title: The selection and characterization of antibodies to minichromosome maintenance proteins that highlight cervical dysplasia.
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.
  
  
  CONCLUSIONS
  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.
authors: Becton Dickinson Diagnostics, Woman's Health and Cancer, 4025 Stirrup Creek, Durham, NC 27703, United States.
authors: Dorian
authors: D
authors: Henderson
authors: Laura
authors: L
authors: Hall
authors: Nikki
authors: N
authors: Prpic
authors: Jan
authors: J
authors: Hessling
authors: Margaret
authors: M
authors: Parker
authors: Susan
authors: S
authors: Sampson
authors: Stephen
authors: S
authors: Simkins
authors: George
authors: G
authors: Brough
authors: Eric
authors: E
authors: Dixon
authors: Karen
authors: K
authors: Lenz
authors: Steve
authors: S
authors: Knapp
authors: Patricia
authors: P
authors: Murphy
authors: Adriann
authors: A
authors: Taylor
authors: Tim
authors: T
authors: Fischer
authors: Douglas P
authors: DP
authors: Malinowski