ABSTRACT
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.
Subject(s)
Antibodies, Monoclonal/analysis , Blotting, Western/methods , Cell Cycle Proteins/immunology , DNA-Binding Proteins/immunology , Epitope Mapping/methods , Immunohistochemistry/methods , Nuclear Proteins/immunology , Uterine Cervical Dysplasia/chemistry , Amino Acid Sequence , Antibodies, Monoclonal/immunology , Antibody Specificity , Biopsy , Cell Cycle Proteins/analysis , DNA-Binding Proteins/analysis , Enzyme-Linked Immunosorbent Assay , Epitopes/analysis , Epitopes/chemistry , Epitopes/immunology , Female , Humans , Minichromosome Maintenance Complex Component 6 , Minichromosome Maintenance Complex Component 7 , Molecular Sequence Data , Nuclear Proteins/analysis , Uterine Cervical Dysplasia/immunology , Uterine Cervical Dysplasia/pathologyABSTRACT
The objective of this longitudinal study was to determine the feasibility of a model for disseminating community-based strength training programs for older adults through leadership training of laypersons or "peers" and health and fitness professionals. The intervention consisted of a progressive strength training, balance, and flexibility exercise program and a leader training and certification workshop. Feasibility was defined as 75% or more of individuals who completed leader training establishing or teaching at least two 12-week strength training classes within 1 year. Program dissemination was quantified as the number of classes established between January 2005 and December 2006. Demographic characteristics and health status of leaders and program participants were evaluated. Two hundred forty-four leaders (peers, n=149; professionals, n=95) were trained and certified. Seventy-nine percent of all leaders (n=193) met the feasibility criteria of establishing or teaching strength training classes. There was no difference in the percentage of peer leaders (80%, n=119) and professional leaders (78%, n=74) who established or taught classes (P5.71) despite significant differences in their demographic and health profiles. Ninety-seven self-sustaining strength training classes were established in senior and community centers, and 2,217 older adults (women, n=1,942; men, n=275) aged 50 to 97 with multiple chronic medical conditions enrolled. In conclusion, training peer and professional leaders is a feasible and successful model for disseminating a community strength training program for older adults. Widespread dissemination of this program has significant public health implications for increasing physical activity participation by older adults.