HPV IgG抗体,并负载于基质上,干燥,得到胶体金垫;
(2)将FITC单克隆抗体、TAMRA单克隆抗体、DIG单克隆抗体或CY5单克隆抗体中的至少两种包被到硝酸纤维素膜上,或者将FAM单克隆抗体、TAMRA 单克隆抗体、DIG单克隆抗体或CY5单克隆抗体中的至少两种包被到硝酸纤维素膜上,作为检测线;将羊抗鼠抗体包被到硝酸纤维素膜上,做为质控线上;
(3)将胶体金垫、硝酸纤维素膜与底板、样品垫和吸水垫进行组装。
根据本发明具体实施方式的HPV病毒分型检测试剂盒,步骤(2)中,将包被浓度为0.3mg/mL的FITC单克隆抗体、0.3mg/mL的TAMRA单克隆抗体、 0.2mg/mL的DIG抗体单克隆抗体、0.5mg/mL的CY5单克隆抗体或0.3mg/mLFAM 单克隆抗体,按1μL/cm的速度喷于硝酸纤维素膜上,作为检测线。。
根据本发明具体实施方式的HPV病毒分型检测试剂盒的制备方法,将生物素单克隆抗体加入到金标缓冲液中,加入封闭液,混匀,离心,得到沉淀,将沉淀复溶,得到胶体金标记的生物素单克隆抗体。
根据本发明具体实施方式的HPV病毒分型检测试剂盒的制备方法,将胶体金与0.2M碳酸钾混合,得到所述金标缓冲液,其中,胶体金的金颗粒为40nm。
根据本发明具体实施方式的HPV病毒分型检测试剂盒的制备方法,所述封闭液为10%的牛血清白蛋白。
本发明的检测试剂盒的使用方法:
一、样本采集:
宫颈细胞的采集:
1.窥阴器扩张***后,将宫颈刷头中央较长刷丝***宫颈管内,两端较短刷丝抵住***粘膜面,直到两翼刷毛和宫颈口两侧紧密接触。
2.大拇指和食指捏住刷柄,以***为圆心,始终朝一个方向(顺时针或逆时针)旋转至少3-5圈。旋转时需加适当压力。若白带过多,应先用无菌干棉轻轻擦拭去,再刷取标本。
3.用镊子或剪刀取下刷头放入标本收集的提取液中,拧紧瓶盖,用力摇晃数次后取出刷头。
二、样本检验:
1.在进行测试前必须先完整阅读使用说明书,并严格按照说明书进行操作;
2.取出检测卡,将其平放;
3.用吸管在标本提取液中液体离瓶底深度三分之一处吸取2-3滴已处理过的样本(60-90μL)垂直滴入检测卡的加样孔内;
4.室温放置10-20分钟后观察检测结果
四、检验结果解释:
1.阳性:两条***条带,检测线(T16线)和质控线(C线)均显色;或检测线(T18线)和质控线(C线)均显色;
2.阴性:一条***条带,仅质控线(C线)显色;
3.无效:质控线(C线)无显色,检测无效,建议另取检测卡重新检测。
本发明的有益效果:
本试剂盒可直接对HPV病毒进行抗原检测,具有较高的敏感性和特异性,能够快速分型检测,无需PCR仪和高水平技术人员,简便快捷。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1显示本发明的试剂盒中试纸条的结构示意图,其中,1-样品垫、2-结合垫、3-检测线T16线、4-检测线T18线、5-质控线C线、6-吸水垫、7-硝酸纤维素膜、8-PVC底板。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将对本发明的技术方案进行详细的描述。显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其它实施方式,都属于本发明所保护的范围。
实施例1制备本发明的双标记的胶体金免疫层析试纸条
1.胶体金制备:按柠檬酸三钠还原法制备胶体金
取50mL 0.01%的氯金酸溶液,边加热边搅拌直至沸腾,沸腾后迅速加入 2mL 1%的柠檬酸三钠溶液,快速均匀搅拌15min,直至液体变成红色不再变化,室温下自然冷却后补充超纯水至50mL,4℃保存备用。
2.金标蛋白制备:
待标记蛋白:HPV IgG抗体、羊抗人HPV IgG抗体
用0.1M碳酸钠调节胶体金pH 7.9-8.5,按每毫升胶体金溶液缓慢加入5-25 ug待标记蛋白,混匀,静置10min,然后加入BSA至终浓度0.5-1%,混匀,静置10min,9000rpm离心2min,去沉淀,将上层溶液转至新管,12500离心10min,去上清,加入重悬液至原量,将溶液移至新管,12500rpm再次离心10min,加入用十分之一初始体积的保存液将沉淀重悬,置4℃备用。
待标记金标蛋白包括但不限于鼠抗人HPVIgG抗体、兔抗人。
3.胶体金试纸条的制备:
用包被缓冲液稀释将链霉亲和素、检测待包被蛋白至0.5-1.5mg/mL浓度,用划膜喷金仪,距离结合垫1cm处,以0.5-1.5uL/cm喷涂于膜上,构成检测带 4,质控带5,检测带与质控带距离约5mm,质控带同时距离吸水垫约1cm。
分析膜37℃烘干,封装备用。
所使用的包被缓冲液是由硼酸盐、碳酸盐、磷酸盐、Tris-HCl或Tris-磷酸盐等,缓冲液的pH值为7.0-7.5范围内。
4.检测带T16线:将鼠抗人HPV16单克隆抗体与pH为7.2的10mM PBS缓冲溶液混合配制成为浓度为0.4mg/mL混合溶液,喷在硝酸纤维素膜上,涂布量 1uL/cm。
5.质控带C线:将羊抗鼠IgG多克隆抗体与pH为7.5的10m MPBS缓冲溶液混合配制成为浓度为0.5mg/mL混合溶液,喷在硝酸纤维素膜上,涂布量为 1uL/cm。
试纸条的结构如图1所示,其包括样品垫(1)、结合垫(2)、检测线T16线 (3)、检测线T18线(4)质控线C线(5)、吸水垫(6)硝酸纤维素膜(7)、和PVC底板(8)。
将试纸条与卡壳组装成检测卡,并配制标本提取液、无菌采样拭子、吸管和说明书一份,即可完成本发明的检测试剂盒。
其中,标本提取液包括以下原料:100mmol Tris-Hcl(pH8.0)、25mmol EDTA(pH8.0)、500mmol NaCl和1%SDS。
实施例2样本的检测
本实施例中制备的试剂盒对临床样本进行检测,总计纳入98例临床样本。实验过程包括样品的采集和检测:
样品的采集:
1.窥阴器扩张***后,将宫颈刷头中央较长刷丝***宫颈管内,两端较短刷丝抵住***粘膜面,直到两翼刷毛和宫颈口两侧紧密接触。
2.大拇指和食指捏住刷柄,以***为圆心,始终朝一个方向(顺时针或逆时针)旋转至少3-5圈。旋转时需加适当压力。若白带过多,应先用无菌干棉轻轻擦拭去,再刷取标本。
3.用镊子或剪刀取下刷头放入标本收集的提取液中,拧紧瓶盖,用力摇晃数次后取出刷头。
样品检测和结果分析:
1.在进行测试前必须先完整阅读使用说明书,并严格按照说明书进行操作;
2.取出检测卡,将其平放;
3.用吸管在标本提取液中液体离瓶底深度三分之一处吸取2-3滴已处理过的样本(60-90μL)垂直滴入检测卡的加样孔内;
4.室温放置10-20分钟后观察检测结果:
1)阳性:两条***条带,检测线(T16线)和质控线(C线)均显色;或检测线(T18线)和质控线(C线)均显色;
2)阴性:一条***条带,仅质控线(C线)显色;
3)无效:质控线(C线)无显色,检测无效,建议另取检测卡重新检测。
其中,19例确诊为阳性,79例为阴性,高危型HPV16型6例,高危型HPV18 型7例,将利用本实施例中的试剂盒的检测结果和PCR仪器HPV-DNA检测结果、组织免疫组化结果进行一致性分析,结果如下表所示:
表1临床样本检测结果
通过对108例样本,分别采用本发明试剂盒与HPV-DNA方法,对照组织学检测实验结果相符,故本发明试剂盒可以达到临床要求。
实施例3对比实验
①常规胶体金方法
1.1将HPV16和HPV18病毒株的标本按1:1000,1:500,1:200,1:100,1:500 稀释标准同时加入目前医院常用PCR方法的标本提取液(标本提取液的成分为三羟甲基氨基甲烷10mol/L,乙二胺四乙酸1mol/L;乙基苯基聚乙二醇0.02%,氯化钠0.06%)。
其他操作步骤同实施例1。
用吸管吸取2-3滴样品溶液(60-90μL)分别垂直滴入检测卡的加样孔内。
结果显示,不同稀释比例的样品溶液对应的质控线C全显色,只有按1:50 稀释的样品溶液检测T才有轻微显色,其余检测线T皆不显色。
②本发明方法
2.1将HPV16和HPV18病毒株的标本分别按 1:1000,1:500,1:200,1:100,1:500稀释标准同时加入本发明的标本提取液中 (100mmol Tris-Hcl(pH8.0)、25mmol EDTA(pH8.0)、500mmol NaCl和1%SDS)。
其他操作步骤同实施例1。
结果显示,不同稀释比例下,质控C线、检测T线皆显色,其中配比1:1000 的标本卡T线显色相对较浅。
常规保存液大都是对细胞的固定防腐等处理,而病毒在胞核内很难与抗体结合,更换为本发明的保存液后,其可使胞核上出现无数穿孔,从而大大提高了抗原抗体结合密度,从而,在不同的稀释浓度下,T线仍能够显色。
以上所述,仅为本发明的具体实施方式,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揭露的技术范围内,可轻易想到变化或替换,都应涵盖在本发明的保护范围之内。因此,本发明的保护范围应以所述权利要求的保护范围为准。
Claims (10)
1.HPV病毒分型检测试剂盒,其特征在于,所述试剂盒包括试纸条和标本提取液,所述试纸条包括底板、样品垫、金标垫、硝酸纤维素膜和吸水垫,所述硝酸纤维素膜上包被有检测线和质控线,所述质控线上包被有胶体金标记的鼠抗人HPV16蛋白单克隆抗体和鼠抗人HPV18蛋白单克隆抗体,所述质控线上包被有抗鼠HPV16 IgG抗体和HPV18 IgG抗体。
2.根据权利要求1所述的HPV病毒分型检测试剂盒,其特征在于,将抗鼠HPV16 IgG抗体和HPV18 IgG抗体与包被缓冲液混合,配制成为浓度为0.5mg/mL质控混合溶液,以涂布量为1uL/cm的速度喷涂在硝酸纤维素膜上,得到质控线。
3.根据权利要求1所述的HPV病毒分型检测试剂盒,其特征在于,将鼠抗人HPV16单克隆抗体与包被缓冲溶液混合,配制成为浓度为0.4mg/mL混合溶液,喷在硝酸纤维素膜上,得到检测线T16。
4.根据权利要求1所述的HPV病毒分型检测试剂盒,其特征在于,将鼠抗人HPV18单克隆抗体与包被缓冲溶液混合,配制成为浓度为0.4mg/mL混合溶液,喷在硝酸纤维素膜上,得到检测线T18。
5.根据权利要求2-4任一项所述的HPV病毒分型检测试剂盒,其特征在于,所述包被缓冲液为硼酸盐、碳酸盐、磷酸盐、Tris-HCl或Tris-磷酸盐缓冲液,缓冲液的pH值为7.0-7.5。
6.权利要求1所述的HPV病毒分型检测试剂盒的制备方法,其特征在于,所述制备方法包括以下步骤:
(1)将HPV IgG抗体加入到金标缓冲液中,加入BSA,混匀,静置,得到沉淀,将沉淀复溶得到胶体金标记的HPV IgG抗体,并负载于基质上,干燥,得到胶体金垫;
(2)将FITC单克隆抗体、TAMRA单克隆抗体、DIG单克隆抗体或CY5单克隆抗体中的至少两种包被到硝酸纤维素膜上,或者将FAM单克隆抗体、TAMRA单克隆抗体、DIG单克隆抗体或CY5单克隆抗体中的至少两种包被到硝酸纤维素膜上,作为检测线;将羊抗鼠抗体包被到硝酸纤维素膜上,做为质控线上;
(3)将胶体金垫、硝酸纤维素膜与底板、样品垫和吸水垫进行组装。
7.根据权利要求6所述的HPV病毒分型检测试剂盒的制备方法,其特征在于,步骤(2)中,将包被浓度为0.3mg/mL的FITC单克隆抗体、0.3mg/mL的TAMRA单克隆抗体、0.2mg/mL的DIG抗体单克隆抗体、0.5mg/mL的CY5单克隆抗体或0.3mg/mLFAM单克隆抗体,按1μL/cm的速度喷于硝酸纤维素膜上,作为检测线。
8.根据权利要求6所述的HPV病毒分型检测试剂盒的制备方法,其特征在于,将生物素单克隆抗体加入到金标缓冲液中,加入封闭液,混匀,离心,得到沉淀,将沉淀复溶,得到胶体金标记的生物素单克隆抗体。
9.根据权利要求6所述的HPV病毒分型检测试剂盒的制备方法,其特征在于,将胶体金与0.2M碳酸钾混合,得到所述金标缓冲液,其中,胶体金的金颗粒为40nm。
10.根据权利要求8所述的HPV病毒分型检测试剂盒的制备方法,其特征在于,所述封闭液为10%的牛血清白蛋白。
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Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101576561A (zh) * | 2009-06-16 | 2009-11-11 | 重庆理工大学 | 检测高危型人***瘤病毒的免疫胶体金测试卡及其测试方法 |
CN103865883A (zh) * | 2014-03-26 | 2014-06-18 | 重庆理工大学 | 抗高危型人***瘤病毒蛋白的单克隆抗体及其应用 |
CN106885902A (zh) * | 2017-03-03 | 2017-06-23 | 四川大学华西第二医院 | 以ncam‑1为检测指标的金标试剂盒及其制备方法和应用 |
US20170315124A1 (en) * | 2014-09-26 | 2017-11-02 | Attogen Biomedical (Suzhou) Inc. Ltd. | Cervical cancer-related hpv e7 protein monoclonal antibody and use thereof |
CN111073997A (zh) * | 2018-10-19 | 2020-04-28 | 重庆博艾迈迪森生物科技有限公司 | 一种早期快速筛查hpv16-e6和hpv18-e6的试剂盒 |
CN112062837A (zh) * | 2020-06-19 | 2020-12-11 | 上海健康医学院 | 一种hpv16型病毒抗体的快速检测试纸 |
CN113063943A (zh) * | 2021-04-02 | 2021-07-02 | 淄博市中心医院 | 用于检测高危型hpv病毒的试剂盒及检测方法 |
-
2022
- 2022-10-31 CN CN202211344872.0A patent/CN117074668A/zh active Pending
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101576561A (zh) * | 2009-06-16 | 2009-11-11 | 重庆理工大学 | 检测高危型人***瘤病毒的免疫胶体金测试卡及其测试方法 |
CN103865883A (zh) * | 2014-03-26 | 2014-06-18 | 重庆理工大学 | 抗高危型人***瘤病毒蛋白的单克隆抗体及其应用 |
US20170315124A1 (en) * | 2014-09-26 | 2017-11-02 | Attogen Biomedical (Suzhou) Inc. Ltd. | Cervical cancer-related hpv e7 protein monoclonal antibody and use thereof |
CN106885902A (zh) * | 2017-03-03 | 2017-06-23 | 四川大学华西第二医院 | 以ncam‑1为检测指标的金标试剂盒及其制备方法和应用 |
CN111073997A (zh) * | 2018-10-19 | 2020-04-28 | 重庆博艾迈迪森生物科技有限公司 | 一种早期快速筛查hpv16-e6和hpv18-e6的试剂盒 |
CN112062837A (zh) * | 2020-06-19 | 2020-12-11 | 上海健康医学院 | 一种hpv16型病毒抗体的快速检测试纸 |
CN113063943A (zh) * | 2021-04-02 | 2021-07-02 | 淄博市中心医院 | 用于检测高危型hpv病毒的试剂盒及检测方法 |
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