1994

Objectives
 
 (1) To define the epitopes within TOP2A recognized by the mAb Clone SWT3D1

(2) To determine the affinity constants of the TOP2A mAb using TOP2A antigen and TOP2A peptide specific epitope

Background:

Topoisomerase II alpha is an important nuclear protein which controls DNA topology during DNA replication and chromosome separation and has been shown to be a useful biomarker that reflects cell cycle status. Over-expression of TOP2A occurs in many human cancers including cervical carcinoma and has been linked to the grade of cervical intraepithelial neoplasia (CIN) and high-risk human papillomavirus (Branca, Giorgi et al. 2006). We initiated characterization of TOP2A expression in cervical neoplasia using the TOP2A monoclonal antibody Clone SWT3D1 obtained from EMD Biosciences (San Diego, CA). Epitope mapping and affinity measurements of the TOP2A antibody Clone SWT3D1 are described in this study.

Methods:

Cervical neoplasia was obtained as either biopsy specimens or cytology specimens under IRB approval. Western blotting using recombinant truncated fragments of TOP2A identified the exact epitope recognized by Clone SWT3D1. All gene fragments were expressed using the Roche in vitro Rapid Translation System. The dissociation constants (Kd) were determined using a solution phase equilibrium system where free antibody concentration was determined by ELISA. Analysis of TOP2A expression in formalin fixed cervical tumor specimens and cervical cytology specimens were performed using standard immunochemistry methods.

Results: Over-expression of TOP2A was confirmed in cervical carcinoma specimens, high-grade CIN lesions and within atypical cells from cervical cytology specimens by immunochemistry methods. Over-expression of TOP2A was localized to the nucleus of cervical neoplastic cells. Western blot analysis of recombinant fragments of TOP2A indicated that the Clone SWT3D1 binds the C-terminal region of the TOP2A protein within the 11 amino acid sequence PRETEPRRAAT (amino acids 1312 to 1322). The Kd values for monoclonal antibody binding to the TOP2A antigen and the synthetic peptide epitope were 5.5 x 10-10 M and 3.5 x 10-10 M, respectively.

Conclusions: This monoclonal antibody Clone SWT3D1 is a sensitive and specific reagent for monitoring TOP2A over expression in human cervical cancer cells. The amino acid residues P1312 to T1322 represent the core epitope for detection of the TOP2A protein in both cervical cytology and cervical histology specimens.

Branca, M., C. Giorgi, et al. (2006). Int J Gynecol Pathol25(4): 383-92.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA