Development of a Monoclonal Antibody Against Estrogen Quinone-Adducted Proteins as Potential Biomarkers of Breast Cancer Risk
Abstract
The purpose of this research is to develop a Specific monoclonal antibody (MAb) for intracellular detection and quantification of E2-3,4-Q adducted to proteins, as a potential biomarker of breast cancer risk. An established, protein-coupling chemistry was applied successfully to production of an immunogen, via linkage of E2-3,4-Q to cationized protein, through a short linker. A coating complex for indirect ELISA and a low molecular-weight conjugate for competitive ELISA were prepared by spontaneous adduction of E2-3,4-Q to an unmodified protein and a short-chain molecule with a primary amino group respectively. ELISA screening identified a hybridoma producing relatively large amounts of MAb with high signal-to- noise ratio. The affinity constant (0.5 x 10(exp 8) M(exp -1) for binding to E2-3,4-Q was determined by competitive ELISA. In specifically designed immunohistochemical (IHC)-staining protocols, the antibody produced intense and specific staining with no detectable cross-reactivity to E2-2,3-Q and little or no background. Failure to detect IHC staining in paraffin sections of ACI rat breast-tumors or human breast tumors is associated with loss of the epitope during microwave recovery or formalin fixation. Optimization of tissue fixation is required for validation studies that can be based on Quantitative Fluorescence Imaging Analysis or Surfaced-Enhanced Laser Desorption Ionization/tandem mass spectrometry.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jun 01, 2002
- Accession Number
- ADB282748
Entities
People
- George P. Casale
Organizations
- University of Nebraska Medical Center