CN104004067B - 用于检测抗人乳头瘤病毒抗体的抗原及相关免疫检测试剂盒 - Google Patents
用于检测抗人乳头瘤病毒抗体的抗原及相关免疫检测试剂盒 Download PDFInfo
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Abstract
本发明涉及一种人乳头瘤病毒HPV致癌基因蛋白作为抗原，用于检测抗人乳头瘤病毒HPV的抗体，其选自如SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5所示的氨基酸序列或者与它们具有80%同源性的氨基酸序列中的一种或几种。还提供了包括上述抗原的免疫检测试剂盒。以及上述抗原和试剂盒的应用。本发明的用于检测抗HPV抗体的抗原可以高灵敏度、高特异性和高稳定性地检测血液或唾液中的HPV抗体，从而可以为医生准确诊断病人有HPV病毒致癌基因转化的宫颈上皮内瘤变和子宫颈癌带来依据，进而及时采取治疗，以防止癌症发生，或癌症扩散，及时减轻病人痛苦，由其制成的免疫检测试剂盒可以快速简单准确地检测血液或唾液中的HPV抗体，适于大规模推广应用。
Description
技术领域
本发明涉及抗体检测技术领域，特别涉及抗人乳头瘤病毒（HumanPapillomavirus，HPV）抗体检测技术领域，具体是指一种用于检测抗人乳头瘤病毒抗体的抗原及相关免疫检测试剂盒，尤其可用于国内流行的高危险型人乳头瘤病毒（HPV）亚型HPV16和HPV18。
背景技术
子宫颈癌是第二位常见女性恶性肿瘤，人乳状瘤病毒（HPV）是其发生的必要条件。尽管人群HPV感染很普遍，如：宫颈癌高发区妇女35岁以后感染率仍高达20.8%。但超过80%的HPV DNA阳性妇女不会进展为癌症或癌前病变。因此HPV DNA检测缺陷是无法分辨一过性感染和可引起宫颈病变的感染，导致较高假阳性率。大量研究显示，HPV致癌基因表达产物（癌蛋白oncoprotein）有很高的致癌活性，因此这些生物标志物有可以作为比HPV DNA更为准确有特异性的宫颈癌预测指标。高危HPV E2、E6、E7癌蛋白(oncoprotein)的表达水平和宫颈癌前病变不同阶段直接相关。利用这一特性可以构建全新的宫颈癌分子筛查指标。
对HPV致癌机理的研究表明，高危型HPV的早期表达蛋白E6，E7蛋白在宫颈癌的发生中起着重要作用。在宫颈高度病变和癌症患者的宫颈上皮细胞中HPV E6及E7蛋白一般呈过度表达，持续表达和过量表达的E6，E7致癌蛋白可与宿主细胞调控蛋白（如，P53和Rb蛋白等）相互作用，使肿瘤抑制因子失活，导致细胞永生化和恶性转化。
除了HPV DNA检测和HPV致癌蛋白检测，血液（体液）中HPV抗体也证明对HPV感染及宫颈癌发病史有很大的相关性。HPV L1血液抗体可用标准ELISA方法检测,提供HPV感染信息。随着HPV感染时间的延长，HPV诱导产生了抗HPV抗体，故可检测到血清中抗HPV抗体。Wikstrom等检测了47例男性现症尖锐湿疣患者和32例曾有尖锐湿疣病史患者的男性对HPV6型和HPV11型衣壳蛋白的血清IgG抗体，并与205例无生殖器尖锐湿疣病史的男性比较，结果，血清中抗HPV6型的IgG抗体在曾有尖锐湿疣病史的男性中为35%，在现症尖锐湿疣中为27%，而在对照组为10%。这表明抗HPV6IgG抗体的出现发生在尖锐湿疣病期的晚些时候，同时IgG阳性也反映曾感染过这种病毒。
Stone等报道了利用此方法对于美国普通人群7218例HPV-16的感染率研究，结果表明女性阳性率17.9%，男性阳性率7.9%（Stone，K.M.,et al.,Seroprevalence ofHumanPapillomavirus Type16Infection in the United States.2002.The Journal ofInfectious Diseases2002;186:1396-402）。HPV L1抗体ELISA方法在配合HPV疫苗（Merck公司Gardasil预防性疫苗）的研发及临床试验过程中曾被广泛采用。Luevano等用蛋白芯片例宫颈癌病人的血清样品进行了高通量检测分析（Luevano,M.,et al.,High-ThroughputProfling of the Humoral Immune Responses Against Thirteen HumanPapillomavirusTypes by Proteome Microarrays.2010.Virology.2010;405(1):31–40），分析13个HPV型的总共54604种蛋白的抗体profiling，结果表明了血液中致癌蛋白的抗体与宫颈癌、高度病变、低度病变、及无症状普通人有较强的相关性（Luevano,M.,et al.,High-Throughput Profling of the Humoral Immune Responses Against ThirteenHuman Papillomavirus Types by Proteome Microarrays.2010.Virology.2010;405(1):31–40）。
因此，为了实现HPV抗体检测的高灵敏度、高特异性和高稳定性，需要提供新的用于检测抗人乳头瘤病毒抗体的抗原。
发明内容
为了解决上述存在的问题，本发明的目的是提供一种用于检测抗人乳头瘤病毒抗体的抗原及相关免疫检测试剂盒，该用于检测抗人乳头瘤病毒抗体的抗原可以高灵敏度、高特异性和高稳定性地检测血液或唾液中的HPV抗体，从而可以为医生准确诊断病人宫颈上皮内瘤变和子宫颈癌带来依据，进而及时采取治疗，以防止癌症发生，或癌症扩散，及时减轻病人痛苦，由其制成的免疫检测试剂盒可以快速简单准确地检测血液或唾液中的HPV抗体，适于大规模推广应用。
为了实现上述目的，在本发明的第一方面，提供了一种用于检测抗人乳头瘤病毒抗体的抗原，其特点是，所述的用于检测抗人乳头瘤病毒抗体的抗原选自如SEQ ID NO:1所示的氨基酸序列、如SEQ ID NO:2所示的氨基酸序列、如SEQ ID NO:3所示的氨基酸序列、如SEQ ID NO:4所示的氨基酸序列、如SEQ ID NO:5所示的氨基酸序列、与SEQ ID NO:1所示的氨基酸序列具有80%同源性的氨基酸序列、与SEQ ID NO:2所示的氨基酸序列具有80%同源性的氨基酸序列、与SEQ ID NO:3所示的氨基酸序列具有80%同源性的氨基酸序列、与SEQID NO:4所示的氨基酸序列具有80%同源性的氨基酸序列以及与SEQ ID NO:5所示的氨基酸序列具有80%同源性的氨基酸序列中的一种或几种。
较佳地，所述的用于检测抗人乳头瘤病毒抗体的抗原的第m+2位、第m+3位、第m+5位、第m+6位以及第m+8-m+12位氨基酸依次是Asp、Ser、Glu、Glu、Asp、Glu、Ile、Asp和Gly，其中m≥0。
较佳地，所述的用于检测抗人乳头瘤病毒抗体的抗原采用化学合成或生物工程方法获得。例：重组蛋白，融合蛋白。
在本发明的第二方面，提供了一种免疫检测试剂盒，其特点是，包括上述的用于检测抗人乳头瘤病毒抗体的抗原。
较佳地，所述免疫检测试剂盒还包括载体，所述的用于检测抗人乳头瘤病毒抗体的抗原包被在所述载体上。
较佳地，所述免疫检测试剂盒还包括标记的抗人乳头瘤病毒抗体的二抗。二抗用来检测人血清中的抗体IgG，IgM，或IgA.所用标记物可以是辣根过氧化物酶，碱性磷酸酶，荧光分子FITC（或其它荧光标记），化学发光检测。检测方法可以是显色法，荧光方法，化学发光，电化学发光法进行定性、定量分析。
更佳地，所述的标记的抗人乳头瘤病毒抗体的二抗是辣根过氧化物酶标记的抗人乳头瘤病毒抗体的二抗。
在本发明的第三方面，提供了上述的用于检测抗人乳头瘤病毒抗体的抗原或上述的免疫检测试剂盒在检测人体液中抗人乳头瘤病毒抗体中的应用。
较佳地，所述体液是血液或唾液。
本发明的有益效果在于：本发明的用于检测抗人乳头瘤病毒抗体的抗原选自如SEQ ID NO:1所示的氨基酸序列、如SEQ ID NO:2所示的氨基酸序列、如SEQ ID NO:3所示的氨基酸序列、如SEQ ID NO:4所示的氨基酸序列、如SEQ ID NO:5所示的氨基酸序列、与SEQID NO:1所示的氨基酸序列具有80%同源性的氨基酸序列、与SEQ ID NO:2所示的氨基酸序列具有80%同源性的氨基酸序列、与SEQ ID NO:3所示的氨基酸序列具有80%同源性的氨基酸序列、与SEQ ID NO:4所示的氨基酸序列具有80%同源性的氨基酸序列以及与SEQ ID NO:5所示的氨基酸序列具有80%同源性的氨基酸序列中的一种或几种，病人在被病毒感染后，可在血清和口腔唾液中高特异性、高灵敏度、高特异性地地检测出现的特异性的抗体IgM，IgA或IgG分子，从而可以为医生准确诊断病人宫颈上皮内瘤变和子宫颈癌带来依据，进而及时采取治疗，以防止癌症发生，或癌症扩散，及时减轻病人痛苦，以此制成的免疫检测试剂盒，可以快速简单准确地检测血液或唾液中的HPV抗体，适于大规模推广应用。
具体实施方式
为了能够更清楚地理解本发明的技术内容，特列举以下具体实施例详细说明。然而，具体实施例仅仅是用于举例说明，而不是对本发明的限制。
一、材料与方法
1、试剂和药品
辣根过氧化物酶标记羊抗人（Goat Anti-Human IgG，FC），购自Priece Product#31413。
氯化钠、氯化钾、磷酸氢二钠、磷酸二氢钾购自国药集团化学试剂有限公司。Tween-20购自AMRESCO。胎牛血清购自北京元亨金马生物技术开发有限公司。
2、仪器和耗材
电子天平（梅特勒-托利多仪器上海有限公司）；85-1恒温磁力搅拌器（江苏金坛市杰瑞尔电器有限公司）；MULTISKAN MK3酶标仪（Thermo公司）；超纯水制备装置（美国MILLPORE公司）；GZX-9240MBE数显鼓风干燥箱（上海博迅实业有限公司医疗设备厂）；WellWASK4MK2洗板机(购自Thermo公司)；酶标板（COSTAR公司）；单道、多道移液器（德国eppendorf公司）。
3、主要溶液系统
·包被缓冲液（PBS磷酸盐溶液）：磷酸二氢钾0.2g，12水磷酸氢二钠2.9g，氯化钠8.0g，氯化钾0.2g，加蒸馏水至1L，4度保存。
·洗涤缓冲液（PBS-Tween20，0.1%）：Tween-201mL，加入到1L稀释缓冲液（PBS）中，搅拌混匀，调pH值到7.0～7.2之间。
·封闭液：5%milk-PBS-Tween
·血清稀释液：10%FBS-PBS-Tween
·TMB显示液：Amresc，CAT NO.64285730，用前TMB底色显色A液和B液等体积混合。
○底物显色A液：乙酸钠13.6g，柠檬酸1.6g，双氧水（30%）0.3ml，蒸馏水加至500ml。
○底物显色B液：乙二胺四乙酸二钠0.2g柠檬酸0.95g甘油50ml四甲基联苯胺0.2g蒸馏水加至500ml。
·终止液（2mol/L H2SO4）：双蒸水178.3mL，滴加浓硫酸（98%）21.7mL。
4、间接ELISA方法的建立
1）用PBS稀释HPV多肽抗原HPV16-1/HPV16-2/HPV16-3/HPV18-1/HPV18-2浓度为4ug/ml，包被costar板条100ul/孔，4℃过夜。
2）PBS洗涤一次，拍干。
3）加入5%奶粉-PBS封闭，250ul/孔，37度3小时。
4）PBST洗涤一次，拍干。
5）10%FBS-PBST稀释血清1：50稀释，振荡器混匀。
6）加入稀释好的血清，空白对照，阴性对照以及阳性对照，50ul/孔，37度孵育1小时。
7）0.1%tween-PBS洗涤5次，拍干。
8）加入辣根过氧化物酶标记羊抗人，用10%FBS-PBST稀释（1：40000），100ul/孔，37度孵育30分钟。
9）0.1％tween-PBS洗涤5次，拍干。
10）TMB显色:TMBA液和B液等体积混合，100ul/孔，37度20min。
11）2M H2SO4，50ul/孔终止，用酶标仪450nm读数。
5、试剂盒组成：
本试剂盒由以下几个部分组成：
1.包被本发明的用于检测抗人乳头瘤病毒抗体的抗原的costar酶标板一块
2.辣根过氧化物酶标记羊抗人一管（10ul）
3.10*PBST一瓶（10ml）
4.TMB显色液A液一瓶（6ml），B液一瓶（6ml）
5.终止液一瓶（6ml）
6、包被抗原
多肽抗原如下
| HPV16-1 | KCDSTLRLCVQSTHVDIRTLE | SEQ ID NO：1 |
| HPV16-2 | PTLHEYMLDLQPETTDLYCYEQLNDSSEEE | SEQ ID NO：2 |
| HPV16-3 | LNDSSEEEDEIDGPAGQAEPDRAH | SEQ ID NO：3 |
| HPV18-1 | IDGVNHQHLPARRAEPQR | SEQ ID NO：4 |
| HPV18-2 | SDSEEENDEIDGVNHQHLPARRAEPQRH | SEQ ID NO：5 |
二、检测结果
试剂盒选择5条多肽（SEQ ID NO：1，SEQ ID NO：2，SEQ ID NO：3，SEQ ID NO：4，SEQID NO：5）作为包被选择，通过ELISA方法检测血清。
1、用包被抗原并封闭过的酶标板通过ELISA方法检测健康人血清，检测的原始数据结果如下；
表一：HPV血液ELISA检测正常人血清结果表
2、选择2条多肽（SEQ ID NO：3，SEQ ID NO：5）作为包被选择并封闭过的酶标板通过ELISA方法检测正常人血清，检测的原始数据结果如下；
表二：HPV血液ELISA检测正常人血清结果表
3、选择5条多肽（SEQ ID NO：1，SEQ ID NO：2，SEQ ID NO：3，SEQ ID NO：4，SEQ IDNO：5）作为包被选择包被并封闭过的酶标板通过ELISA方法检测病人血清，病人的病理诊断结果如表三中所示，其中部分病人经过HC2病理检验，结果如下：
表三：HPV血液ELISA检测病人血清结果表
4、选择3条多肽（SEQ ID NO：1，SEQ ID NO：3，SEQ ID NO：5）作为包被选择包被并封闭过的酶标板通过ELISA方法检测病人血清，病人的病理诊断结果如表四中所示，其中部分病人经过HC2病理检验，结果如下：
表四：HPV血液ELISA检测病人血清结果表
三、数据分析
1、根据正常人检测值，计算出平均值，标准方差，CUTOFF值。分别将表一中的44例正常人血清在HPVE7多肽上的反应OD值取平均数，并计算标准方差SD，本试剂盒在确定CUTOFF值时选择正常人平均值加上两倍的标准方差作为HPV阴阳性的判断标准，结果如下：
Cutoff计算公式为：cutoff=平均值OD+2*SD标准方差
1.1根据表一结果，HPVE7（SEQ ID NO：1，SEQ ID NO：2，SEQ ID NO：3，SEQ ID NO：4，SEQ ID NO：5）5条多肽检测正常人血清结果统计分析得出，结果如下表：
表五：HPV E7的5条多肽血清ELISA检测数据表
| SEQ ID NO：1 | SEQ ID NO：2 | SEQ ID NO：3 | SEQ ID NO：4 | SEQ ID NO：5 | |
| 平均值 | 0.292 | 0.279 | 0.382 | 0.301 | 0.383 |
| 标准方差 | 0.098 | 0.100 | 0.111 | 0.076 | 0.111 |
| cutoff | 0.487 | 0.480 | 0.604 | 0.452 | 0.605 |
1.2根据表二结果，HPVE7（SEQ ID NO：3和SEQ ID NO：5）2条多肽检测正常人血清结果统计分析得出，结果如下表：
表六：HPV E7的2条多肽血清ELISA检测数据表
| SEQ ID NO：3 | SEQ ID NO：5 | |
| 平均值 | 0.393 | 0.387 |
| 标准方差 | 0.104 | 0.108 |
| cutoff | 0.601 | 0.603 |
1.3根据表五和表六的结果，对比分析得出SEQ ID NO：3和SEQ ID NO：5这2条多肽在不同实验时得出的cutoff值一致。
2、通过cutoff值，判断检测的病人血清HPV的阴性、阳性，超过这个cutoff值的即认为是阳性，且只要一个多肽检测为阳性，即认为有HPV感染，同时与HC2诊断结果对比。
2．1在检测的病人血清中291例血清有HC2诊断结果，有病理诊断阳性的病例84例.在病理阳性病人组(临床诊断”金标准”),血清抗体检测结果与HC2分子检测结果相对比（见表7），将其与本试剂盒检测HPV结果进行对比如下表：（本检测结果中统计为阳性的包括：（i）两个抗原检测结果有任何一个是阳性结果，（ii）两个抗原检测结果都是阳性结果）
表七：根据表三的结果，对于病人的血清ELISA诊断结果与HC2诊断结果对比表
（“+”表示检测结果阳性；“-”表示检测结果阴性）
表八：根据表四的结果，对于病人的血清ELISA诊断结果与HC2诊断结果对比表
（“+”表示检测结果阳性；“-”表示检测结果阴性）
将表七结果的HC2结果和血液ELISA检查结果进行人数统计分析，结果如下表：
表九：HC2结果与血液ELISA检测准确率人数及比例对比表
其他妇科疾病主要为盆腔炎，卵巢囊肿，子宫肌瘤等妇科疾病。
将表八结果的HC2结果和血液ELISA检查结果进行人数统计分析，结果如下表：
表十：HC2结果与血液ELISA检测准确率人数及比例对比表
其他妇科疾病主要为盆腔炎，卵巢囊肿，子宫肌瘤等妇科疾病。
2．2检测病人血清中有10例血清为没有HC2检测报告，但病理报告为CIN和宫颈癌，本试剂盒检测HPV结果，病人数及血液检测阳性率对比结果如表。
表十一：对于没有HC2诊断结果，但有病理结论的病人与HPV血液ELISA检测结果总结
（“+”表示检测结果阳性；“-”表示检测结果阴性）
表十二：无HC2结果的CIN，宫颈癌及宫颈病变的病人血液ELISA检测的人数表
| 病理 | 人数（10） | SEQ ID NO：1阳性 | SEQ ID NO：3阳性 | SEQ ID NO：5阳性 |
| CINⅠ | 2 | 2 | 1 | 1 |
| CINⅡ，Ⅲ | 7 | 4 | 5 | 5 |
| 宫颈癌 | 1 | 0 | 1 | 1 |
2．3将所有CINⅠⅡⅢ和宫颈癌的病人血液ELISA检测结果统计人数及阳性比例结果如表十三。
表十三：所有CIN，宫颈癌及宫颈病变的病人血液ELISA检测的人数及准确率比例表
本发明的上述多肽是经过发明人的选择并在病人样品及正常人样品中进行了检测验证，而直接采用GST-HPV16-E7和GST-HPV118E7全长融合蛋白（分别为SEQ ID NO：6、SEQID NO：7所示序列）检测血清，效果不佳，请参见下列实验数据。或
四、实验材料和ELISA方法同前面第一点，只是采用多肽（SEQ ID NO：3，SEQ IDNO：5）检测的同时，还采用GST-HPV16-E7和GST-HPV118E7全长融合蛋白作为检测抗原，在用PBS稀释GST-HPV16-E7、GST-HPV18-E7全长融合蛋白时，浓度均为2ug/ml，检测结果如下：
1.根据上面实验步骤，检测部分病人血清和普通人血清，检测结果和病人病理报告如下表所示：
五．实验分析
1.将使用全长蛋白的检测结果与之前采用HPV多肽SEQ ID:3和SEQ ID:5两种多肽联合检测结果对比，对比如下表：
总结：总结：HPV16-E7，HPV18-E7全长融合蛋白在检测CIN，宫颈癌病人时有一定的阳性病人漏检，灵敏性比多肽检测差。在检测普通妇科炎症病人和普通人群时有一定的假阳性率，敏感性比多肽检测略差。所以，HPV16-E7和HPV18-E7全长蛋白对于检测CIN，宫颈癌等不是很适合。
本发明的血液酶标免疫分析法(ELISA)以免疫生化原理为基础，提供高灵敏度，高特异性和高稳定性的抗原-抗体检测方法。病人在被病毒感染后，可在血清和口腔唾液中出现特异性的抗体IgM,IgA或IgG分子。用酶标免疫分析法ELISA检测病人血清或唾液中特异性抗HPV的抗体分子。本发明通过对HPV病毒高危型HPV16，HPV18基因序列的分析，用生物信息学分析对抗原性的理论预测，并用实验方法筛选、证明临床应用的意义。通过制备多个高纯度的抗原多肽，并以国内人群健康者血清和感染者血清对制备抗原进行反复筛选、验证，获得了有高灵敏度，高特异性和高稳定性的目的抗原HPV16-1、HPV16-2、HPV16-3、HPV18-1、HPV18-2。并证明用这些多肽抗原结合到某种固相载体表面，能保持其免疫反应活性。在测定时，使受检标本与固相载体表面的抗原起反应。用洗涤的方法使固相载体上形成的抗原、抗体复合物与其他物质分开，最后是结合在固定载体上的酶量与标本中受检物质的量成一定的比例。加入酶反应的底物后，然后通过显色来检测，故可根据颜色反应的深浅作定性或定量分析。本发明的血液ELISA检测试剂盒与DNA检测(HC2,CareHPV)相比,取样均匀，非侵略性，操作更为快速，简单；费用便宜，容易推广，更适合中国市场；可避免PCR方法带来的假阳性和假阴性，大大提高了检测结果的精确度和准确度；与传统的巴氏涂片、醋酸染色VIA，阴道镜检查相比，更为准确、容易、快速。ELISA检测方法经各种参数和实验程上的优化，可作为大规模筛选的工具用于宫颈癌的早期筛查。
综上，本发明的用于检测抗人乳头瘤病毒抗体的抗原可以高灵敏度、高特异性和高稳定性地检测血液或唾液中的HPV抗体，从而可以为医生准确诊断病人宫颈上皮内瘤变和子宫颈癌带来依据，进而及时采取治疗，以防止癌症发生，或癌症扩散，及时减轻病人痛苦，由其制成的免疫检测试剂盒可以快速简单准确地检测血液或唾液中的HPV抗体，适于大规模推广应用。
在此说明书中，本发明已参照其特定的实施例作了描述。但是，很显然仍可以作出各种修改和变换而不背离本发明的精神和范围。因此，说明书应被认为是说明性的而非限制性的。
Claims (9)
1.一种用于检测抗人乳头瘤病毒抗体的抗原，其特征在于，所述的用于检测抗人乳头瘤病毒抗体的抗原的氨基酸序列如SEQ ID NO:3所示。
2.一种用于检测抗人乳头瘤病毒抗体的抗原，其特征在于，所述的用于检测抗人乳头瘤病毒抗体的抗原包括如SEQ ID NO.3所示的氨基酸序列、和选自下组的一种或几种氨基酸序列：如SEQ ID NO:1所示的氨基酸序列、如SEQ ID NO:4所示的氨基酸序列、和如SEQ IDNO:5所示的氨基酸序列。
3.一种用于检测抗人乳头瘤病毒抗体的抗原，其特征在于，所述的用于检测抗人乳头瘤病毒抗体的抗原包括如SEQ ID NO:3所示的氨基酸序列、和如SEQ ID NO:5所示的氨基酸序列。
4.根据权利要求1所述的用于检测抗人乳头瘤病毒抗体的抗原，其特征在于，所述的用于检测抗人乳头瘤病毒抗体的抗原采用化学合成或生物工程方法获得。
5.一种用于检测抗人乳头瘤病毒抗体的抗原，其特征在于，所述的用于检测抗人乳头瘤病毒抗体的抗原包括如SEQ ID NO:1所示的氨基酸序列、如SEQ ID NO:3所示的氨基酸序列、如SEQ ID NO:4所示的氨基酸序列、和如SEQ ID NO:5所示的氨基酸序列。
6.一种免疫检测试剂盒，其特征在于，包括根据权利要求1-5任一所述的用于检测抗人乳头瘤病毒抗体的抗原。
7.根据权利要求6所述的免疫检测试剂盒，其特征在于，所述免疫检测试剂盒还包括载体，所述的用于检测抗人乳头瘤病毒抗体的抗原包被在所述载体上。
8.根据权利要求1-5任一所述的用于检测抗人乳头瘤病毒抗体的抗原在制备检测试剂中的应用，所述检测试剂用于检测人体液中抗人乳头瘤病毒抗体。
9.根据权利要求8所述的应用，其特征在于，所述体液是血液或唾液。
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| US7026443B1 (en) * | 1999-12-10 | 2006-04-11 | Epimmune Inc. | Inducing cellular immune responses to human Papillomavirus using peptide and nucleic acid compositions |
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| US7026443B1 (en) * | 1999-12-10 | 2006-04-11 | Epimmune Inc. | Inducing cellular immune responses to human Papillomavirus using peptide and nucleic acid compositions |
| CN1433428A (zh) * | 2000-04-05 | 2003-07-30 | 冲击诊断公司 | 辨别人乳头瘤病毒的免疫学方法 |
| US7314630B2 (en) * | 2005-01-07 | 2008-01-01 | Yao-Xiong Hu | Compounds and methods of early diagnosis of cervical cancer and genital condyloma with HPV, CHSP60 tumor suppressor H-Ras, K-Ras and PTEN derived peptides modified |
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