CN112362874A - 一种宫颈癌筛查试剂盒 - Google Patents
一种宫颈癌筛查试剂盒 Download PDFInfo
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- CN112362874A CN112362874A CN202011185161.4A CN202011185161A CN112362874A CN 112362874 A CN112362874 A CN 112362874A CN 202011185161 A CN202011185161 A CN 202011185161A CN 112362874 A CN112362874 A CN 112362874A
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Abstract
本发明公开了一种宫颈癌筛查试剂盒,涉及生物检测技术领域。所述试剂盒包括HPV E6单克隆抗体和HPV E7单克隆抗体,所述HPV E6单克隆抗体由保藏号为CCTCC NO:C2020153的杂交瘤细胞株产生;所述HPV E7单克隆抗体由保藏号为CCTCC NO:C2020152的杂交瘤细胞株产生。本发明检测同一载玻片上的宫颈脱落细胞中的HPV E6/E7蛋白总含量及DNA倍体含量;并且检测宫颈上皮内瘤变(CIN1及以上)灵敏度高,特异性强,稳定性好;通过同时分析两项数据对患者进行宫颈癌风险评估,减少漏诊,避免过度诊疗。
Description
技术领域
本发明属于生物检测领域,具体涉及一种宫颈癌筛查试剂盒。
背景技术
DNA作为遗传物质,其结构和功能的异常,是细胞恶变的分子基础,不少文献报道细胞DNA异常与临床行为有关联(细胞分裂的实质是DNA的复制,肿瘤细胞由于调节和生长控制失调而获得无限增殖能力。肿瘤细胞的增生需要合成大量的核酸以满足其迅速增长的物质需要,因此DNA含量的变化可作为直接反映肿瘤增殖能力的重要生物学指标。薛凤霞等对100例子宫内膜癌中异倍体及二倍体患者的生存率进行比较,两者的生存率差异有显著性(P<0.01),异倍体患者的生存率低于二倍体患者。国外也有研究表明,卵巢癌异倍体者的预后较二倍体者差。温宏武等对31例可能影响卵巢癌预后的六个因素包括临床分期、DNA倍体、病理分级、组织学类型、术后残余瘤大小及化疗情况进行多因素分析,结果显示预后与DNA倍体情况关系最密切,其次是术后残余瘤的大小。这可能是为什么病理分级相同的肿瘤在临床上可表现出明显不同的生物学行为及临床预后的原因。说明对肿瘤DNA含量的定量分析,可获得单纯从形态上难以得到的肿瘤生物学特征的信息,不仅为用形态学估价肿瘤生物学特征提供了有价值的补充,而且有助于进一步提高对形态学的认识水平。
宫颈癌是所有癌症中目前唯一明确病因的癌症,其主要原因为高危型HPV的持续感染。据报道,女性罹患宫颈癌呈逐年上升趋势,宫颈癌已经成为威胁女性健康的重要疾患,由此造成的生活质量的降低以及死亡已经引起世界卫生组织的关注。因此,宫颈癌的早期诊断以及预防显得越来越重要。在一些发达国家,宫颈癌的早期筛查已经普及。其原因归功于巴氏涂片的广泛应用,从而极大地推动了宫颈癌筛查项目的进展,然而该项技术本身存在局限性,影响了临床诊断效果,如所取的细胞量不稳定,诊断结果受取材的影响而不能重复等。近年来随着液基细胞学的临床应用使细胞量不足的问题得到了改善,然而其与巴氏涂片一样要依赖细胞病理学医生的主观判断,而细胞病理学医生的诊断水准不尽一致,从而降低了细胞学诊断的敏感性。并且,由于细胞学形态异常改变发生比较晚,不能早期预示病变情况,制约了其早期诊断意义。基于以上种种因素,HPV的检测就成为了宫颈癌早期筛查的切入点,这也被国内外同行所认可。
随着HPV HC2方法的普及,其高的检测敏感性以及检测的稳定性使之成为美国FDA认证的检测的金标准,其对宫颈癌的筛查起到了不可替代的作用。然而,由于HPV存在的普遍性和的终生累计感染风险已经达到了80%,使得该方法对宫颈病变诊断的特异性降低。有很多报道显示,其检测的特异性较低,容易引起假阳性,其结果往往会给临床医生进一步治疗带来困惑,相当一部分医师会采取过度的治疗方式,这样会给病人的身体和心理带来许多的伤害。特异性不高的根本原因在于感染普遍性,以及感染后有一过性消除的特点,检测阳性结果只能证明曾经有过的感染,目前体内是否已经清除病毒,是否持续感染,这些并不能得到证实。即使病毒依然存在于体内,其是否整合入宿主基因中,是否会导致上皮内病变,是否有活性的复制与转录,都无法从的实验结果中判读出来,因而方法也存在局限性。在这样的前提下,检测是否能够成为一个新的生物标记,其所检测的是否可以反映当前体内病毒的活性情况,从而对临床起到更大指导意义成为一个值得探讨的问题。
环状HPV基因组包括七个早期区和两个晚期区,其中,由E6、E7基因编码的致癌蛋白是导致宫颈上皮癌变的重要因子。在HPV感染的最初阶段,HPV病毒会随着细胞凋亡而被清除,但若HPV病毒持续感染,可能会导致病毒随机将E6E7基因整合入宿主细胞中,进而引起E6/E7癌蛋白的过度表达。E7癌蛋白可与抑癌因子Rb结合,代替周期蛋白激酶CDK4/6的作用机制,进而导致细胞DNA合成期失控,进而导致细胞癌变。E6癌蛋白通过E6AP复合物与细胞凋亡因子p53结合,是p53降解,导致细胞凋亡程序失控,因此,E6/E7癌蛋白是宫颈上皮癌变的最重要的生物标志物,其中E7蛋白与早期肿瘤有关,E6蛋白与晚期肿瘤有密切关系。
2015年CFDA批准的Aptima HPV E6/E7 mRNA检测是E6/E7作为宫颈癌筛查特异性生物标志物应用的重要里程碑。然而,mRNA体内易于降解,其检测需要昂贵的仪器、严格的实验室环境及繁琐的程序,因此基于E6/E7mRNA检测的测试可能在常规妇科实践中具有有限的临床应用。而基于E6或E7蛋白检测的试剂将比检测HPV DNA或HPV E6/E7 mRNA具有优势。
因此有必要研究一种HPV多种蛋白检测试剂盒,结合DNA染色技术用以检测同一载玻片上的宫颈脱落细胞中的HPV E6/E7蛋白总含量及DNA倍体含量,并且实现灵敏度高,特异性强,稳定性好;并且通过同时分析多项数据对患者进行宫颈癌风险评估,减少漏诊,避免过度诊疗。
发明内容
针对于现有技术的不足,本发明的目的在于提供一种宫颈癌筛查试剂盒,提高整个检测系统的客观性,提高宫颈癌筛查的灵敏度和特异度,减少漏诊,避免过度诊疗。
鉴于此,首先,本发明提出一种HPV多种蛋白检测试剂盒,由HPV E6单克隆抗体和HPV E7单克隆抗体作为一抗试剂;所述HPV E6单克隆抗体由保藏号为CCTCC NO:C2020153的杂交瘤细胞株产生;所述HPV E7单克隆抗体由保藏号为CCTCC NO:C2020152的杂交瘤细胞株产生。
进一步地,所述试剂盒还包括样本稀释液及二抗检测系统。
进一步地,所述样本稀释液包括三(2-羧乙基)膦盐酸盐和Tris缓冲液。
进一步地,所述二抗检测系统包括封闭液,生物素标记试剂,过氧化物酶标记链霉亲和素试剂和DAB显色剂。
进一步地,所述生物素标记试剂采用生物素标记羊抗鼠IgG。
进一步地,所述封闭液为质量分数1%山羊血清,质量分数5%牛血清白蛋白,10mMPBS。
进一步地,所述DAB显色剂包括DAB显色剂A液和B液;所述A液为质量分数0.1%吐温20,质量分数0.05%过氧化脲,20mM咪唑,所述B液为质量分数1%DAB,10mM PBS。
本发明的另一个目的在于提供一种宫颈癌筛查试剂盒,包括以上所述HPV多种蛋白检测试剂盒。
进一步地,所述宫颈癌筛查试剂盒还包括DNA倍体染色液。
进一步地,所述DNA倍体染色液按质量分数计,组分包括:0.1%硫堇,0.05%苏木精,0.2%硫酸铝钾,1%丙三醇,0.05%碘酸钠,1%亚硫酸氢钠,0.6%冰乙酸。
相比现有技术,本发明的有益效果为:
1.本发明提供的HPV多种蛋白检测试剂盒中的一抗试剂为非HPV亚型特异性的抗体,可识别不同HPV类型的E6蛋白和E7蛋白,生产抗E6抗体优选编号为CCTCC NO:C2020153的抗体细胞株;生产抗E7抗体优选编号为CCTCC NO:C2020152的抗体细胞株,这些细胞来自本公司专利US8865162中的四株细胞,非HPV亚型特异性抗体识别临床样品中存在的大多数流行的HPV类型的标本,与现有市场上采用单个亚型的HPV E6/E7蛋白检测试剂相比有很大的优势,可大大提高本试剂盒的灵敏度。
2.本发明提供的宫颈癌筛查试剂盒包括样本稀释液,HPV E6/E7蛋白检测试剂,DNA倍体染色液,该试剂盒可用上述两种试剂检测同一载玻片上的宫颈脱落细胞中的HPVE6/E7蛋白总含量及DNA倍体含量;并且检测宫颈上皮内瘤变(CIN1及以上)灵敏度高,特异性强,稳定性好;通过同时分析两项数据对患者进行宫颈癌风险评估,减少漏诊,避免过度诊疗。
3.由于宫颈脱落细胞样本的特殊性,其样本中大部分含有组织粘液等干扰物质,本发明提供的宫颈癌筛查试剂盒首次在试剂盒中加入样本处理液,使样本检测前所有的细胞团包括腺细胞团分散,减少非特异性染色,增加DNA倍体分析中腺细胞的检出率,更有利于后续的样本结果判断。
4.本发明提供的筛选试剂盒将Schiff试剂硫堇与苏木素共同染色细胞染色体,两种核酸染色剂协同染色细胞核,既保证了试剂的特异度,又缩短了试剂的染色时间,可实现5~10分钟以内的快速染色。
具体实施方式
本发明的检测原理:
1)HPVE6/E7蛋白检测技术利用免疫学抗原抗体分子由于结构的互补和彼此亲和性所发生特异性结合原理,并通过氧化还原化学反应使标记抗体的酶显色剂显色来确定组织细胞内抗原,对其进行定位、定性。
首先联结鼠抗人HPV E6/E7单克隆抗体和细胞上的HPV E6/E7抗原;第二,生物素标记羊抗小鼠IgG识别已经连接上的HPV E6/E7抗体;第三,加入过氧化物酶标记的链霉亲和素,识别生物素;第四,加入底物,聚合物上的辣根过氧化物酶部分可以催化DAB显色液中的H2O2分解,使联苯胺氧化变成联苯亚胺,使细胞沉降片中抗原位点上出现黄色或棕黄色着色;最后对样本进行复染和封片。通过显微镜观测显色情况,推断细胞沉降片上HPV E6/E7蛋白的存在位点和情况。
2)DNA染色技术:细胞核内所含有的DNA酸性条件的分解成游离的醛基,暴露的醛基可与Schiff试剂硫堇特异性结合形成醌基后着色,苏木素属于碱性试剂,可与呈酸性的核酸结合而使细胞着色,由于在前期酸性分解过程中,RNA分子在分解过程中已从细胞中脱落下来,因此苏木素最后着色的仅为DNA分子。
基于以上原理,我们首先筛选出特异性的抗人HPV E6单克隆抗体与抗人HPV E7单克隆抗体,然后使用免疫细胞化学的方法对宫颈脱落细胞样本进行染色,免疫细胞染色后再使用DNA倍体染色液对细胞核染色。
本发明所涉及的宫颈脱落细胞样本为志愿者病例,以下结合实施例对本发明作进一步说明,但是本发明的保护范围并不限于这些实施例。凡是不背离本发明构思的改变或等同替代均包括在本发明的保护范围之内。下列实施例中未特别说明的具体条件的实验方法是按常规实验方法。
实施例1.抗人HPV E6单克隆抗体筛选
使用HPV16 E6或HPV18 E6按照正常的免疫程序免疫BALB/C小鼠后(本公司专利US8865162),检测小鼠血清效价,当血清效价达到最佳状态时,按照标准的杂交瘤细胞融合程序生产筛选单克隆杂交瘤细胞,将筛选出的单克隆细胞株同时使用HPV16 E6和HPV18 E6筛选细胞上清,选择双项阳性的单克隆细胞株进行扩培,选择抗体效价高,细胞状态好的4株单克隆细胞株(根据筛选先后顺序分别命名为Mab1,Mab2,Mab13,Mab14)按照标准的单克隆细胞株生产腹水,收集的腹水按照标准的抗体纯化程序使用ProteinG纯化。
将上述所述分泌得到的抗体使用抗体稀释液(10mM PBS,4%BSA,0.05%吐温-20)稀释至4μg/ml,分别检测Caski,C-33A,及10例样本。检测结果如表1所示。
表1:抗人HPV E6单克隆抗体筛选结果
根据以上结果发现,Mab14所得抗体检测有非特异性显色,Mab13所得特异性显色较弱,Mab1和Mab2显色基本一致,但Mab1所得抗体会引起某些样本白细胞显色,但不影响最终结果的判断,因此这两个细胞株分泌得到的抗体择其一可作为本发明试剂盒的一抗试剂;因Mab1所得抗体会引起某些样本白细胞显色,故优选Mab2所得抗体作为本发明试剂盒的一抗试剂。两株细胞株于2020年08月24日送交中国.武汉.武汉大学中国典型培养物保藏中心保藏。Mab1提交保藏信息为,培养物名称(分类命名):杂交瘤细胞株P2B2-1-D4-1G4,保藏编号:CCTCC NO:C2020149。Mab2提交保藏信息为,培养物名称(分类命名):杂交瘤细胞株P1B7-2-D8-1F10,保藏编号为:CCTCC NO:C2020153。
实施例2.抗人HPV E7单克隆抗体筛选
使用HPV16 E7或HPV18 E7按照正常的免疫程序免疫BALB/C小鼠后(本公司专利US8865162),检测小鼠血清效价,当血清效价达到最佳状态时,按照标准的杂交瘤细胞融合程序生产筛选单克隆杂交瘤细胞,将筛选出的单克隆细胞株同时使用HPV16 E7和HPV18 E7筛选细胞上清,选择双项阳性的单克隆细胞株进行扩培,选择抗体效价高,细胞状态好的4株单克隆细胞株(根据筛选先后顺序分别命名为Mab3,Mab4,Mab8)按照标准的腹水生产程序生产腹水,收集的腹水按照标准的抗体纯化程序使用ProteinG纯化。
将上述所述三种抗体使用抗体稀释液(10mM PBS,4%BSA,0.05%吐温-20,除有说明之外,本发明涉及的组分的百分比均为质量百分比,下同)稀释至4μg/ml,分别检测Caski,C-33A,及10例样本。检测结果如表2。
表2:抗人HPV E7单克隆抗体筛选结果
Mab3(比例%,显色度) | Mab4(比例%,显色度) | Mab8(比例%,显色度) | |
Caski | 90%+++ | 40%+++ | 90%++ |
C-33A | N | N | N |
样本11 | 20%+ | 20%+(白细胞100%+) | 20%+ |
样本12 | N | N | N |
样本13 | 30%++ | 20%++ | 30%+ |
样本14 | 10%+ | 40%+ | 10%+ |
样本15 | 50%+ | 50%+ | 50%+ |
样本16 | N | 100%+ | N |
样本17 | 100+ | 100+ | 100+ |
样本18 | N | N | N |
样本19 | 5%++ | 50%+(白细胞100%+) | 5%+ |
样本20 | N | N(白细胞100%+) | N |
根据以上结果,Mab4抗体检测细胞,会引起白细胞非特异性显色,以及某些样本非特异性染色,Mab3和Mab8显色基本一致,满足试剂盒需求,Mab8显色较Mab3显色弱,因此优选Mab3抗体作为本发明试剂盒一抗试剂。Mab3和Mab8与2020年08月24日送交中国.武汉.武汉大学中国典型培养物保藏中心保藏,Mab3提交保藏信息为,培养物名称(分类命名):杂交瘤细胞株P1F5-3-B10,保藏编号:CCTCC NO:C2020152。Mab8提交保藏信息为,培养物名称(分类命名):杂交瘤细胞株P3C1-H5-1-C11,保藏编号:CCTCC NO:C2020154。
实施例3.抗人HPV E6单克隆抗体与抗人HPV E7单克隆抗体组合筛选
将实施例1中的优选抗体Mab1,Mab2与实施例2中优选抗体Mab3,Mab8分别组合,使用抗体稀释液(10mM PBS,4%BSA,0.05%吐温-20)稀释至2μg/ml,分别检测Caski,C-33A,及10例样本。结果见表3:
表3:抗人HPV E6单克隆抗体与抗人HPV E7单克隆抗体组合筛选结果
根据以上结果,Mab1+Mab3与Mab1+Mab8组合抗体检测细胞,会引起白细胞非特异性显色,以及某些粘液团的非特异性染色,其他两种抗体组合检测细胞阴阳性一致,但Mab2+Mab3组合信噪比更高,因此选用Mab2+Mab3抗体组合作为本发明试剂盒一抗试剂。
实施例4.检测试剂盒的制备
用于宫颈癌筛查的试剂盒,包括样本稀释液,如上实施例3中所述的抗HPV E6单克隆抗体和抗HPV E7单克隆抗体,二抗检测系统及DNA倍体染色液。
样本稀释液为20mM Tris缓冲液,3%TCEP·4HCl;一抗试剂共包含两个单克隆抗体,Mab2抗体按照1:400稀释,Mab3抗体按照1:400稀释,稀释液为4%牛血清白蛋白,10mMPBS缓冲液;二抗检测系统包括封闭液(1%山羊血清,5%牛血清白蛋白,10mM PBS),生物素标记羊抗鼠IgG试剂(1:1000稀释,稀释液为1%山羊血清,5%牛血清白蛋白,10mM PBS),过氧化物酶标记链霉亲和素试剂(1:500稀释,稀释液同封闭液),DAB显色剂A液(0.1%吐温20,0.05%过氧化脲,20mM咪唑),DAB显色剂B液(1%DAB,10mM PBS);DNA倍体染色液(0.1%硫堇,0.05%苏木精,0.2%硫酸铝钾,1%丙三醇,0.05%碘酸钠,1%亚硫酸氢钠,0.6%冰乙酸)。
实施例5.用本发明试剂盒对宫颈脱落细胞的检测方法
使用实施例4中制备的试剂盒进行检测,具体如下:
1)检测所需仪器、设备:
卧式染缸、移液器、免疫组化油笔、计时器、孵育盒、染色架、盖玻片、光学显微镜(10×~40×)、制片板、洗瓶、载玻片、10mm套管、离心机、恒温水浴培养箱、冰箱。
2)溶液配制:
PBS溶液、PBST溶液参见各产品说明书;DAB显色液的配制:DAB显色液需要在使用前30分钟内配制。配制方法:在配备的小试管中依次加入DAB显色剂A液1mL、DAB显色剂B液40μL;配置好的DAB显色液混匀后避光存放,30分钟内有效。
AF固定液的配制:40%(v/v)甲醛10ml+95%(v/v)酒精90ml(甲醛水溶液:95%酒精=1:9)。
分化液的配制:270ml蒸馏水+30ml盐酸(蒸馏水:盐酸=9:1)。
3)实验室温度条件:22℃~28℃。
4)试验步骤:
a.取适量细胞离心去上清,加次缓500μL重悬(对于粘液类标本可用样本稀释液Ⅲ型处理后再进行沉降)。
b.于10mm卡好套管中沉降15分钟。
c.沉降结束后用50%(v/v)乙醇清洗两遍,取下套管。(实验各步骤避免玻片细胞干燥)。
d.将沉降好的细胞玻片放入卧式染缸中,用PBS溶液清洗5分钟×2次。
e.弃去PBS溶液,95%(v/v)酒精固定10分钟。
f.阻断内源性过氧化物酶:弃去PBS溶液,加入内源性过氧化物酶阻断剂,室温下阻断10分钟。PBS溶液清洗5分钟×3次。
g.加入封闭液:弃去PBS溶液,在油笔圈定的细胞区域内加150μL封闭液,37℃恒温箱1小时。
h.加入抗体或空白对照试剂:弃去封闭液,加入150μL抗体或空白对照试剂后4℃冰箱放置过夜(12-16小时)。
i.弃去抗体液体,PBST溶液清洗5分钟×3次。
j.加入生物素标记物:弃去清洗液,加入150μL生物素标记羊抗鼠IgG,室温或25℃恒温箱温育15分钟,PBST溶液清洗5分钟×3次。
k.加入酶标记链霉亲和素:弃去清洗液,加入150μL HRP酶标记链霉亲和素,室温或25℃恒温箱温育15分钟,PBST溶液清洗5分钟×3次。
l.显色:除去清洗液,加150μL新鲜配制的DAB显色液,孵育5~10分钟。
m.AF固定液20分钟;流水洗涤4~5次,沥干。
n.分化(酸解)液20分钟,流水洗涤4~5次,沥干。
o.DNA倍体染色液加热至45度左右,染色10分钟,流水洗涤4~5次,沥干。
p.脱水、透明、封片。
5)结果判断:
该染色液会在相应的抗原存在区域沉淀,出现棕黄色物质。E6/E7染色结果和DNA倍体染色结果由武汉呵尔多光谱扫描系统扫描分析。
实施例6.试剂盒灵敏度和特异度测定
使用实施例4中的试剂盒,按照实施例5的检测方法检测500例宫颈脱落细胞样本,其中有200例样本追踪到活检结果,以活检结果为金标准,计算本发明试剂盒灵敏度和特异度。
其中DNA倍体参考值为异常细胞最高DI值大于4.5或异常细胞数大于10。
本发明提供的试剂盒与市售宫颈癌筛查试剂HPV DNA检测结果如表4-5示。
表4:本发明试剂盒检测结果
表5:现有宫颈癌筛查试剂HPV DNA检测结果
不难看出,本发明检测试剂盒灵敏度和特异度分别为99%和70%(表4所示),相比于现有宫颈癌筛查试剂HPV DNA检测所得灵敏度96%,特异度40%(表5),灵敏度和特异度均有所提高,在临床应用上大幅度减少了患者恐慌及过度诊疗。
本发明并不仅仅限于说明书和实施方式中所描述,因此对于熟悉领域的人员而言可容易地实现另外的优点和改进,故在不背离权利要求及等同范围所限定的一般概念的精神和范围的情况下,本发明并不限于特定的细节、代表性的方案和这里示出与的实施例。
Claims (10)
1.一种HPV多种蛋白检测试剂盒,其特征在于,包括HPV E6单克隆抗体和HPV E7单克隆抗体,所述HPV E6单克隆抗体由保藏号为CCTCC NO:C2020153的杂交瘤细胞株产生;所述HPV E7单克隆抗体由保藏号为CCTCC NO:C2020152的杂交瘤细胞株产生。
2.根据权利要求1所述的检测试剂盒,其特征在于,还包括样本稀释液、二抗检测系统。
3.根据权利要求2所述的检测试剂盒,其特征在于,所述样本稀释液包括三(2-羧乙基)膦盐酸盐和Tris缓冲液。
4.根据权利要求2所述的检测试剂盒,其特征在于,所述二抗检测系统包括封闭液,生物素标记试剂,过氧化物酶标记链霉亲和素试剂和DAB显色剂。
5.根据权利要求4所述的检测试剂盒,其特征在于,所述生物素标记试剂采用生物素标记羊抗鼠IgG。
6.根据权利要求4所述的检测试剂盒,其特征在于,所述封闭液包括:质量分数1%的山羊血清、质量分数5%的牛血清白蛋白,以及10mM PBS。
7.根据权利要求4所述的检测试剂盒,其特征在于,所述DAB显色剂包括DAB显色剂A液和B液;所述A液为质量分数0.1%的吐温20,质量分数0.05%过氧化脲,20mM咪唑;所述B液为质量比1%DAB,10mM PBS。
8.一种宫颈癌筛查试剂盒,其特征在于包括权利要求1-7任意一项所述HPV多种蛋白检测试剂盒。
9.根据权利要求8所述的宫颈癌筛查试剂盒,其特征在于,还包括DNA倍体染色液。
10.根据权利要求9所述的宫颈癌筛查试剂盒,其特征在于,所述DNA倍体染色液按质量分数计,组分包括:0.1%硫堇,0.05%苏木精,0.2%硫酸铝钾,1%丙三醇,0.05%碘酸钠,1%亚硫酸氢钠,0.6%冰乙酸。
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Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20050031589A (ko) * | 2003-09-30 | 2005-04-06 | 메타볼랩(주) | 경부암 관련 hpv 감염의 인 비트로 면역염색 검사방법및 이를 위한 키트 |
KR100671825B1 (ko) * | 2006-04-05 | 2007-01-19 | 주식회사 바이오포커스 | 인유두종바이러스에 의한 자궁경부암 진단키트 |
CN103130890A (zh) * | 2011-11-28 | 2013-06-05 | 常小迦 | Hpv16e7单克隆抗体、相关杂交瘤细胞株和应用 |
CN103254307A (zh) * | 2012-02-16 | 2013-08-21 | 艾托金生物医药(苏州)有限公司 | Hpv18e7单克隆抗体、相关杂交瘤细胞株和应用 |
CN103865883A (zh) * | 2014-03-26 | 2014-06-18 | 重庆理工大学 | 抗高危型人乳头瘤病毒蛋白的单克隆抗体及其应用 |
CN105131114A (zh) * | 2015-10-26 | 2015-12-09 | 无锡傲锐东源生物科技有限公司 | 抗p16蛋白单克隆抗体杂交瘤细胞及其产生的抗p16单克隆抗体和应用 |
CN106318912A (zh) * | 2016-08-23 | 2017-01-11 | 广州瑞博奥生物科技有限公司 | 杂交瘤细胞株scca1及其分泌的单克隆抗体和应用 |
CN112430583B (zh) * | 2020-10-30 | 2022-02-15 | 武汉呵尔医疗科技发展有限公司 | 一种抗hpv e7的单克隆抗体及其细胞株和应用 |
CN112458061B (zh) * | 2020-10-30 | 2022-03-22 | 武汉呵尔医疗科技发展有限公司 | 一种抗hpv e6的单克隆抗体及其细胞株和应用 |
-
2020
- 2020-10-30 CN CN202011185161.4A patent/CN112362874B/zh active Active
Patent Citations (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20050031589A (ko) * | 2003-09-30 | 2005-04-06 | 메타볼랩(주) | 경부암 관련 hpv 감염의 인 비트로 면역염색 검사방법및 이를 위한 키트 |
KR100671825B1 (ko) * | 2006-04-05 | 2007-01-19 | 주식회사 바이오포커스 | 인유두종바이러스에 의한 자궁경부암 진단키트 |
CN103130890A (zh) * | 2011-11-28 | 2013-06-05 | 常小迦 | Hpv16e7单克隆抗体、相关杂交瘤细胞株和应用 |
CN103254307A (zh) * | 2012-02-16 | 2013-08-21 | 艾托金生物医药(苏州)有限公司 | Hpv18e7单克隆抗体、相关杂交瘤细胞株和应用 |
CN103865883A (zh) * | 2014-03-26 | 2014-06-18 | 重庆理工大学 | 抗高危型人乳头瘤病毒蛋白的单克隆抗体及其应用 |
CN105131114A (zh) * | 2015-10-26 | 2015-12-09 | 无锡傲锐东源生物科技有限公司 | 抗p16蛋白单克隆抗体杂交瘤细胞及其产生的抗p16单克隆抗体和应用 |
CN106318912A (zh) * | 2016-08-23 | 2017-01-11 | 广州瑞博奥生物科技有限公司 | 杂交瘤细胞株scca1及其分泌的单克隆抗体和应用 |
WO2018036435A1 (zh) * | 2016-08-23 | 2018-03-01 | 广州瑞博奥生物科技有限公司 | 杂交瘤细胞株scca1及其分泌的单克隆抗体和应用 |
CN112430583B (zh) * | 2020-10-30 | 2022-02-15 | 武汉呵尔医疗科技发展有限公司 | 一种抗hpv e7的单克隆抗体及其细胞株和应用 |
CN112458061B (zh) * | 2020-10-30 | 2022-03-22 | 武汉呵尔医疗科技发展有限公司 | 一种抗hpv e6的单克隆抗体及其细胞株和应用 |
Non-Patent Citations (3)
Title |
---|
WEN-JING, SHI;HAO, LIU;DAN, WU;ZHEN-HUA, TANG;YU-CHEN, SHEN;LIN, GUO: "E6/E7 proteins are potential markers for the screening and diagnosis of cervical pre-cancerous lesions and cervical cancer in a Chinese population.", ONCOLOGY LETTERS, vol. 14, no. 5, 31 December 2017 (2017-12-31) * |
原亚莉;贺桂芳;马莉;张浩军;卞美璐;杨文艳;张媛;: "人乳头瘤病毒16/18型E6和E7蛋白在宫颈上皮内病变中的表达水平及临床价值", 中日友好医院学报, no. 06, 15 December 2018 (2018-12-15) * |
张莉;占超;: "人类白细胞抗原-E、人类白细胞抗原-G蛋白在早期宫颈癌及癌前病变中的表达与人乳头瘤病毒感染的相关性研究", 癌症进展, no. 14, 25 July 2020 (2020-07-25) * |
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