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High throughput cell-based HPV immunoassays for diagnosis and screening of HPV-associated cancers

一种细胞、癌症的技术，应用在用于诊断和筛选与HPV有关的癌症的高通量细胞基HPV免疫测定领域

Active Publication Date: 2012-12-12
HEER MEDICAL TECH DEV CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, prior art antibodies have only limited utility for in situ detection of E6 / E7 oncoproteins

Method used

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  • High throughput cell-based HPV immunoassays for diagnosis and screening of HPV-associated cancers
  • High throughput cell-based HPV immunoassays for diagnosis and screening of HPV-associated cancers
  • High throughput cell-based HPV immunoassays for diagnosis and screening of HPV-associated cancers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0145] Example 1. Expression, purification and preparation of recombinant HPV proteins for use as immunogens to generate antisera and screen monoclonal antibodies from hybridoma cell lines

[0146] The HPV recombinant protein can be any kind of HPV protein, HPV protein of early gene and / or late gene, including but not limited to: E2, E6, E7, L1, L2, and can be from various HPV types. One aspect of the present invention provides a recombinant protein, such as a recombinant hybrid protein, which comprises a partial sequence or a full-length sequence of an HPV oncogene protein. For example, full length E6, E7 and / or L1 polypeptide sequences, which have been found difficult to obtain and purify due to undesired aggregation during protein purification, protein instability, low level expression, low immunogenic response of purified protein . For example, many early E6 oncoproteins contain many cysteine ​​amino acids, thus correct topology of E6 oncoproteins requires proper formatio...

Embodiment 2

[0154] Example 2. Anti-HPV antibody preparation. Recombinant HPV proteins produced using the techniques described in Example 1 were used as immunogens to generate antisera, polyclonal antibodies, and monoclonal antibodies.

[0155] Basic techniques for performing cloning and performing immunoassays can be found in: "Antibodies: A Laboratory Manual", Harlow and Lane, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1989;" Molecular Cloning", A Laboratory Manual, eds. Sambrook, Fritsch and Maniatis, Cold Spring Harbor Laboratory Press, 1989 and other books and technical manuals known in the art. Details of our antibody production process to characterize the antibodies produced and certain assays are described in our co-owned U.S. Application Serial No. 12 / 456,053 - filed June 10, 2009, entitled "Novel Monoclonal Antibodies against HPV Proteins (for Novel Monoclonal Antibodies to HPV Proteins), U.S. Application Serial No. 12 / 456,054 - filed June 10, 2009, entitled "In si...

Embodiment 26

[0157] Example 2.6. Specificity of anti-HPV antibodies.

[0158]One or more immunoassays can be used to test the specificity of monoclonal antibodies produced by screening hybridoma cell lines with two or more HPV recombinant proteins. EIA (enzyme immunoassay) and / or Western blot were used as assay formats to test the specificity of the HPV antibodies described herein. Various purified recombinant HPV proteins—including the initial screening proteins used to obtain anti-HPV antibodies and other proteins not used in the screening—were used to coat microtiter plates to test the anti-HPV antibodies obtained by EIA. specificity. Proteins in cell lysates of cervical cancer cell lines (with or without HPV infection) were used to test the specificity of anti-HPV antibodies by Western blot. To determine the binding and reactivity of HPV antibodies to proteins from HPV-infected cell lines, Western blots are very helpful in showing specific protein bands corresponding to proteins pre...

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PUM

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Abstract

Methods for quantifying an HPV protein expression in a clinical sample are disclosed. The quantifying methods include the process for obtaining the clinical sample. Such a clinical sample is consisted of a population of cells that are susceptible to infection by an HPV. The quantifying methods also include the process for depositing the clinical sample into a container. The clinical sample is contacted with the first antibody that specifically binds to an HPV protein which is expressed by an HPV-infected cell under a condition that promotes specific binding of the first antibody to the HPV protein expressed by the population of cells. The methods further include the process for quantifying the specific binding of the first antibody and thereby quantifying the HPV protein expression in the clinical sample. The assay provides an objective test to identify patients with high-grade precursor from cytology samples before biopsy.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of U.S. Provisional Application Serial No. 61 / 335,540, entitled "High Throughput Cell-Based HPV Immunoassay for Diagnosis and Screening of HPV-Associated Cancers," filed January 8, 2010, the contents of which are adopted by References are incorporated herein for all purposes. Background of the invention [0003] Cervical cancer is the second most common cause of cancer death in women worldwide, with approximately 500,000 new cases and approximately 250,000 cumulative deaths each year. In the United States, cervical cancer death rates have fallen dramatically thanks to cervical cancer screening programs, which detect precancerous conditions so they can be treated before they develop into cancer. Currently, the paradigm for cervical cancer screening is based on the Pap test, a cytology-based test in which cells are scraped from the cervix and examined under a microscope to detect change...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53C12Q1/70
CPCG01N33/57411G01N33/56983G01N2333/025
Inventor S・成
Owner HEER MEDICAL TECH DEV CO LTD
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