The selection and characterization of antibodies to minichromosome maintenance proteins that highlight cervical dysplasia
Screening efforts using the Papanicolaou test have significantly reduced the incidence of cervical cancer in countries with an active screening program. However, this test does not accurately identify all abnormal cases. Significant effort has been expended investigating molecular markers that could...
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| Published in | Journal of immunological methods Vol. 370; no. 1; pp. 1 - 13 |
|---|---|
| Main Authors | , , , , , , , , , , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
Amsterdam
Elsevier B.V
29.07.2011
Elsevier |
| Subjects | |
| Online Access | Get full text |
| ISSN | 0022-1759 1872-7905 1872-7905 |
| DOI | 10.1016/j.jim.2011.04.008 |
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| Abstract | Screening efforts using the Papanicolaou test have significantly reduced the incidence of cervical cancer in countries with an active screening program. However, this test does not accurately identify all abnormal cases. Significant effort has been expended investigating molecular markers that could improve the sensitivity and specificity of detection of high-grade disease. In this study, we describe the selection and characterization of a set of antibodies to the minichromosome maintenance proteins MCM6 and MCM7 that highlight cervical disease in an immunoassay.
Antibodies to MCM6 or MCM7 proteins were identified from hybridoma clones screened against tissue microarrays containing different grades of diseased cervical tissue along with normal controls. We determined epitopes by western blotting against nested truncations of either the MCM6 or MCM7 proteins fused to GFP protein. We also determined specificity by western blotting against a panel of major MCM proteins (MCM2-MCM8). Affinity to recombinant antigen and epitope-only peptides was determined using solution-phase binding and determination of free antibody concentration by ELISA. Optimization studies resulted in the selection of antibodies specific to MCM6 and MCM7 for use in immunocytochemistry (ICC) with cervical cytology samples.
Four antibodies were identified that demonstrated strong nuclear staining of abnormal cervical epithelial cells in immunohistochemistry (IHC) of cervical biopsies with minimal background staining of normal cervical tissues. Of these four antibody clones, 2E6.7 (MCM7) and 9D4.3 (MCM6) were chosen for further study. Linear epitopes of at most 12 amino acids were identified and verified by binding to epitope-only peptides. Affinities of at least 4
×
10
−
9
M were determined for these two antibodies and both were found to be specific for their respective antigens by western blotting. Clones 9D4.3 and 2E6.7 were also determined to stain abnormal cells in high-grade squamous intraepithelial lesion cytology samples, with minimal background staining of normal cells.
In this study, we present a method for selecting antibodies that perform well in IHC and ICC applications and characterize two antibodies generated by this method that effectively stain abnormal cells in cervical cancer tissue and cervical cytology samples.
► We present the development of anti-MCM6 and anti-MCM7 antibody clones. ► These MCM antibodies demonstrate robust performance in immunological assays. ► These antibodies stain abnormal cells in cervical cancer tissue and cytology samples. ► ICC analysis of cervical specimens holds promise for improving the sensitivity/specificity of the Pap test. |
|---|---|
| AbstractList | Screening efforts using the Papanicolaou test have significantly reduced the incidence of cervical cancer in countries with an active screening program. However, this test does not accurately identify all abnormal cases. Significant effort has been expended investigating molecular markers that could improve the sensitivity and specificity of detection of high-grade disease. In this study, we describe the selection and characterization of a set of antibodies to the minichromosome maintenance proteins MCM6 and MCM7 that highlight cervical disease in an immunoassay.BACKGROUNDScreening efforts using the Papanicolaou test have significantly reduced the incidence of cervical cancer in countries with an active screening program. However, this test does not accurately identify all abnormal cases. Significant effort has been expended investigating molecular markers that could improve the sensitivity and specificity of detection of high-grade disease. In this study, we describe the selection and characterization of a set of antibodies to the minichromosome maintenance proteins MCM6 and MCM7 that highlight cervical disease in an immunoassay.Antibodies to MCM6 or MCM7 proteins were identified from hybridoma clones screened against tissue microarrays containing different grades of diseased cervical tissue along with normal controls. We determined epitopes by western blotting against nested truncations of either the MCM6 or MCM7 proteins fused to GFP protein. We also determined specificity by western blotting against a panel of major MCM proteins (MCM2-MCM8). Affinity to recombinant antigen and epitope-only peptides was determined using solution-phase binding and determination of free antibody concentration by ELISA. Optimization studies resulted in the selection of antibodies specific to MCM6 and MCM7 for use in immunocytochemistry (ICC) with cervical cytology samples.METHODSAntibodies to MCM6 or MCM7 proteins were identified from hybridoma clones screened against tissue microarrays containing different grades of diseased cervical tissue along with normal controls. We determined epitopes by western blotting against nested truncations of either the MCM6 or MCM7 proteins fused to GFP protein. We also determined specificity by western blotting against a panel of major MCM proteins (MCM2-MCM8). Affinity to recombinant antigen and epitope-only peptides was determined using solution-phase binding and determination of free antibody concentration by ELISA. Optimization studies resulted in the selection of antibodies specific to MCM6 and MCM7 for use in immunocytochemistry (ICC) with cervical cytology samples.Four antibodies were identified that demonstrated strong nuclear staining of abnormal cervical epithelial cells in immunohistochemistry (IHC) of cervical biopsies with minimal background staining of normal cervical tissues. Of these four antibody clones, 2E6.7 (MCM7) and 9D4.3 (MCM6) were chosen for further study. Linear epitopes of at most 12 amino acids were identified and verified by binding to epitope-only peptides. Affinities of at least 4×10(-9) M were determined for these two antibodies and both were found to be specific for their respective antigens by western blotting. Clones 9D4.3 and 2E6.7 were also determined to stain abnormal cells in high-grade squamous intraepithelial lesion cytology samples, with minimal background staining of normal cells.RESULTSFour antibodies were identified that demonstrated strong nuclear staining of abnormal cervical epithelial cells in immunohistochemistry (IHC) of cervical biopsies with minimal background staining of normal cervical tissues. Of these four antibody clones, 2E6.7 (MCM7) and 9D4.3 (MCM6) were chosen for further study. Linear epitopes of at most 12 amino acids were identified and verified by binding to epitope-only peptides. Affinities of at least 4×10(-9) M were determined for these two antibodies and both were found to be specific for their respective antigens by western blotting. Clones 9D4.3 and 2E6.7 were also determined to stain abnormal cells in high-grade squamous intraepithelial lesion cytology samples, with minimal background staining of normal cells.In this study, we present a method for selecting antibodies that perform well in IHC and ICC applications and characterize two antibodies generated by this method that effectively stain abnormal cells in cervical cancer tissue and cervical cytology samples.CONCLUSIONIn this study, we present a method for selecting antibodies that perform well in IHC and ICC applications and characterize two antibodies generated by this method that effectively stain abnormal cells in cervical cancer tissue and cervical cytology samples. BACKGROUND: Screening efforts using the Papanicolaou test have significantly reduced the incidence of cervical cancer in countries with an active screening program. However, this test does not accurately identify all abnormal cases. Significant effort has been expended investigating molecular markers that could improve the sensitivity and specificity of detection of high-grade disease. In this study, we describe the selection and characterization of a set of antibodies to the minichromosome maintenance proteins MCM6 and MCM7 that highlight cervical disease in an immunoassay. METHODS: Antibodies to MCM6 or MCM7 proteins were identified from hybridoma clones screened against tissue microarrays containing different grades of diseased cervical tissue along with normal controls. We determined epitopes by western blotting against nested truncations of either the MCM6 or MCM7 proteins fused to GFP protein. We also determined specificity by western blotting against a panel of major MCM proteins (MCM2-MCM8). Affinity to recombinant antigen and epitope-only peptides was determined using solution-phase binding and determination of free antibody concentration by ELISA. Optimization studies resulted in the selection of antibodies specific to MCM6 and MCM7 for use in immunocytochemistry (ICC) with cervical cytology samples. RESULTS: Four antibodies were identified that demonstrated strong nuclear staining of abnormal cervical epithelial cells in immunohistochemistry (IHC) of cervical biopsies with minimal background staining of normal cervical tissues. Of these four antibody clones, 2E6.7 (MCM7) and 9D4.3 (MCM6) were chosen for further study. Linear epitopes of at most 12 amino acids were identified and verified by binding to epitope-only peptides. Affinities of at least 4×10⁻⁹ M were determined for these two antibodies and both were found to be specific for their respective antigens by western blotting. Clones 9D4.3 and 2E6.7 were also determined to stain abnormal cells in high-grade squamous intraepithelial lesion cytology samples, with minimal background staining of normal cells. CONCLUSION: In this study, we present a method for selecting antibodies that perform well in IHC and ICC applications and characterize two antibodies generated by this method that effectively stain abnormal cells in cervical cancer tissue and cervical cytology samples. Screening efforts using the Papanicolaou test have significantly reduced the incidence of cervical cancer in countries with an active screening program. However, this test does not accurately identify all abnormal cases. Significant effort has been expended investigating molecular markers that could improve the sensitivity and specificity of detection of high-grade disease. In this study, we describe the selection and characterization of a set of antibodies to the minichromosome maintenance proteins MCM6 and MCM7 that highlight cervical disease in an immunoassay. Antibodies to MCM6 or MCM7 proteins were identified from hybridoma clones screened against tissue microarrays containing different grades of diseased cervical tissue along with normal controls. We determined epitopes by western blotting against nested truncations of either the MCM6 or MCM7 proteins fused to GFP protein. We also determined specificity by western blotting against a panel of major MCM proteins (MCM2-MCM8). Affinity to recombinant antigen and epitope-only peptides was determined using solution-phase binding and determination of free antibody concentration by ELISA. Optimization studies resulted in the selection of antibodies specific to MCM6 and MCM7 for use in immunocytochemistry (ICC) with cervical cytology samples. Four antibodies were identified that demonstrated strong nuclear staining of abnormal cervical epithelial cells in immunohistochemistry (IHC) of cervical biopsies with minimal background staining of normal cervical tissues. Of these four antibody clones, 2E6.7 (MCM7) and 9D4.3 (MCM6) were chosen for further study. Linear epitopes of at most 12 amino acids were identified and verified by binding to epitope-only peptides. Affinities of at least 4×10(-9) M were determined for these two antibodies and both were found to be specific for their respective antigens by western blotting. Clones 9D4.3 and 2E6.7 were also determined to stain abnormal cells in high-grade squamous intraepithelial lesion cytology samples, with minimal background staining of normal cells. In this study, we present a method for selecting antibodies that perform well in IHC and ICC applications and characterize two antibodies generated by this method that effectively stain abnormal cells in cervical cancer tissue and cervical cytology samples. Screening efforts using the Papanicolaou test have significantly reduced the incidence of cervical cancer in countries with an active screening program. However, this test does not accurately identify all abnormal cases. Significant effort has been expended investigating molecular markers that could improve the sensitivity and specificity of detection of high-grade disease. In this study, we describe the selection and characterization of a set of antibodies to the minichromosome maintenance proteins MCM6 and MCM7 that highlight cervical disease in an immunoassay. Antibodies to MCM6 or MCM7 proteins were identified from hybridoma clones screened against tissue microarrays containing different grades of diseased cervical tissue along with normal controls. We determined epitopes by western blotting against nested truncations of either the MCM6 or MCM7 proteins fused to GFP protein. We also determined specificity by western blotting against a panel of major MCM proteins (MCM2-MCM8). Affinity to recombinant antigen and epitope-only peptides was determined using solution-phase binding and determination of free antibody concentration by ELISA. Optimization studies resulted in the selection of antibodies specific to MCM6 and MCM7 for use in immunocytochemistry (ICC) with cervical cytology samples. Four antibodies were identified that demonstrated strong nuclear staining of abnormal cervical epithelial cells in immunohistochemistry (IHC) of cervical biopsies with minimal background staining of normal cervical tissues. Of these four antibody clones, 2E6.7 (MCM7) and 9D4.3 (MCM6) were chosen for further study. Linear epitopes of at most 12 amino acids were identified and verified by binding to epitope-only peptides. Affinities of at least 410-9 M were determined for these two antibodies and both were found to be specific for their respective antigens by western blotting. Clones 9D4.3 and 2E6.7 were also determined to stain abnormal cells in high-grade squamous intraepithelial lesion cytology samples, with minimal background staining of normal cells. In this study, we present a method for selecting antibodies that perform well in IHC and ICC applications and characterize two antibodies generated by this method that effectively stain abnormal cells in cervical cancer tissue and cervical cytology samples. Screening efforts using the Papanicolaou test have significantly reduced the incidence of cervical cancer in countries with an active screening program. However, this test does not accurately identify all abnormal cases. Significant effort has been expended investigating molecular markers that could improve the sensitivity and specificity of detection of high-grade disease. In this study, we describe the selection and characterization of a set of antibodies to the minichromosome maintenance proteins MCM6 and MCM7 that highlight cervical disease in an immunoassay. Antibodies to MCM6 or MCM7 proteins were identified from hybridoma clones screened against tissue microarrays containing different grades of diseased cervical tissue along with normal controls. We determined epitopes by western blotting against nested truncations of either the MCM6 or MCM7 proteins fused to GFP protein. We also determined specificity by western blotting against a panel of major MCM proteins (MCM2-MCM8). Affinity to recombinant antigen and epitope-only peptides was determined using solution-phase binding and determination of free antibody concentration by ELISA. Optimization studies resulted in the selection of antibodies specific to MCM6 and MCM7 for use in immunocytochemistry (ICC) with cervical cytology samples. Four antibodies were identified that demonstrated strong nuclear staining of abnormal cervical epithelial cells in immunohistochemistry (IHC) of cervical biopsies with minimal background staining of normal cervical tissues. Of these four antibody clones, 2E6.7 (MCM7) and 9D4.3 (MCM6) were chosen for further study. Linear epitopes of at most 12 amino acids were identified and verified by binding to epitope-only peptides. Affinities of at least 4 × 10 − 9 M were determined for these two antibodies and both were found to be specific for their respective antigens by western blotting. Clones 9D4.3 and 2E6.7 were also determined to stain abnormal cells in high-grade squamous intraepithelial lesion cytology samples, with minimal background staining of normal cells. In this study, we present a method for selecting antibodies that perform well in IHC and ICC applications and characterize two antibodies generated by this method that effectively stain abnormal cells in cervical cancer tissue and cervical cytology samples. ► We present the development of anti-MCM6 and anti-MCM7 antibody clones. ► These MCM antibodies demonstrate robust performance in immunological assays. ► These antibodies stain abnormal cells in cervical cancer tissue and cytology samples. ► ICC analysis of cervical specimens holds promise for improving the sensitivity/specificity of the Pap test. |
| Author | Hessling, Jan Parker, Margaret Simkins, Stephen Henderson, Dorian Sampson, Susan Fischer, Tim Hall, Laura Knapp, Steve Taylor, Adriann Dixon, Eric Malinowski, Douglas P. Brough, George Lenz, Karen Prpic, Nikki Murphy, Patricia |
| Author_xml | – sequence: 1 givenname: Dorian surname: Henderson fullname: Henderson, Dorian organization: Becton Dickinson Diagnostics, Woman's Health and Cancer, 4025 Stirrup Creek, Durham, NC, 27703, United States – sequence: 2 givenname: Laura surname: Hall fullname: Hall, Laura organization: Becton Dickinson Diagnostics, Woman's Health and Cancer, 4025 Stirrup Creek, Durham, NC, 27703, United States – sequence: 3 givenname: Nikki surname: Prpic fullname: Prpic, Nikki organization: Becton Dickinson Diagnostics, Woman's Health and Cancer, 4025 Stirrup Creek, Durham, NC, 27703, United States – sequence: 4 givenname: Jan surname: Hessling fullname: Hessling, Jan organization: Carilion-Spectrum, 4380 Federal Drive, Suite 100, Greensboro, NC, 27410, United States – sequence: 5 givenname: Margaret surname: Parker fullname: Parker, Margaret organization: Becton Dickinson Diagnostics, Woman's Health and Cancer, 4025 Stirrup Creek, Durham, NC, 27703, United States – sequence: 6 givenname: Susan surname: Sampson fullname: Sampson, Susan organization: Becton Dickinson Diagnostics, Woman's Health and Cancer, 4025 Stirrup Creek, Durham, NC, 27703, United States – sequence: 7 givenname: Stephen surname: Simkins fullname: Simkins, Stephen organization: Becton Dickinson Diagnostics, Woman's Health and Cancer, 4025 Stirrup Creek, Durham, NC, 27703, United States – sequence: 8 givenname: George surname: Brough fullname: Brough, George organization: Becton Dickinson Diagnostics, Woman's Health and Cancer, 4025 Stirrup Creek, Durham, NC, 27703, United States – sequence: 9 givenname: Eric surname: Dixon fullname: Dixon, Eric organization: Becton Dickinson Diagnostics, Woman's Health and Cancer, 4025 Stirrup Creek, Durham, NC, 27703, United States – sequence: 10 givenname: Karen surname: Lenz fullname: Lenz, Karen organization: Becton Dickinson Diagnostics, Woman's Health and Cancer, 4025 Stirrup Creek, Durham, NC, 27703, United States – sequence: 11 givenname: Steve surname: Knapp fullname: Knapp, Steve organization: Becton Dickinson Diagnostics, Woman's Health and Cancer, 4025 Stirrup Creek, Durham, NC, 27703, United States – sequence: 12 givenname: Patricia surname: Murphy fullname: Murphy, Patricia organization: Becton Dickinson Diagnostics, Woman's Health and Cancer, 4025 Stirrup Creek, Durham, NC, 27703, United States – sequence: 13 givenname: Adriann surname: Taylor fullname: Taylor, Adriann organization: Becton Dickinson Diagnostics, Woman's Health and Cancer, 4025 Stirrup Creek, Durham, NC, 27703, United States – sequence: 14 givenname: Tim surname: Fischer fullname: Fischer, Tim organization: Becton Dickinson Diagnostics, Woman's Health and Cancer, 4025 Stirrup Creek, Durham, NC, 27703, United States – sequence: 15 givenname: Douglas P. surname: Malinowski fullname: Malinowski, Douglas P. email: doug_malinowski@bd.com organization: Becton Dickinson Diagnostics, Woman's Health and Cancer, 4025 Stirrup Creek, Durham, NC, 27703, United States |
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| Keywords | ASC-US Pap test SurePath IHC Immunocytochemistry Minichromosome maintenance protein ICC LSIL TMA HSIL HPV ASC-H MCM Diagnostic epitopes NILM RIMMS Cervical cancer Premalignant lesion Antigenic determinant Antibody Malignant tumor Protein Female genital diseases Immunological method Cervical dysplasia Minichromosome Uterine cervix diseases Cancer |
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| Snippet | Screening efforts using the Papanicolaou test have significantly reduced the incidence of cervical cancer in countries with an active screening program.... BACKGROUND: Screening efforts using the Papanicolaou test have significantly reduced the incidence of cervical cancer in countries with an active screening... |
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| SubjectTerms | Amino Acid Sequence amino acids antibodies Antibodies, Monoclonal - analysis Antibodies, Monoclonal - immunology Antibody Specificity Biological and medical sciences Biopsy Blotting, Western - methods Cell Cycle Proteins - analysis Cell Cycle Proteins - immunology Cervical cancer clones Diagnostic epitopes DNA-Binding Proteins - analysis DNA-Binding Proteins - immunology Enzyme-Linked Immunosorbent Assay epithelial cells Epitope Mapping - methods epitopes Epitopes - analysis Epitopes - chemistry Epitopes - immunology Female Fundamental and applied biological sciences. Psychology Fundamental immunology genetic markers Humans hybridomas Immunocytochemistry immunohistochemistry Immunohistochemistry - methods microarray technology Minichromosome Maintenance Complex Component 6 Minichromosome Maintenance Complex Component 7 Minichromosome maintenance protein Molecular immunology Molecular Sequence Data Nuclear Proteins - analysis Nuclear Proteins - immunology Pap test peptides proteins screening SurePath Techniques tissues Uterine Cervical Dysplasia - chemistry Uterine Cervical Dysplasia - immunology Uterine Cervical Dysplasia - pathology uterine cervical neoplasms Western blotting |
| Title | The selection and characterization of antibodies to minichromosome maintenance proteins that highlight cervical dysplasia |
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