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hpv检查参考值高
您好，就是在这次妇女检查出来的，没什么症状，就是做了个HPV检查才知道的还没治呢想想问问怎么治疗
病情分析：你好！妇科检查发现有HPV感染，一般上都是没有症状的，其高危型与女性的宫颈癌有很大的关系，其可以增加宫颈癌的发生率。指导意见：正常人是没有感染HPV的或已经是痊愈的，一般上选用干扰素治疗，建议积极接受治疗，以后定期的做宫颈癌的筛查。如果出现CIN二级及以上建议做宫颈锥切或全子宫切除。
病情分析：HPV正常应该是没有的哈，也就是阴性哈指导意见：但是如果这个是阳性的话，就比较不好了哈，这个感染与宫颈糜烂疾病相关性是比较高的，所以首先还是要去检查一下宫颈再说额
Hpv核酸检测值127.34，包含13种高危亚型，医生说需要做tct检查，还有病毒分析，如果不是1Hpv核酸检测值127.34，包含13种高危亚型，医生说需要做tct检查，还有病毒分析，如果不是16和18就做普通的抗病毒处理，如果是16和18就需要做手术请问，1hpv检测话费350元，只检测出是高危亚型，检测不出具体的病毒种类吗？如果做病毒分析，费用一般是多少？2.如果是前两种类型的病毒，一定需要手术吗？需要做什么手术呢？我未婚未生育，会影响怀孕吗？3.为什么会感染hpv呢？无不洁性经历。有宫颈糜烂四五年了。之前没测过，是否一直有？4.测出有病毒之后还能和男友同房吗？5.二级甲等市人民医院可以做这些检查吗
病情分析：您好！看来您不一定是在正规医院做的检查。Hpv核酸检测值127.34，包含13种高危亚型，就已经明确了HPV得分型；hpv的感染出不洁性交外，做妇科检查、游泳等均有可能被感染。宫颈糜烂多是因为hpv的感染所致。做tct检查，是为了更加明确您的宫颈糜烂情况，查出病因，做出病情诊断，是否处于癌前病变等。一般的二级甲等市人民医院可以做这些检查的。指导意见：1、您现在已经是hpv的高危型病毒感染，需要尽快采用干扰素等治疗。
2、现在不能与男友同房。
3、您的男友也需要到医院做hpv的检查。
4、做好做tct检查。根据检查结果尽快激光或leep刀治疗宫颈糜烂。
5、定期复查。
6、治愈后一般不
病情分析：轻度宫颈糜烂可以采用药物治疗。中重度药物治疗不容易治愈，指导意见：建议采用物理治疗如利普刀，利普刀治疗定位准确、无痛无创、不会破坏宫颈的正常组织，没有瘢痕形成，不会影响性生活的质量和将来的生育。 提问人的追问
17:12:09Leep刀费用一般是多少？
病情描述（主要症状、发病时间）:2011年8月体检时宫颈刮片呈阳性。（时间为经期结束后头一天）无想体检医院建议复查，请问需间隔多长时间进行复查比较合适。化验、检查结果:检测值2.5。
病情分析:高危型hpv主要导致高度宫颈上皮内瘤变和宫颈癌的发生，目前无特效药，治疗主要是增强身体抵抗力。指导意见:建议每年复查一次TCT，平时注意个人卫生和性生活卫生。
病情分析:宫颈癌前筛查是需要检查HPV和TCT的。您的报告内容提供不详。如果是HPV阳性，需要4-6个月后复查HPV。指导意见:建议4-6个月后复查HPV，如果是CIN，需要做物理治疗或宫颈锥切手术。
我今年40岁，2009.7月&&作人乳头瘤病毒分型检测，HPV显阳性，并有宫颈糜烂，连续用胸腺五肽30针（2针/天）后，再作检查，HPV显阴性，接着做了LEEP刀宫颈环切手术治疗宫颈糜烂。2010.8月换了家医院复查，赛泰HPV-L1显阳性液基检查为正常，阴道镜:宫颈光滑，SPI？白带每次检查结果均为:清洁度Ⅲ。2011．7月&HPV检查&&HPV16&&&阳性（+）&&&HPV52&&&&&阳性（+）&&&TCT宫颈中度糜烂（检查报告是这样写的，但医生说没问题，哈哈）赛泰HPV-L1显阳性2012.2月&&HPV检查&HPV52&阳性（+）&&HPV1阴性(-)&&&&&HPV2阴性（-）&&&&&&&&TCT检查未见癌细胞和上皮内病变细胞&&&&阴道镜检查未见异常2012．9&&TCT正常2013.3&&HC2&HPV&DNA检测为:阳性，检测值为:43.33&&&&&&&&&&&TCT结果:大量上皮细胞及炎细胞，未见上皮内病变细胞及恶性细胞。每次只是治一下阴道炎，随访想我知道HPV52属于高危，现在HPV检测值:43.33&&&到底是严重了？还是好点了？
病情分析：HPV持续阳性，TCT正常，指导意见：HPV检测值：43.33这个是正常的数值啊，还是这个数值后面还有个单位，这个很重要的。如是果没有那就是正常的数值。 提问人的追问
07:48:44HC2HPVDNA检测检测结果：阳性正常参考值是&1.00检测值：43.33
病情分析：你好,HPV阳性是生殖器官病毒感染，通常伴发于宫颈糜烂、慢性宫颈炎等疾病，指导意见：原来没有数值，看不出来好点没有。确诊后进行规范的抗病毒治疗，常用的药物有干扰素等。
主要症状:白带过多,呈浅黄色发病时间:7月初化验检查结果:HPV-DNA检测结果:送检标本:宫颈粘液脱落细胞标本状态:正常执行科室:病理科检验结果:阳性正常参考值≤1.00pg/ml
检测值:1.84第一次补充提问:( 15:56:30)医生您好,在做HPV-DNA检查之前,我分别做了白带常规以及子宫抹片检查,结果是:1,清洁度:3；念珠菌,滴虫子未见；白细胞+++/HP；细菌性阴道病试验（BV）阴性（-）2,子宫抹片检查:电脑抹片分析:非典型鳞状细胞（ASU-US）中度发炎；细胞项目:有鳞状细胞,有内宫颈细胞,有化生细胞；微生物项目:未见念珠菌,未见滴虫；病毒项目:未见泡疱疹毒感染,未见乳头瘤病毒感染；抹片质素:满意；炎症细胞/遮盖比率:在50%-75%之间第二次补充提问:( 16:00:58)还有医生说我患有中度宫颈糜烂,综合以上结果,请问医生:我主要是得了什么病?有没有患上尖锐湿庞?应该作怎样的治疗?在日常生活中应该注意哪些问题?第三次补充提问:( 23:10:35)请问尖锐湿庞的症状是什么?目前我除了白带过多,没发现其他症状,请问怎么判断我有无患尖锐湿庞呢?
您好!如果进行HPV检查存在阳性的情况考虑是存在人乳头瘤感染的情况,建议您选择进行血液HPV检查明确是否存在高危因素的情况?每年需要进行TCT检查明确宫颈粘液细胞的情况.
尖锐湿疣的临床症状主要为：
尖锐湿疣大小不等,形态各异,颜色不同,基本没有感觉.
其形态有：细小的丘疹,丘疹,丝状,乳头状,鸡冠状,采花状,巨大形等.
颜色有：白色,灰色,红色,棕色,黑色等.一般没有自觉症状. 尖锐湿疣的诊断主要以临床症状为主,配合醋酸白试验或病理检查.而HPV的检测只能作临床参考.HPV是尖锐湿疣的抗体,是抽血化验的.
医生?你好为什么我女朋友检查到HPV2?是有问题我自己去检查?但是HPV?上面没有显示HPV2?这个数字？需要医生帮助提供远程诊断:了解下为什么{[OnlyCharacteristic!!!No.1
病情分析：您好，尖锐湿疣又称生殖器疣或性病疣，是一种由人类乳头瘤病毒引起的性传播疾病。
指导意见：提高机体免疫力。应用抗病药物。可以激光治疗：通常用CO2激光，采用烧灼法治疗尖锐湿疣，本疗法最适用女阴、阴茎或肛周的湿疣。对单发或少量多发湿疣可行一次性治疗，对多发或面积大的湿疣可行2-3次治疗，间隔时间一般为一周。
病情分析：你好！根据你描述的情况来看，做了hpv感染，可以对照结果参考值是做的定量还是做的分析指导意见：如果大于参考值或者是做的分型，可以看一下是高危型还是低危型，如果有问题建议你要到医院去检查一下。
后在检查会不会变成晚期宫颈癌
病情分析：HPV高危型3个月后在检查会不会变成晚期宫颈癌&病情指导：一般不会的，Hpv检测是对付人乳头瘤病毒的一种手段,病毒是一种DNA病毒,人类是HPV唯一的宿主.HPV进入机体皮肤粘膜后,主要潜伏于表皮内基底细胞间,一旦时机成熟它就会致病。目前科学家已证明了经性生活传染的HPV病毒是引起宫颈癌的主要病因,高危-HPV检测捕获早期宫颈癌是近几年医学界刚刚发明并开展起来的一种快速、有效的检测方法,可一次检测所有引致宫颈癌的13个高危型HPV病毒,使宫颈癌检出率达99%以上.平时饮食要规律，新鲜的为主，不要吃油炸，烧烤，腌制，烟熏的东西，其他没什么大碍，心情乐观，生活规律点，不要睡太晚
病情分析：你好，HPV高危型只是表示你感染的HPV病毒是高危型的指导意见：HPV高危型有致癌的危险性，你现在做妇科检查只是说明你有感染，并不一定说你已经有宫颈癌了，除非你已经做了宫颈的病理检查。
HC2HPVDNA阳性，正常参考值《1.00检测值:1962.46，是不是太高了。。。子宫颈抹片:轻度发炎，其他未见病毒什么的。炎症细胞低于50%产后不到一年想病情分析下，为什么HPV那么高，超出正常值那么高
病情分析：你好，这说明你有HPV的感染，而且比较严重。指导意见：这个值确实很高，但是如果TCT没有问题的话，规律服药就可以的，建议口服更昔洛韦，阴道用保妇康栓，共3个月，定期复查。
病情分析：您好，该检查仅提示您宫颈HPV病毒数量较多，且传染性较强。指导意见：1、您的抹片结果大致正常，无需担心，药物治疗即可。建议您尽早到正规医疗机构行免疫治疗。
2、增加体育锻炼，增强免疫力。
3、治愈前禁性生活。
4、该病毒存在于自然界，一般免疫力低的人感染，请不要增加不必要的担心。
hpv检查参考值高相关文章推荐
hpv检查参考值高相关热门回答ANTIBODIES SPECIFIC TO E6 PROTEINS OF HPV AND USE THEREOF
United States Patent Application
The subject invention provides an antibody composition for detecting E6 protein of at least one HPV strain in a sample. The subject antibodies may be used to detect oncogenic HPV E6 proteins in a sample, and the antibodies find use in a variety of diagnostic and therapeutic applications, including methods of diagnosing and treating cancer. Kits for performing the subject methods and containing the subject antibodies are also provided. Also disclosed in the present invention is a method of generating an antibody that specifically binds to amino-terminus of E6 proteins of at least two HPV strains.
Inventors:
Dixon, Eric (Cary, NC, US)
Blaesius, Rainer (Chapel Hill, NC, US)
Simkins, Stephen (Watertown, CT, US)
Knapp, Steven L. (Apex, NC, US)
Brough, George (Wake Forest, NC, US)
Lenz, Karen (Apex, NJ, US)
Schweizer, Johannes (San Jose, CA, US)
Lu, Peter (Pato Alto, CA, US)
Garman, David (Thornhill, CA)
Silver, Jon (San Jose, CA, US)
Mahoney, Charles (Sunnyvale, CA, US)
Diaz-sarmiento, Chamorro Somoza (Santa Clara, CA, US)
Application Number:
Publication Date:
06/07/2012
Filing Date:
04/20/2010
Export Citation:
DIXON ERIC
BLAESIUS RAINER
SIMKINS STEPHEN
KNAPP STEVEN L.
BROUGH GEORGE
LENZ KAREN
SCHWEIZER JOHANNES
GARMAN DAVID
SILVER JON
MAHONEY CHARLES
DIAZ-SARMIENTO CHAMORRO SOMOZA
Primary Class:
Other Classes:
International Classes:
A61K39/395; A61P31/20; C12Q1/70; C40B30/04; C40B40/10
View Patent Images:
&&&&&&PDF help
Related US Applications:
April, 2010Burwell et al.May, 2007RileyOctober, 2005LandschulzApril, 2004SchurAugust, 2009Omura et al.April, 2010Taylor et al.September, 2005Schiffelers et al.July, 2008PatelNovember, 2004LiebermanJuly, 2007Hahn et al.February, 2008White et al.
Other References:
Wlazlo AP et al. Generation and characterization of monoclonal antibodies against the E6 and E7 oncoproteins of HPV. Hybridoma. ):257-63.
Karoonuthaisiri N et al. Development of antibody array for simultaneous detection of foodborne pathogens. Biosens Bioelectron. 2009 Feb 15;24(6):1641-8. Epub 2008 Aug 26.
What is claimed is:
1. 1-162. (canceled)
An antibody composition for detecting E6 protein of a plurality of HPV strains in a sample, the composition comprising a first antibody and a second antibody, wherein the first antibody binds to an E6 protein of a first HPV strain, and the second antibody binds to an E6 protein of a second HPV strain wherein the first and second antibodies are arranged on a solid support such that the E6 protein of the first HPV strain is detectable at a first region on the solid support and the E6 protein of the second HPV strain is detectable at a second region on the solid support, and wherein the first and second regions comprise different sets of antibodies.
The antibody composition of claim 163, wherein the first HPV strain is an oncogenic HPV strain.
(canceled)
The antibody composition of claim 163, wherein the first HPV strain is selected from the group consisting of HPV strains 16, 18, 26, 30, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 73, and 82.
The antibody composition of claim 163, wherein the first HPV strain is HPV-6 or HPV-11.
The antibody composition of claim 163, wherein the first antibody specifically binds to E6 protein of HPV strain 16, 18, 31, 33, 45, 52 or 58.
(canceled)
(canceled)
The antibody composition of claim 163, wherein the first and the second antibodies bind to E6 proteins of HPV-16, HPV-18, HPV-45 or a combination thereof.
(canceled)
The antibody composition of claim 163, wherein the sample is a cervical scrape, cervical biopsy, cervical lavage, blood or urine.
(canceled)
The antibody composition of claim 163, wherein the first and the second antibodies are monoclonal.
The antibody composition of claim 163, wherein the first or second antibody binds to the N-terminus of an E6 protein and further comprising a third antibody that binds to the C-terminus of the E6 protein.
The antibody composition of claim 163, wherein the first or second antibody binds to the C-terminus of an E6 protein and further comprising a third antibody that binds to the N-terminus of the E6 protein.
The antibody composition of claim 176, wherein the third antibody is labeled.
The antibody composition of claim 176, wherein the third antibody is conjugated to an enzyme.
180. 180-184. (canceled)
The antibody composition of claim 163, wherein the antibody composition enhances signal-to-noise ratio of detecting an oncogenic E6 protein as compared to using a PDZ domain containing polypeptide for the detection of an oncogenic E6 protein in a sample.
(canceled)
The antibody composition of claim 163, wherein the antibody composition can be used to detect oncogenic E6 protein in a sample with a false positive rate that is less than 1.7%.
(canceled)
The antibody composition of claim 163, wherein two or more antibodies recognizing different epitopes of E6 protein are used for capture and/or detection of the E6 protein.
A diagnostic kit for detection of a plurality of strains of HPV in a sample, the kit comprising an antibody composition of claim 163.
The diagnostic kit of claim 190, wherein the kit detects E6 protein of at least one oncogenic HPV strain.
(canceled)
The diagnostic kit of claim 190, wherein the kit detects E6 proteins of HPV-16, HPV-18, HPV-45 or a combination thereof.
The diagnostic kit of claim 190 further comprising a strip on which the first and the second antibodies bind to E6 protein of at least one oncogenic HPV strain.
The diagnostic kit of claim 190, wherein the kit further contains reagents for detection of the second antibody that is bound to an E6 protein by an enzyme-linked immunosorbent assay (ELISA).
A method for detecting E6 protein of a plurality of HPV strains in a sample, comprising: (a) contacting a first antibody which specifically binds to a first E6 protein of at least one first strain of HPV (b) contacting a second antibody which specifically binds to a second E6 protein of at least one second strain of HPV and (c) detecting binding of the first or second antibody to the first or second E6 protein, thereby detecting the first or second E6 p wherein the first and second antibodies are arranged on a solid support such that the first E6 protein is detectable at a first region on the solid support and the second E6 protein is detectable at a second region on the solid support, and wherein the first and second regions comprise different sets of antibodies.
The method of claim 196, wherein the first HPV strain is an oncogenic HPV strain.
(canceled)
The method of claim 196, wherein the first HPV strain is selected from the group consisting of HPV strains 16, 18, 26, 30, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 73, and 82.
200. 200-225. (canceled)
A method for ameliorating an HPV disease, comprising administering to a subject in need thereof an effective amount of an antibody which specifically binds an HPV E6 protein.
227. 227-240. (canceled)
The antibody composition of claim 163, wherein the solid support is a strip.
The antibody composition of claim 163, wherein the first region and the second region each forms a test line across the solid support.
The antibody composition of claim 163, wherein the composition is capable of detecting E6 proteins from a sample containing less than about 10,000 HPV positive cells.
The antibody composition of claim 163, wherein the first and second antibodies are not separated in different vessels.
The antibody composition of claim 163, wherein the solid support is not a well.
The antibody composition of claim 163, wherein the antibody composition can be used to detect oncogenic E6 protein in a sample with a false positive rate that is less than 10%.
The antibody composition of claim 163, wherein the antibody composition can be used to detect oncogenic E6 protein in a sample with a specificity of at least about 85%.
The antibody composition of claim 176, wherein the E6 protein or a portion of the E6 protein is bound to the first antibody prior to contacting the third antibody.
The antibody composition of claim 177, wherein the E6 protein or a portion of the E6 protein is bound to the second antibody prior to contacting the third antibody.
Description:
RELATED APPLICATIONSThis application claims the benefit of U.S. Provisional Application No. 61/171,025, filed Apr. 20, 2009, U.S. Provisional Application No. 61/171,032, filed Apr. 20, 2009, U.S. Provisional Application No. 61/171,039, filed Apr. 20, 2009, U.S. Provisional Application No. 61/175,362, filed May 4, 2009, and U.S. Provisional Application No. 61/175,365, filed May 4, 2009, each of which is incorporated by reference herein in its entirety.BACKGROUND OF THE INVENTIONCervical cancer is the second most common cancer diagnosis in women and is linked to high-risk human papillomavirus infection 99.7% of the time. Currently, 12,000 new cases of invasive cervical cancer are diagnosed in US women annually, resulting in 5,000 deaths each year. Furthermore, there are approximately 400,000 cases of cervical cancer and close to 200,000 deaths annually worldwide. Human papillomaviruses (HPVs) are one of the most common causes of sexually transmitted disease in the world. Overall, 50-75% of sexually active men and women acquire genital HPV infections at some point in their lives. An estimated 5.5 million people become infected with HPV each year in the US alone, and at least 20 million are currently infected. The more than 100 different isolates of HPV have been broadly subdivided into high-risk and low-risk subtypes based on their association with cervical carcinomas or with benign cervical lesions or dysplasias.A number of lines of evidence point to HPV infections as the etiological agents of cervical cancers. Multiple studies in the 1980's reported the presence of HPV variants in cervical dysplasias, cancer, and in cell lines derived from cervical cancer. Further research demonstrated that the E6-E7 region of the genome from oncogenic HPV 18 is selectively retained in cervical cancer cells, suggesting that HPV infection could be causative and that continued expression of the E6-E7 region is required for maintenance of the immortalized or cancerous state. Further research demonstrated that the E6-E7 genes from HPV 16 are sufficient to immortalize human keratinocytes in culture. It was also demonstrated that although E6-E7 genes from high risk HPVs could transform cell lines, the E6-E7 regions from low risk, or non-oncogenic variants such as HPV 6 and HPV11 are unable to transform human keratinocytes. HPV 16 and 18 infection was examined by in situ hybridization and E6 protein expression by immunocytochemistry in 623 cervical tissue samples at various stages of tumor progression and a significant correlation was found between histological abnormality and HPV infection.A significant unmet need exists for early and accurate diagnosis of oncogenic HPV infection as well as for treatments directed at the causative HPV infection, preventing the development of cervical cancer by intervening earlier in disease progression. Human papillomaviruses characterized to date are associated with lesions confined to the epithelial layers of skin, or oral, pharyngeal, respiratory, and, most importantly, anogenital mucosae. Specific human papillomavirus types, including HPV 6 and 11, frequently cause benign mucosal lesions, whereas other types such as HPV 16, 18, and a host of other strains, are predominantly found in high-grade lesions and cancer. Individual types of human papillomaviruses (HPV) which infect mucosal surfaces have been implicated as the causative agents for carcinomas of the cervix, breast (Yu et al. (1999) Anticancer Res. 19:1; Liu et al. (2001) J. Hum. Virol. 44:329-334), anus, penis, prostate (De Villiers et al. (1989) Virology 171:248:253), larynx and the buccal cavity, tonsils (Snijders et al. (1994) J. Gen. Virol. 75(Pt 10):), nasal passage (Trujillo et al. (1996) Virus Genes 12:165-178; Wu et al. (1993) Lancet 341:522-524), skin (Trenfield et al. (1993) Australas. J. Dermatol. 34:71-78), bladder (Baithun et al. (1998) Cancer Surv. 31:17-27), head and neck squamous-cell carcinomas (Braakhuis et al. (2004) J. Natl. Cancer Inst. 96:978-980), occasional periungal carcinomas, as well as benign anogenital warts. The identification of particular HPV types is used for identifying subjects with premalignant lesions who are at risk of progression to malignancy. Although visible anogenital lesions are present in some persons infected with human papillomavirus, the majority of individuals with HPV genital tract infection do not have clinically apparent disease, but analysis of cytomorphological traits present in cervical smears can be used to detect HPV infection. Papanicolaou tests are a valuable screening tool, but they miss a large proportion of HPV-infected persons due to the unfortunate false positive and false negative test results. In addition, they are not amenable to worldwide testing because interpretation of results requires trained pathologists.HPV infection is also associated with Netherton's syndrome (Weber et al. (2001) Br. J. Dermatol. 144:) and epidermolysis verruciformis (Rubaie et al. (1998) Int. J. Dermatol. 37:766-771). HPV can also be transmitted to a fetus by the mother (Smith et al. (2004) Sex. Transm. Dis. 31:57-62; Xu et al. (1998) Chin. Med. Sci. J. 13:29-31; Cason et al. (1998) Intervirology 41:213-218).The detection and diagnosis of disease is a prerequisite for the treatment of disease. Numerous markers and characteristics of diseases have been identified and many are used for the diagnosis of disease. Many diseases are preceded by, and are characterized by, changes in the state of the affected cells. Changes can include the expression of pathogens or proteins in infected cells, changes in the expression patterns of genes or proteins in affected cells, and changes in cell morphology. The detection, diagnosis, and monitoring of diseases can be aided by the accurate assessment of these changes. Inexpensive, rapid, early and accurate detection of pathogens can allow treatment and prevention of diseases that range in effect from discomfort to death.LiteratureLiterature of interest includes the following references: Zozulya et al., (Genome Biology 2:8.12, 2001; Mombairts (Annu. Rev. Neurosci 22:487-509, 1999); Raining et al., (Nature 361: 353-356, 1993); Belluscio et al., (Neuron 20: 69-81, 1988); Ronnet et al., (Annu. Rev. Physiol. 64:189-222, 2002); Lu et al., (Traffic 4: 416-533, 2003); Buck (Cell 100:611-618, 2000); Malnic et al., (Cell 96:713-723, 1999); Firestein (Nature 413:211-218, 2001); Zhao et al., (Science 279: 237-242, 1998); Touhara et al., (Proc. Natl. Acad. Sci. 96: , 1999); Sklar et al., (J. Biol. Chem. 261:, 1986); Dryer et al., (TiPS 20:413-417, 1999); Ivic et al., (J. Neurobiol. 50:56-68, 2002); Munger (2002) Front. Biosci. 7:d641-9; Glaunsinger (2000) Oncogene 19:5270-80; Gardiol (1999) Oncogene 18:5487-96; Pim (1999) Oncogene 18:7403-8; Meschede (1998) J. Clin. Microbiol. 36:475-80; Kiyono (1997) Proc. Natl. Acad. Sci. 94:11612-6; and Lee (1997) Proc. Natl. Acad. Sci. 94:6670-5; Banks (1987) J. Gen. Virol. 68:; Fuchs et al., (Hum. Genet. 108:1-13, 2001); and Giovane et al. (1999) Journal of Molecular Recognition 12:141-152 and published US patent applications
and ; and U.S. Pat. Nos. 6,610,511, 6,492,143 6,410,249, 6,322,794, 6,344,314, 5,415,995, 5,753,233, 5,876,723, 5,648,459, 6,391,539, 5,665,535 and 4,777,239.SUMMARY OF THE INVENTIONIn one aspect, the present invention provides an antibody which specifically binds to amino-terminus (N-terminus) of oncognenic E6 proteins of at least two strains of human papilloma virus (HPV) with enhanced binding affinity and sensitivity of detecting the E6 proteins of at least two HPV strains. In some embodiments, the antibody specifically binds to E6 proteins of at least three different oncogenic HPV strains. In some embodiments, the antibody specifically binds to E6 proteins of HPV strains 16, 18, and 45. In some embodiments, the antibody specifically binds to E6 proteins of at least six different oncogenic HPV strains. In some embodiments, the antibody specifically binds to E6 proteins of HPV strains 16, 18, 31, 33, 45, 52, and 58. The antibody can also specifically bind to E6 proteins of HPV strains 16, 18, 26, 30, 31, 34, 39, 45, 51, 52, 53, 58, 59, 66, 68, 69, 70, 73, or 82 or a combination thereof. In some embodiments, the antibody specifically binds to E6 proteins in a sample. The sample can be a cervical scrape, cervical biopsy, cervical lavage, blood or urine. The sample can also be a histological sample. In some embodiments, the antibody binds to E6 protein with a binding affinity of less than 10-8 M, less than 10-9 M, less than 10-10 M, less than 10-11 M, or less than 10-12 M. In some embodiments, the antibody detects E6 protein with increased sensitivity. In some embodiments, the antibody is monoclonal. The antibody can also be labeled. In some embodiments, the antibody is a mixture of two or more monoclonal antibodies specific against oncogenic E6 proteins. In some embodiments, the antibody is used as a part of a test for cervical cancer. Also provided by the present invention is a kit for detection of an E6 protein of an oncogenic HPV strain in a sample, comprising the subject antibody disclosed herein. In some embodiments, the kit further comprises reagents for detection of the antibody. The detection can be by an enzyme-linked immunosorbent assay (ELISA).In another aspect, the present invention provides a method of detecting an E6 protein of at least two HPV strains in a sample, comprising: contacting an antibody which specifically binds to amino-terminus (N-terminus) of E6 proteins of at least two HPV str and detecting any binding of the antibody to the E6 p wherein binding of the antibody to the E6 protein in the sample indicates the presence of at least one HPV
and wherein the binding affinity of the antibody to the E6 protein is increased. In some embodiments of the subject method, the antibody specifically binds to E6 proteins of at least three different oncogenic HPV strains. In some embodiments, the antibody specifically binds to E6 proteins of HPV strains 16, 18, and 45. In some embodiments, the antibody specifically binds to E6 proteins of at least six different oncogenic HPV strains. The antibody may specifically bind to E6 proteins of HPV strain 16, 18, 26, 30, 31, 34, 39, 45, 51, 52, 53, 58, 59, 66, 68, 69, 70, 73, or 82 or a combination thereof. In some embodiments of the subject method, the sample is a cervical scrape, cervical biopsy, cervical lavage, blood or urine. The sample can be a histological sample. In some embodiments, the sample is from a human. In some embodiments, the antibody binds to E6 protein with a binding affinity of less than 10-8 M, less than 10-9 M, less than 10-10 M, less than 10-11 M, or less than 10-12 M. In some embodiments of the subject method, the antibody detects E6 protein with increased sensitivity. The antibody can be monoclonal or labeled. In some embodiments, the antibody is used as a part of a test for cervical cancer. In some embodiments, the antibody is a mixture of two or more monoclonal antibodies specific against oncogenic E6 proteins. In some embodiments, the antibody is used as a capture antibody to capture E6 protein in an enzyme-linked immunosorbent assay (ELISA).In some embodiments, the subject method is an enzyme-linked immunosorbent assay (ELISA), comprising: contacting the subject anti contacting the E6 protein that is bound to the subject antibody with another E6 binding partner that specifically binds to the E6 protein at a binding site that is different from that of
and detecting binding of the E6 binding partner to the E6 protein, thereby detecting the presence of the E6 protein in the sample. In some embodiments, the antibody is used as a detector antibody to detect E6 protein that is bound to an E6 binding partner specific for the E6 protein in an enzyme-linked immunosorbent assay (ELISA). In some embodiments, the method is an enzyme-linked immunosorbent assay (ELISA), comprising: contacting the sample with an E6 binding partner that specifically binds to E6 protein at a binding site that is different from that of
contacting the E6 protein that is bound to the E6 binding partner with
and detecting binding of the subject antibody to the E6 protein, thereby detecting the presence of the E6 protein in the sample. In some embodiments, the detection of E6 protein is via an immunological based assay selected from the group consisting of enzyme immunoassays (EIA), Ramon spectroscopy, lateral flow, and cytometric bead array (CBA). In some embodiments, the antibody is immobilized. In some embodiments, the E6 binding partner is immobilized. In some embodiments, the E6 binding partner is a PDZ domain containing polypeptide. The E6 binding partner can be ah antibody specific against the E6 protein. In some embodiments, the antibody is used in combination with an antibody that specifically binds to C-terminal region of an E6 protein for detection of the E6 protein. In some embodiments, the antibody is used in combination with a PDZ domain containing polypeptide that binds to C-terminal region of an E6 protein for detection of the E6 protein.In yet another aspect, the present invention provides a method of generating an antibody that binds to amino-terminus (N-terminus) of E6 proteins of at least two HPV strains in a sample, the method comprising: (a) immunizing animal with a peptide which has a T cell epitope sequence fused with an N-terminal sequence of an E6 (b) obtaining B lymphocytes from
(c) fusing the B lymphocytes obtained from the immunized animal with myeloma cells to generate hybridoma cells
and (d) screening the hybridoma cells for antibodies that specifically bind to the N-terminus of E6 proteins of at least two HPV strains. In some embodiments, the T cell epitope amino acid sequence is F-I-S-E-A-I-I-H-V-L-H-S-R. In some embodiments, the immunizing peptide is a consensus peptide present in HPV-16 E6 protein. In some embodiments, the immunizing peptide is a consensus peptide present in HPV-18 E6 protein. In some embodiments, the immunizing peptide step contains an amino acid sequence F-Q-D-P-A-E-R-P-R-K-L-H-D-L-C-T-E-L or F-Q-D-P-A-E-R-P-Y-K-L-P-D-L-C-T-E-L. In some embodiments, the method further comprises cloning hybridoma cells that secret antibodies specific for the N-terminus of oncognenic E6 proteins. In some embodiments, the method further comprises purifying antibodies that specifically bind to the N-terminus of oncognenic E6 proteins. In some embodiments of the subject method, the antibody is monoclonal. In some embodiments, the antibody specifically binds to E6 proteins of at least three different oncogenic HPV strains. In some embodiments, the antibody specifically binds to E6 proteins of HPV strains 16, 18, and 45. In some embodiments, the antibody specifically binds to E6 proteins of at least six different oncogenic HPV strains. In some embodiments, the antibody specifically binds to E6 proteins of HPV strains 16, 18, 26, 30, 31, 34, 39, 45, 51, 52, 53, 58, 59, 66, 68, 69, 70, 73, or 82 or a combination thereof. In some embodiments of the subject method, the sample is a cervical scrape, cervical biopsy, cervical lavage, blood or urine, or a histological sample. In some embodiments, the antibody is used as a diagnostic or therapeutic agent for cervical cancer. In some embodiments, the antibody binds to E6 protein with a binding affinity of less than 10-8 M, less than 10-9M, less than 10-10 M, less than 10-11 M, or less than 10-12 M. In some embodiments, the antibody detects E6 protein with higher sensitivity.INCORPORATION BY REFERENCEAll publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGSThe novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:FIGS. 1a and 1b show peptide antigens that closely mimic sequences of high-risk HPV for generating antibodies that are specific against N-terminal region of E6 proteins of at least two high-risk HPV strains. Black dots denote residues in all oncogenic strains and gray dots denote positions with only conservative differences. The differences between the two consensus peptide sequences shown in FIGS. 1a and 1b are highlighted in stripes.FIG. 2 shows cross-reactivity of consensus peptide antibodies to HPV E6 types in ELISA. Recombinant HPV E6 proteins were purified and coated directly to microtiter plates (gray bars) or captured with a PDZ domain containing protein (black bars). Primary antibodies to the consensus peptides were diluted to 1 μg/ml and added to the wells. Binding was detected by addition of a secondary goat anti-mouse IgG:HRP followed by the substrate TMB. Signal to noise (S/N) ratios were calculated by dividing the OD450 of test wells by the OD450 from wells with no consensus peptide antibody. Cross-reactivity profiles varied greatly between antibodies. Clone 6H5.3 demonstrated monospecific binding, while clone 4E9.7 had the ability to bind 8 HPV E6 types in the direct ELISA format.FIG. 3 shows cross-reactivity of consensus peptide antibodies to HPV E6 types in Western blot. Recombinant HPV E6 proteins were resolved by SDS-PAGE. Western blots were probed with consensus peptide antibodies diluted to 1 μg/ml in blocking buffer. Goat anti-mouse IgG:AP was used to detect binding. Antibody cross-reactivity profiles ranged from single type specific to 5 or more HPV types. Cross-reactivity to low risk HPV types 6b and 11 was not observed.FIG. 4 shows immunoprecipitation of recombinant HPV16 E6 by consensus peptide antibodies. Antibodies were linked to protein-G Dynabeads and incubated with 1 μg of recombinant maltose binding protein (MBP) tagged HPV-16 E6 (~60 kDa). After washing, immune complexes were separated by SDS-PAGE followed by Western blotting with an HPV-16 E6 specific mouse antibody. An alkaline phosphatase conjugated anti-mouse light chain specific antibody was used to detect immunoprecipitated HPV16 E6:MBP. Mouse Ig light chain is indicated by “LC”. Clone 4E9.7 was able to immunoprecipitate detectable levels of HPV16 E6:MBP.FIG. 5 shows detection of HPV-16 E6 from SiHa cell lysates by sandwich ELISA using a consensus peptide capture antibody. HPV-16 positive SiHa cells were lysed in RIPA buffer and applied to microtiter plates according to cell equivalents (black circles). Consensus peptide antibody 4E9.7 was used as the capture antibody with an HPV-16 E6 specific monoclonal detector antibody. Lysates were prepared similarly for the HPV negative C33A- cell line (gray squares). HPV-16 E6 was detected from less than 5,000 SiHa cell equivalents.FIG. 6 shows Western blot probed with HPV oncopeptide and anti-HIS monoclonal antibodies demonstrating the specificity of the antibody of the present invention for the HPV E6 oncopeptide.FIG. 7 shows characterization of an anti-HPV 16 and 18 E6 monoclonal antibody by ELISA and Western blot. An intrasplenic immunization with T cell epitope-6mer peptides and CD40 agonist treatment resulted in a 5mer (RRETQ)-specific Mab. This Mab (2H9.15) was epitope mapped with a high degree of specificity to the C-terminus of oncogenic E6 proteins from HPV 16 and HPV 18. 2H9.15 has a potential utility as a pan-antibody that detects both HPV types 16 and 18.FIG. 8 shows inhibition of HPV E6 binding to the MAGI-1 PDZ binding domain by competitive blocking with the C-terminal HPV E6 oncopeptide antibodies.FIG. 9 shows sandwich ELISA pairings. Various anti-HPV E6 capture antibodies were used to develop sandwich ELISA assays to detect HPV16 E6 protein. Combining antibody libraries (capture C1, C2, and C3 MAb) with the HPV Oncopeptide PDZ antibodies identified capture/detector pairs.FIG. 10 shows immunocytochemistry (ICC) for the detection of HPV E6 in cancer cell lines. In ICC, the MAb stained SiHa and HeLa cells expressing HPV E6 but not the HPV-negative cell line C-33A. Shown is the staining with MAb 1A9.1.FIG. 11 is a scheme showing detection of E6 protein via anti-E6 antibody sandwich assay in comparison with PDZ peptide capture of E6.FIG. 12 shows HPV16 singleplex detection. For HPV16-E6, the anti-E6 antibody sandwich assay results in an improved signal to background ratio and decreased dampening with individual cervical swab sample as compared to the PDZ peptide capture of E6.FIG. 13 shows HPV16 singleplex detection in negative cervical swab samples (NCLS): comparison of anti-E6 mAb sandwich detection with PDZ peptide capture of E6.FIG. 14 shows HPV18 singleplex detection using the anti-E6 mAb sandwich assay as compared to PDZ peptide capture of E6. The results show that for HPV18-E6, antibody capture results in substantially improved signal-to-background ratio.FIG. 15 shows HPV45 singleplex detection in the presence of cervical swab material. The anti-E6 antibody sandwich assay results in improved signal-to-background ratio as compared to PDZ peptide capture of E6. No NCLS specific dampening was observed.FIG. 16 is a scheme showing detection of E6 from multiple HPV strains on a test strip. E6 capture via HPV type specific anti-E6 mAb sandwich detection allows for E6 typing.FIG. 17 shows HPV16+HPV18 multiplex detection via two test-line strip. E6 capture via HPV strain specific mAb allows for E6 typing and the HPV16/18 antibody detector cocktail does not result in higher background or reduced signal when compared to singleplex detection.FIG. 18 shows HPV16+HPV18 multiplex detection via two test-line strip in negative cervical swab samples (NCLS).FIG. 19 shows HPV16/18-E6 detection using a two test-line strip. The results show no HPV16/18 false positives on 60 individual HPV negative cervical swab samples (NCLS), indicating a low false positive rate using the anti-E6 antibody sandwich strip test approach.FIG. 20 shows HPV16/18/45-E6 detection using a three test-line strip. The results show that E6 proteins from three different HPV strains can be detected as three distinct lines simultaneously on one strip.DETAILED DESCRIPTION OF THE INVENTIONThroughout this application, various publications, patents and published patent applications are cited. The disclosures of these publications, patents and published patent applications referenced in this application are hereby incorporated by reference in their entirety into the present disclosure. Citation herein by Applicant of a publication, patent, or published patent application is not an admission by Applicant of said publication, patent, or published patent application as prior art.In one aspect, the present invention provides an antibody which specifically binds to the amino-terminus (N-terminus) of oncognenic E6 proteins of at least two strains of human papilloma virus (HPV). In some embodiments, the antibody specifically binds to E6 proteins of at least three different oncogenic HPV strains, for example, HPV strains 16, 18, and 45. In some embodiments, the antibody specifically binds to E6 proteins of at least six different oncogenic HPV strains, for example, HPV strains 16, 18, 31, 33, 45, 52, and 58. In some embodiments, the antibody binds to E6 protein of at least one HPV strain, preferably, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 HPV strains, with a binding affinity of less than 10-8 M, 10-9 M, 10-10 M, 10-11M, or 10-12 M. In some embodiments, the antibody is monoclonal. The antibody may also be labeled. In some embodiments, the antibody is a mixture of two or more monoclonal antibodies specific against oncogenic E6 proteins.In another aspect, the present invention provides a method of detecting an E6 protein of at least two HPV strains in a sample, comprising: contacting an antibody which specifically binds to amino-terminus (N-terminus) of E6 proteins of at least two HPV str and detecting any binding of the antibody to the E6 protein in the sample. In most embodiments, binding of the antibody to the E6 protein in the sample indicates the presence of at least one HPV strain in the sample. The sample may be a cervical scrape, cervical biopsy, cervical lavage, blood or urine. The sample may also be a histological sample. In some embodiments, the antibody is used as a′ capture antibody to capture E6 protein in an enzyme-linked immunosorbent assay (ELISA). In this case, the method of the present invention can be an enzyme-linked immunosorbent assay (ELISA), comprising: contacting an antibody of the present inven contacting the E6 protein that is bound to the subject antibody with another E6-binding partner that specifically binds to the E6 protein at a binding site that is different from that of the antibody of t and detecting binding of the E6-binding partner to the E6 protein, thereby detecting the presence of the E6 protein in the sample. In other embodiments, the antibody is used as a detector antibody to detect E6 protein that is bound to an immobilized E6-binding partner specific for the E6 protein in an ELISA. For example, the method of the present invention may be an ELISA comprising: contacting the sample with an immobilized E6-binding partner that specifically binds to E6 protein at a binding site that is different from that of the antibody of t contacting the E6 protein that is bound to the immobilized E6-binding partner with an antibody of t and detecting binding of the subject antibody to the E6 protein, thereby detecting the presence of the E6 protein in the sample.In yet another aspect, the present invention a method of generating an antibody that binds to amino-terminus (N-terminus) of E6 proteins of at least two HPV strains in a sample, the method comprising: (a) immunizing animal with a peptide which has a T cell epitope sequence fused with a C-terminal sequence of oncognenic E6 (b) obtaining B lymphocytes from
(c) fusing the B lymphocytes obtained from the immunized animal with myeloma cells to generate hybridoma cells
and (d) screening the hybridoma cells for antibodies that specifically bind to the N-terminus of oncognenic E6 proteins of at least two HPV strains. In some embodiments, the T cell epitope amino acid sequence is F-J-S-E-A-I-I-H-V-L-H-S-R.In some embodiments, a consensus peptide is used to immunize an animal for production of the antibodies of the present invention. A consensus peptide disclosed herein can be used to generate antibodies that may cross-react with E6 proteins from more than one HPV strain with high affinity. Method of generating consensus peptide based on amino acid sequences is known in the art and may involve comparison and substitution of polar and/or nonpolar residues. The immunizing peptide of the present invention can be a consensus peptide of any length based on any portion within the N-terminal region of a HPV E6 protein. The consensus peptide can be based on the N-terminal region of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 HPV strains, preferably oncogenic HPV strains. In some embodiments, the consensus peptide is based on E6 N-terminal region of HPV-16. In other embodiments, the consensus peptide is based on E6 N-terminal region of HPV-18. Examples of the immunizing peptides include amino acid sequences F-Q-D-P-A-E-R-P-R-K-L-H-D-L-C-T-E-L and F-Q-D-P-A-E-R-P-Y-K-L-P-D-L-C-T-E-L. The generated antibody of the present invention may bind to E6 proteins of at least three different oncogenic HPV strains, for example, HPV strains 16, 18, and 45. In some embodiments, the antibody generated via the method of the present invention specifically binds to E6 proteins of at least six different oncogenic HPV strains including but not limited to HPV 16, 18, 26, 30, 31, 34, 39, 45, 51, 52, 53, 58, 59, 66, 68, 69, 70, 73, and 82. In terms of the binding affinity of the subject antibody for E6 protein, the antibody may bind to E6 protein with a binding affinity of less than 10-8 M, less than 10-9M, less than 10-10 M, less than 10-11 M, or less than 10-12 M.In one aspect, the present invention provides an antibody which specifically binds to the carboxyl-terminus (C-terminus) of oncogenic E6 proteins of at least two high-risk strains of human papilloma virus (HPV). In some embodiments, the antibody specifically binds to E6 proteins of at least three different oncogenic HPV strains, for example, HPV strains 16, 18, and 45. In some embodiments, the antibody specifically binds to E6 proteins of at least six different oncogenic HPV strains, for example, HPV strains 16, 18, 31, 33, 45, 52, and 58. In some embodiments, the antibody binds to E6 protein with a binding affinity of less than 10-8 M, 10-9 M, 10-10 M, 10-11 M, or 10-12 M. In some embodiments, the antibody is monoclonal. The antibody may also be labeled. In some embodiments, the antibody is a mixture of two or more monoclonal antibodies specific against oncogenic E6 proteins.In another aspect, the present invention provides a method of detecting an E6 protein of an oncogenic HPV strain in a sample, comprising: contacting an antibody which specifically binds to carboxyl-terminus (C-terminus) of oncognenic E6 proteins of at least two HPV str and detecting any binding of the antibody to the E6 protein in the sample. In most embodiments, binding of the antibody to the E6 protein in the sample indicates the presence of at least one oncogenic HPV strain in the sample. The sample may be a cervical scrape, cervical biopsy, cervical lavage, blood or urine. The sample may also be a histological sample. In some embodiments, the antibody is used as an antibody to capture E6 protein in an enzyme-linked immunosorbent assay (ELISA). In this case, the method of the present invention can be an enzyme-linked immunosorbent assay (ELISA), comprising: contacting an antibody of the present inven contacting the E6 protein that is bound to the subject antibody with a second antibody that specifically binds to the E6 protein at a binding site that is different from that of the antibody of t and detecting binding of the second antibody to the E6 protein, thereby detecting the presence of the E6 protein in the sample. In other embodiments, the antibody is used as a detector antibody to detect E6 protein that is bound to another E6 binding partner in an ELISA. An “E6 protein binding partner” can be any molecule that specifically binds to an oncogenic E6 protein. Suitable oncogenic E6 protein binding partners include a PDZ domain polypeptide (as described below), other antibodies against oncogenic E6 proteins (such as those described below); other proteins that recognize oncogenic E6 protein (e.g., p53, E6-AP or E6-BP); DNA (i.e., cruciform DNA); and other binding partners such as aptamers. In some embodiments, detection of more than one oncogenic E6 protein (e.g., all oncogenic E6 proteins, E6 proteins from HPV strains 16 and 18, or E6 proteins from HPV strains 16 and 45 etc.) is desirable, and, as such, an oncogenic E6 protein binding partner may be an antibody that binds to these proteins, as described below, or a mixture of antibodies that each binds to different oncogenic HPV E6 proteins. As is known in the art, such binding partners may be labeled to facilitate their detection. In general, binding partners bind E6 with a binding affinity of less then 10-5 M, e.g., less than 10-6, less than 10-7 M, less than 10-8 M (e.g., less than 10-9 M, 10-10, 10-11, 10-12 etc.).In some examples, the method of the present invention may be an ELISA comprising: contacting the sample with a binding partner that specifically binds to E6 protein at a binding site that is different from that of the antibody of t contacting the E6 protein that is bound to the antibody with an antibody of t and detecting binding of the antibody of the present invention to the E6 protein, thereby detecting the presence of the E6 protein in the sample. The subject antibody may or may not be immobilized to a substrate in practicing the subject method.In yet another aspect, the present invention a method of generating an antibody that binds to carboxyl-terminus (C-terminus) of oncognenic E6 proteins of at least two HPV strains, the method comprising: (a) immunizing an animal with chimeric peptide, which contains a T cell epitope sequence fused with a C-terminal sequence of oncognenic E6 (b) obtaining B lymphocytes from
(c) fusing the B lymphocytes obtained from the immunized animal with myeloma cells to generate hybridoma cells
and (d) screening the hybridoma cells for antibodies that specifically bind to the C-terminus of oncognenic E6 proteins of at least two oncogenic HPV strains. An example of a T cell epitope amino acid sequence is F-J-S-E-A-I-I-H-V-L-H-S-R. In some embodiments, the C-terminal sequence of an oncognenic E6 protein used in the immunizing step contains a PDZ domain binding motif. In some embodiments, the C-terminal sequence of an oncogenic E6 protein used in the immunizing step contains a conserved amino acid motif E-(T/S)-X-(V/L), i.e. the C-terminal consensus sequence. Examples of the consensus C-terminal sequence of an oncognenic E6 protein used in the immunizing step include amino acid sequences E-T-Q-L and E-T-Q-V. The generated antibody of the present invention may bind to E6 proteins of at least three different oncogenic HPV strains, for example, HPV strains 16, 18, and 45. In some embodiments, the antibody generated via the method of the present invention specifically binds to E6 proteins of at least six different oncogenic HPV strains including but not limited to HPV 16, 18, 26, 30, 31, 34, 39, 45, 51, 52, 53, 58, 59, 66, 68, 69, 70, 73, and 82.In one aspect, the present invention provides an antibody composition for detecting E6 protein of at least one HPV strain in a sample, the composition comprising a first antibody and a second antibody, wherein the first antibody binds to E6 protein of an oncogenic HPV strain in the sample, and the second antibody specifically binds to the E6 protein that is bound by the first antibody, and wherein the second antibody is part of a signal producing system for detection of the E6 protein in the sample. In some embodiments, the first antibody is immobilized to a substrate. In other embodiments, the first antibody may be fused or bound to another molecule that is immobilized to a substrate. In some embodiments, the antibodies including polyclonal and monoclonal antibodies bind to E6 proteins from at least one strain of HPV. In some embodiments, the HPV strain is an oncogenic HPV strain. In some embodiments, the HPV strain is a non-oncogenic HPV strain. In other embodiments, the antibodies bind to E6 proteins from more than one oncogenic strain of HPV. In some embodiments, the antibodies specific for E6 proteins bind to amino acid motifs that are conserved between the E6 proteins of different HPV strains, particularly HPV strains 16 and 18. In another aspect, the subject antibodies may be used in a method as described herein, for example, an antibody sandwich binding assay, to detect E6 protein of an oncogenic HPV strain in a sample.Accordingly, the antibodies of the present invention find use in a variety of diagnostic applications, including methods of diagnosing cancer, particularly cervical cancer. In another aspect of the present invention, kits for performing the subject methods and containing the subject antibodies are also provided.Human Papilloma Virus (HPV) and E6 OncoproteinHuman papillomaviruses (HPVs) are small double-stranded DNA viruses that induce hyperproliferative lesions in epithelial tissues. Genomic organization is a well conserved feature among papillomaviruses. There are three main regions in an HPV genome-early, late and the long control regions. In the early region (E) resides the transformation and immortalization potential of HPVs and consists of a number of regulatory genes for viral transcription and replication and cell cycle control. The late region (L) codes for the two capsid genes and the long control region (LCR) contains all the cis-regulatory elements necessary for HPV transcription including the early promoter and the origin of replication (ori). The HPV genome encodes 6 early (E) and two late (L) proteins.E1 and E2 are the two viral proteins that are required for viral DNA replication, together with the host cell DNA replication machinery. E4 and E5 are needed for amplification of the viral genome in the upper layers of the epithelium. E6 and E7 proteins of high-risk HPV types are oncogenic. They cooperate to immortalize cells and also induce genomic instability. E6 and E7 abrogate the activity of the cellular tumor suppressor proteins p53 and Rb, respectively. E6 also increases telomerase activity. L1 and L2 proteins form the viral capsid and are expressed late in infection in the upper layers of the epithelium. The long-control-region (LCR) contains most of the regulatory DNA sequences needed for proper replication of the viral genome (origin of DNA replication) and for the expression of the viral genes (enhancer and promoter regions).There are over 100 different types of HPV, and these HPV types i.e. strains have been separated into those that are more likely to develop into cancer and those that are less likely. The so-called “high risk” HPV types are more likely to lead to the development of cancer, while “low-risk” viruses rarely develop into cancer. Certain “high-risk” HPV strains infect epithelia in the anogenital region and are the etiological agents of cervical cancers. These high-risk HPV strains include but are not limited to HPV-16, HPV-18, HPV-26, HPV-30, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV53, HPV54, HPV-56, HPV-58, HPV-59, HPV-66, HPV-68, HPV-69, HPV-73, and HPV-82. The “low-risk” HPV strains include but are not limited to HPV 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, and 81. The sequence analysis of HPV E6 proteins from various HPV strains with regard to the oncogenic potential of the E6 proteins is shown in U.S. Pat. Nos. 7,312,041 and 7,399,467, both of which are herein incorporated by reference in their entirety.An “oncogenic HPV strain” is an HPV strain that is known to cause cervical cancer as determined by the National Cancer Institute (NCI, 2001). “Oncogenic E6 proteins” are E6 proteins encoded by the above oncogenic HPV strains. The sequences of exemplary oncogenic E6 proteins of interest are disclosed in U.S. Pat. No. 7,399,467, which is herein incorporated by reference in its entirety. The sequences of various HPV proteins are also found as database entries at NCBI's Genbank database, as follows: HPV16-E6: GI:9627100; HPV18-E6: GI:9626069; HPV31-E6: GI:9627109; HPV35-E6: GI:9627127; HPV30-E6: GI:9627320; HPV39-E6: GI:9627165; HPV45-E6: GI:9627356; HPV51-E6: GI:9627155; HPV52-E6: GI:9627370; HPV56-E6: GI:9627383; HPV59-E6: GI:9627962; HPV58-E6: GI:9626489; HPV33-E6: GI:9627118; HPV66-E6: GI:9628582; HPV68b-E6: GI:184383; HPV69-E6: GI:9634605; HPV26-E6: GI:396956; HPV53-E6: GI:9627377; HPV73: GI:1491692; HPV82: GI:9634614, HPV34 GI:396989; HPV67 GI:3228267; and HPV70 GI:1173493.The oncogenic potential of these high-risk HPV strains is dependent on the cooperative action of the two early viral gene products, E6 and E7, which bind and alter the activity of cell cycle-regulatory proteins. E6 gene encodes for a small nuclear protein product of about 16-19 kD (Greenfield, I., et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, ). E6 is found and expressed in all HPV-containing cells (Smotkin, D. & Wettstein, F. O. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, ). The E6 protein contributes most significantly to the malignant conversion of the basal layer of the cervical epithelium. The E6 product from high-risk HPV-16 and 18 interacts with the antioncogenic regulator p53 and the ubiquitin degradation pathway protein E6AP, leading to the degradation of the p53 protein (Werness, B. A., et al. (1990) Science 248, 76-79). Briefly, E6 forms a ternary complex composed of the tumor suppressor protein p53 and E6AP (E6-associated protein), a member of E3 ubiquitin ligase family of proteins, resulting in the ubiquitination and subsequent degradation of p53 (Huibregtse, J. M., et al. 1991. EMBO J. 10:). Low-risk HPV types 6 and 11 E6 protein does not induce p53 degradation correlating with their weak transformation potential. Absence of functional p53 protein makes the cell highly susceptible to DNA damage and prevents the activation of p53-mediated apoptosis. Most HPV-positive tumors have wild-type p53 whereas HPV-negative tumors contain mutant p53 (Crook, T., et al. (1991) Oncogene 6, 873-875). As a result of the activities of the E6 protein, keratinocytes reactivate DNA synthesis and this in turn alters the growth and differentiation of the basal epithelium anogenital mucosa, resulting in their immortalization.E6 forms a ternary complex composed of the tumor suppressor protein p53 and E6AP (E6-associated protein), a member of E3 ubiquitin ligase family of proteins, resulting in the ubiquitination and subsequent degradation of p53 (Huibregtse, J. M., et al. 1991. EMBO J. 10:). E7 binds to and inactivates the retinoblastoma (pRb) family of proteins, thereby alleviating the pRb-mediated repression of E2F transcription factors that are responsible for transactivating many genes involved in progression into S phase (Cheng, S., et al. 1995. Genes Dev. 9:). Selective retention and expression of these two viral oncoproteins is essential for HPV-induced oncogenesis (Androphy, E. J., et al. 1987. EMBO J. 6:989-992).HPV-16 E6 gene has two alternative splicing sites resulting in the production of two additional protein products named E6*I and E6*II. However, only the full-length E6 has the capacity to interact with p53 and thus is the only one with clinical relevance. The E6 protein contains four Cys-X-X-Cys motifs forming zinc-binding structures similar to those present in several transcription factors (Grossman, S. R. & Laimins, L. A. (1989) Oncogene 4, ).Exemplary PDZ domain-containing proteins and PDZ domain sequences may be found in U.S. Pat. Nos. 7,312,041, and 7,399,467, which are herein incorporated by their entirety. The term “PDZ domain” also encompasses variants (e.g., naturally occurring variants) of the sequences (e.g., polymorphic variants, variants with conservative substitutions, and the like) and domains from alternative species (e.g. mouse, rat). Typically, PDZ domains are substantially identical to those shown in U.S. patent application Ser. Nos. 09/724,553 and 10/938,249), e.g., at least about 70%, at least about 80%, or at least about 90% amino acid residue identity when compared and aligned for maximum correspondence. It is appreciated in the art that PDZ domains can be mutated to give amino acid changes that can strengthen or weaken binding and to alter specificity, yet they remain PDZ domains (Schneider et al., 1998, Nat. Biotech. 17:170-5). Unless otherwise indicated, a reference to a particular PDZ domain (e.g. a MAGI-1 domain 2) is intended to encompass the particular PDZ domain and HPV E6-binding variants thereof. In other words, if a reference is made to a particular PDZ domain, a reference is also made to variants of that PDZ domain that bind oncogenic E6 protein of HPV, as described below. In this respect it is noted that the numbering of PDZ domains in a protein may change. For example, the MAGI-1 domain 2, as referenced herein, may be referenced as MAGI-1 domain 1 in other literature. As such, when a particular PDZ domain of a protein is referenced in this application, this reference should be understood in view of the sequence of that domain, as described herein, particularly in the sequence listing. U.S. Pat. Nos. 7,312,041, and 7,399,467 show the sequences, the names and Genbank accession numbers for various PDZ domains, where appropriate. Further description of PDZ proteins, particularly a description of MAGI-1 domain 2 protein, is found in Ser. No. 10/630,590, filed Jul. 29, 2003 and published as US. This publication is incorporated by reference herein in its entirety for all purposes.In the case of the PDZ domains described herein, a “HPV E6-binding variant” of a particular PDZ domain is a PDZ domain variant that retains HPV E6 PDZ ligand binding activity. Assays for determining whether a PDZ domain variant binds HPV E6 are described in great detail below, and guidance for identifying which amino acids to change in a specific PDZ domain to make it into a variant may be found in a variety of sources. In one example, a PDZ domain may be compared to other PDZ domains described herein and amino acids at corresponding positions may be substituted, for example. In another example, the sequence a PDZ domain of a particular PDZ protein may be compared to the sequence of an equivalent PDZ domain in an equivalent PDZ protein from another species. For example, the sequence of a PDZ domain from a human PDZ protein may be compared to the sequence of other known and equivalent PDZ domains from other species (e.g., mouse, rat, etc.) and any amino acids that are variant between the two sequences may be substituted into the human PDZ domain to make a variant of the PDZ domain. In some embodiments, the PDZ domain polypeptide used to capture E6 protein in a sample is MAGI-1. For example, the sequence of the human MAGI-1 PDZ domain 2 may be compared to equivalent MAGI-1 PDZ domains from other species (e.g. mouse Genbank GI numbers 7513782 and
or other homologous sequences) to identify amino acids that may be substituted into the human MAGI-1-PDZ domain to make a variant thereof. Such method may be applied to any of the MAGI-1 PDZ domains described herein. Particular variants may have 1, up to 5, up to about 10, up to about 15, up to about 20 or up to about 30 or more, usually up to about 50 amino acid changes as compared to a sequence set forth in the sequence listing. In making a variant, if a GFG motif is present in a PDZ domain, in general, it should not be altered in sequence. Exemplary PDZ domain peptides are disclosed in U.S. Pat. Nos. 7,312,041 and 7,399,467, which are herein incorporated by reference in their entirety.In general, variant PDZ domain polypeptides have a PDZ domain that has at least about 70 or 80%, usually at least about 90%, and more usually at least about 98% sequence identity with a variant PDZ domain polypeptide described herein, as measured by BLAST 2.0 using default parameters, over a region extending over the entire PDZ domain.As used herein, the term “PDZ protein” refers to a naturally occurring protein containing a PDZ domain. Exemplary PDZ proteins include CASK, MPP1, DLG1, DLG2, PSD95, NeDLG, TIP-33, SYN1a, TIP-43, LDP, LIM, LIMK1, LIMK2, MPP2, NOS1, AF6, PTN-4, prIL16, 41.8 kD, KIAA0559, RGS12, KIAA0316, DVL1, TIP-40, TIAM1, MINT1, MAGI-1, MAGI-2, MAGI-3, KIAA0303, CBP, MINT3, TIP-2, KIAA0561, and TIP-1.As used herein, the term “PL protein” or “PDZ Ligand protein” refers to a protein that forms a molecular complex with a PDZ-domain, or to a protein whose carboxy-terminus, when expressed separately from the full length protein (e.g., as a peptide fragment of 4-25 residues, e.g., 8, 10, 12, 14 or 16 residues), forms such a molecular complex. The molecular complex can be observed in vitro using a variety of assays described infra. As used herein, a “PL sequence” refers to the amino acid sequence of the C-terminus of a PL protein (e.g., the C-terminal 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20 or 25 residues) (“C-terminal PL sequence”) or to an internal sequence known to bind a PDZ domain (“internal PL sequence”).A phenotypic characteristic of all high-risk oncogenic HPV E6 proteins is the presence of a conventional PDZ binding motif (X-S/T-X-φ) where X is any amino acid and φ represents a hydrophobic amino acid. This PDZ domain binding motif is located at the last four carboxy-terminal (C-terminal) amino acids of oncogenic E6 proteins of high-risk HPV strains. In contrast, all low-risk HPV strains do not contain this PDZ domain binding motif. As used herein, the term “PDZ domain” refers to protein sequence of less than approximately 90 amino acids, (i.e., about 80-90, about 70-80, about 60-70 or about 50-60 amino acids), characterized by homology to the brain synaptic protein PSD-95, the Drosophila septate junction protein Discs-Large (DLG), and the epithelial tight junction protein ZO1 (Z01). PDZ domains are also known as Discs-Large homology repeats (“DHRs”) and GLGF repeats. PDZ domains generally appear to maintain a core consensus sequence (Doyle, D. A., 1996, Cell 85: 1067-76). PDZ domains are found in diverse membrane-associated proteins including members of the MAGUK family of guanylate kinase homologs, several protein phosphatases and kinases, neuronal nitric oxide synthase, tumor suppressor proteins, and several dystrophin-associated proteins, collectively known as syntrophins. Based upon PDZ ligand binding, all of the high-risk HPV types can be classified as Class I PDZ binding proteins. HPV E6 is known to interact with six different PDZ domain-containing proteins including but not limited to discs large (Dig), MAGI-1, MAGI-2, MAGI-3, MUPP1, and Scribble (hScrib). These PDZ domain-containing proteins are characterized by having multiple protein-protein interaction motifs and are frequently expressed at sites of cell to cell contact. They function mostly by regulating the formation of multicomponent protein complexes at these sites by interaction of their PDZ domains. Through the PDZ domain binding sequences, E6 protein can bind a single PDZ domain on each target protein and then direct its deregulation by the 26S proteosome. In cervical tumor models, it has been demonstrated that expression of oncogenic E6 proteins of high-risk HPV strains targets hDLg, MAGI-1 and MUPP1. All t