Monoclonal antibodies and methods for their use in the detection of cervical disease

a technology of monoclonal antibodies and cervical disease, applied in the field of cervical disease diagnosis, can solve the problems of many false positives, more serious clinical problems, and many hpv-positive women developing high-grade cervical disease or cancer

Inactive Publication Date: 2007-05-24
TRIPATH IMAGING INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In many developing countries where mass screening programs are not available, the clinical problem is more serious.
However, HPV infection is very common, possibly occurring in 5-15% of women over the age of 30, but few HPV-positive women will ever develop high-grade cervical disease or cancer.
The presence of HPV alone is indicative only of infection, not of high-grade cervical disease, and, therefore, testing for HPV infection alone results in many false positives.
For those individuals who develop persistent infections with one or more oncogenic subtypes of HPV, there is a risk for the development of neoplasia in comparison to patients without an HPV infection.
However, a positive HPV result is not diagnostic of cervical disease; rather it is an indication of infection.
Although the majority of HPV infections is transient and will spontaneously clear within a 12-month period, a persistent infection with a high-risk HPV viral subtype indicates a higher risk for the development of cervical neoplasia.
Furthermore, up to 10% of Pap smears are classified as ASCUS (atypical squamous cells of undetermined significance), i.e., it is not possible to make a clear categorization as normal, moderate or severe lesion, or tumor.
However, experience shows that up to 10% of this ASCUS population has high-grade lesions, which are consequently overlooked.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Production of Mouse Monoclonal Antibodies to MCM2

[0054] Mouse monoclonal antibodies specific for MCM2 were generated. The antigen (an immunogenic polypeptide) was a full-length recombinant hexahistidine-tagged MCM2 protein. The antigen was expressed using a baculovirus expression system in Tni cells. Specifically, the coding sequence for the hexahistidine-tagged MCM2 (SEQ ID NO:10) was cloned into the pFastBac1 plasmid (Invitrogen) for expression in Tni cells. Methods for producing recombinant proteins using baculovirus expression systems are well known in the art. The tagged MCM2 protein was purified using a chelating agarose charged with Ni+2 ions (Ni-NTA from Qiagen) and used as an immunogen. The amino acid sequence of the immunogenic MCM2 polypeptide is provided in SEQ ID NO:11.

[0055] Mouse immunizations and hybridoma fusions were performed essentially as described in Kohler et al. (1975) Nature 256:495-496. Mice were immunized with the immunogenic tagged-MCM2 protein in solut...

example 2

Isolation of Monoclonal Antibodies from Hybridoma Cells

[0057] The following procedure is used to isolate monoclonal antibodies from hybridoma cells:

Media Preparation

[0058] To a sterile 1,000 ml storage bottle, add 100 ml Hyclone Fetal Bovine Serum (FBS). [0059] Add 10 ml of MEM Non-Essential Amino Acids Solution. [0060] Add 10 ml of Penicillin-Streptomycin-L-Glutamine Solution. [0061] QS to approximately 1000 ml with ExCell 610-HSF media. [0062] Place sterile cap on bottle and secure tightly. Swirl gently to mix. [0063] Connect a 1000 ml sterile acetate vacuum filter unit (0.2 μm) to a vacuum pump system. [0064] Gently pour approximately half of the media solution into sterile acetate vacuum filter unit and turn on the vacuum. [0065] Once the first half of the media has been filtered, pour the remaining media into the filter unit and continue filtering. [0066] After all the media has been filtered, disconnect the vacuum hose from the vacuum filter unit and turn off the vacuum pu...

example 3

General Method for Epitope Mapping

General Approach

[0164] Epitope mapping is performed to identify the linear amino acid sequence within an antigenic protein (i.e., the epitope) that is recognized by a particular monoclonal antibody. A general approach for epitope mapping requires the expression of the full-length protein, as well as various fragments (i.e., truncated forms) of the protein, generally in a heterologous expression system. These various recombinant proteins are then used to determine if the specific monoclonal antibody is capable of binding one or more of the truncated forms of the target protein. Through the use of reiterative truncation and the generation of recombinant proteins with overlapping amino acid regions, it is possible to identify the region that is recognized by the monoclonal antibody under investigation. Western blot analysis or ELISA is employed to determine if the specific monoclonal antibody under investigation is capable of binding one or more of...

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Abstract

Compositions and methods for diagnosing high-grade cervical disease in a patient sample are provided. The compositions include novel monoclonal antibodies, and variants and fragments thereof, that specifically bind to MCM2. Monoclonal antibodies having the binding characteristics of an MCM2 antibody of the invention are further provided. Hybridoma cell lines that produce an MCM2 monoclonal antibody of the invention are also disclosed herein. The compositions find use in practicing methods for diagnosing high-grade cervical disease comprising detecting overexpression of MCM2 in a cervical sample from a patient. Kits for practicing the methods of the invention are further provided. Polypeptides comprising the amino acid sequence for an MCM2 epitope and methods of using these polypeptides in the production of antibodies are also encompassed by the present invention.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application is a divisional of U.S. application Ser. No. 1 / 410,272, filed Apr. 24, 2006, which claims the benefit of U.S. Provisional Application Serial No. 60 / 675,305, filed Apr. 27, 2005, and U.S. Provisional Application Ser. No. 60 / 718,082, filed Sep. 16, 2005, all of which are incorporated herein by reference in their entirety.FIELD OF THE INVENTION [0002] The invention relates to antibodies capable of binding to MCM2 and methods of using these antibodies, particularly in the diagnosis of cervical disease. BACKGROUND OF THE INVENTION [0003] Carcinoma of the cervix is the second most common neoplasm in women, accounting for approximately 12% of all female cancers and causing approximately 250,000 deaths per year. Baldwin et al. (2003) Nature Reviews Cancer 3:1-10. In many developing countries where mass screening programs are not available, the clinical problem is more serious. Cervical cancer in these countries is the number on...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/574C12N5/06C07K16/30
CPCC07K14/4738G01N33/57411C07K16/3069C07K2317/34C07K16/18C07K16/00
Inventor MALINOWSKI, DOUGLAS P.FISCHER, TIMOTHY J.TAYLOR, ADRIANN J.
Owner TRIPATH IMAGING INC
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