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Expression and epitope characterization of a recombinant CA 125 repeat: fourth report from the ISOBM TD-1 workshop
David J Warren1, Kjell Nustad, John B Beard
1Department of Medical Biochemistry at Radiumhospitalet, Rikshospitalet Medical Centre, Oslo, Norway. david.warren@radiumhospitalet.no
Background:
CA 125 antigenic domains appear to reside within a region containing 156-amino acid sequence repeats. Surprisingly, anti-CA 125 antibodies can be classified into three families (groups A, B and C) indicating limited epitope diversity. In this study we describe the heterologous expression of a CA 125 repeat unit (R11) and an analysis of its epitope topography.
Methods:
R11 was expressed using a baculovirus approach and purified from culture supernatants by sequential ion exchange chromatography. Monoclonal antibody binding was assessed using antigen capture and cross-inhibition methods.
Results:
The recombinant repeat was purified to 2.5 x 10(7) U/mg. Although a number of group A and B monoclonal antibodies were found to bind R11, the prototype antibody OC125 (group A) showed little reactivity. However, the prior binding of some group B monoclonal antibodies dramatically enhanced subsequent OC125 binding. Low monoclonal antibody reactivity to R11 correlated well with poor binding to SDS-denatured human ascites CA 125.
Conclusion:
The ability to 'activate' R11 epitopes indicates that some may not be displayed optimally on isolated repeats. This observation, together with the concordance between monoclonal antibody binding to R11 and denatured CA 125, suggests that a number of epitopes are preferentially displayed only when contained within multiple repeat domains.
