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1.
J Immunol Methods ; 442: 35-41, 2017 03.
Article in English | MEDLINE | ID: mdl-28093271

ABSTRACT

BACKGROUND: The Papanicolaou (Pap) screen has been successful in reducing cervical cancer; but exhibits low sensitivity when detecting cervical dysplasia. Use of molecular biomarkers in Pap tests may improve diagnostic accuracy. DESIGN: Monoclonal antibodies to Minichromosome Maintenance Protein 2 (MCM2) and DNA Topoisomerase II α (TOP2A) were selected for use in IHC based on their ability to differentiate normal from diseased cervical tissues in tissue microarrays. Enhanced Green Fluorescent Protein Western blot analysis was used to help identify binding epitopes specific to MCM2 and TOP2A antibody clones. Antibody affinity was determined by solution phase affinity measurement and immunohistochemistry was performed using high affinity MCM2 or TOP2A antibodies on serial histological sections. RESULTS: Antibody clones to MCM2 and TOP2A clones were selected based on their ability to detect over expression in abnormal cervical epithelia. In IHC, MCM2-27C5.6 and MCM2-26H6.19 demonstrated superior staining in abnormal cervical tissue over the MCM2-CRCT2.1 antibody. A combination of MCM2 and TOP2A antibodies showed greater staining when compared to staining with any of the antibodies alone on serial histological sections. Distinct linear epitopes were elucidated for each of the MCM2 and TOP2A clones. Affinity values (Kd) for MCM2 or TOP2A antibodies had a similar range. In a research study, the MCM2 and TOP2A (BD ProEx™ C) antibody cocktail showed increased epithelia staining with increasing dysplasia. The use of BD ProEx™ C in combination with H&E staining enhanced immunohistochemical discrimination of dysplastic and non-dysplastic FFPE cervical tissue specimens. CONCLUSIONS: BD ProEx™ C containing MCM2 and TOP2A antibodies showed strong specific nuclear staining that correlated with increased dysplasia and lesion severity. Enhanced performance of the antibodies was linked to their unique topography recognition. BD ProEx™ C incorporates antibodies that enhance detection of CIN2+ cervical disease.


Subject(s)
Antibodies, Monoclonal/immunology , Antigens, Neoplasm/immunology , Cervix Uteri/immunology , DNA Topoisomerases, Type II/immunology , DNA-Binding Proteins/immunology , Immunohistochemistry , Minichromosome Maintenance Complex Component 2/immunology , S Phase , Tissue Array Analysis/methods , Uterine Cervical Dysplasia/diagnosis , Uterine Cervical Neoplasms/diagnosis , Biopsy , Blotting, Western , Cell Nucleus/enzymology , Cell Nucleus/immunology , Cell Nucleus/pathology , Cervix Uteri/enzymology , Cervix Uteri/pathology , Epitope Mapping/methods , Epitopes , Female , Humans , Poly-ADP-Ribose Binding Proteins , Predictive Value of Tests , Severity of Illness Index , Uterine Cervical Dysplasia/enzymology , Uterine Cervical Dysplasia/immunology , Uterine Cervical Dysplasia/pathology , Uterine Cervical Neoplasms/enzymology , Uterine Cervical Neoplasms/immunology , Uterine Cervical Neoplasms/pathology
2.
Hybridoma (Larchmt) ; 30(5): 433-43, 2011 Oct.
Article in English | MEDLINE | ID: mdl-22008070

ABSTRACT

The chromosomal translocation t(8;21) often found in acute myeloid leukemia generates an oncogenic fusion protein AML1-ETO. This chimeric oncoprotein disrupts wild-type AML1 function and dysregulates genes important for normal myelopoiesis. Monoclonal antibodies that can capture and detect the AML1-ETO fusion protein would help with early diagnosis and treatment prognosis of acute myeloid leukemia. We report the development of murine monoclonal antibodies (MAbs) that specifically bind epitopes encoded by either AML1 or ETO. Since alignment to the human ETO protein indicated almost 100% homology to the mouse ortholog, a strategy was needed to instruct humoral immunity in mice to focus and respond to self-epitopes. Our strategy to develop capture/detector reagents involved producing MAbs that would bind to epitopes within the non-fused myelopic protein (i.e., either AML1 or ETO). This included a process to select antibodies for their ability to also recognize the translocated chromosomal AML1-ETO fusion protein and to identify complementary capture/detector antibody pairs. Construction of a peptide hapten-carrier complex and use of a rapid immunization protocol resulted in IgM-IgG ETO specific MAbs. These MAbs bound specifically to a recombinant form of AML1-ETO fusion protein expressed in HEK and to an endogenous AML1-ETO form of the fusion protein expressed in Kasumi-1. We report the development of murine hybridoma MAbs derived from immunizations with a peptide "self-epitope." Our findings provide a potential strategy to instruct humoral immunity in mice to focus and respond to self-epitopes. This strategy has been validated with the oncogenic fusion protein AML1-ETO involved in acute myeloid leukemia.


Subject(s)
Antibodies, Monoclonal, Murine-Derived/biosynthesis , Core Binding Factor Alpha 2 Subunit/immunology , Proto-Oncogene Proteins/immunology , Recombinant Fusion Proteins/immunology , Transcription Factors/immunology , Amino Acid Sequence , Animals , Antibody Affinity , Antibody Specificity , Cloning, Molecular , Core Binding Factor Alpha 2 Subunit/isolation & purification , Enzyme-Linked Immunosorbent Assay , Female , HEK293 Cells , Humans , Hybridomas/metabolism , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Proto-Oncogene Proteins/isolation & purification , RUNX1 Translocation Partner 1 Protein , Recombinant Fusion Proteins/isolation & purification , Sequence Homology, Amino Acid , Transcription Factors/isolation & purification
3.
J Clin Virol ; 48(1): 31-5, 2010 May.
Article in English | MEDLINE | ID: mdl-20233675

ABSTRACT

BACKGROUND: The AMPLICOR HPV Test has been validated for use with cervical cells collected in liquid-based preservative fluids, such as BD SurePath. It is currently recommended, however, that residual BD SurePath samples be stored at 4 degrees C prior to testing. OBJECTIVES: The aim of this study was to demonstrate that DNA isolated from SurePath cervical cytology specimens and stored at ambient temperature was also compatible with the AMPLICOR HPV Test. STUDY DESIGN: DNA was extracted using the AmpliLute Media Sample Preparation Kit. Amplification and detection of HPV was performed both as directed by the manufacturer and with minor protocol modifications. RESULTS: Cervical specimens collected in SurePath preservative fluid remained stable for testing with the AMPLICOR HPV Test for at least 21 days. The performance of DNA extracted from specimens stored at room temperature was equivalent to DNA extracted from specimens stored at 4 degrees C. The beta-globin internal control was detected in all of the 146 residual SurePath cervical cytology specimens tested using the AMPLICOR HPV Test, and high-risk HPV was detected in 46.2% (18/39) of ASCUS cases, in 63.3% (19/30) of LSIL cytology specimens, and 92.3% (24/26) of HSIL cases. Concordance of AMPLICOR HPV Test results with Hybrid Capture II was 83.9%. CONCLUSIONS: The AMPLICOR HPV Test can be successfully and reproducibly performed from DNA isolated from residual SurePath cervical cytology specimens stored at ambient temperature for at least 21 days. This provides clinical laboratories flexible storage conditions for residual SurePath cytology specimens.


Subject(s)
Cervix Uteri/virology , DNA, Viral/isolation & purification , Nucleic Acid Amplification Techniques/methods , Papillomaviridae/genetics , Specimen Handling/methods , Virology/methods , Female , Humans , Reagent Kits, Diagnostic , Temperature , Vaginal Smears
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