Research

Characterization of a monoclonal antibody with specificity for holo-transcobalamin

Lars Orning¹ , Anne Rian¹ , Andrew Campbell¹ , Jeff Brady¹ , Sergey N Fedosov² , Birgit Bramlage^1,5 , Keith Thompson³ and Edward V Quadros⁴

¹Axis-Shield AS, POB 206 Økern, N-0510 Oslo, Norway

²Protein Chemistry Laboratory, Department of Molecular and Structural Biology, University of Aarhus, Science Park, Gustav Wieds Vej 10, 8000 Aarhus

³Institute of Immunology, Rikshospitalet University Hospital, University of Oslo, N-0027 Oslo, Norway

⁴Division of Hematology/Oncology, State University of New York, Health Science Center, Brooklyn, NY 11203, USA

⁵Alnylam Europe AG, Fritz-Hornschuch-Str. 9, 95326 Kulmbach, Germany

author email corresponding author email

Nutrition & Metabolism 2006, 3:3doi:10.1186/1743-7075-3-3

Published:	4 January 2006

Abstract

Background

Holotranscobalamin, cobalamin-saturated transcobalamin, is the minor fraction of circulating cobalamin (vitamin B12), which is available for cellular uptake and hence is physiologically relevant. Currently, no method allows simple, direct quantification of holotranscobalamin. We now report on the identification and characterization of a monoclonal antibody with a unique specificity for holotranscobalamin.

Methods

The specificity and affinity of the monoclonal antibodies were determined using surface plasmon resonance and recombinant transcobalamin as well as by immobilizing the antibodies on magnetic microspheres and using native transcobalamin in serum. The epitope of the holotranscobalamin specific antibody was identified using phage display and comparison to a de novo generated three-dimensional model of transcobalamin using the program Rosetta. A direct assay for holotrnscobalamin in the ELISA format was developed using the specific antibody and compared to the commercial assay HoloTC RIA.

Results

An antibody exhibiting >100-fold specificity for holotranscobalamin over apotranscobalamin was identified. The affinity but not the specificity varied inversely with ionic strength and pH, indicating importance of electrostatic interactions. The epitope was discontinuous and epitope mapping of the antibody by phage display identified two similar motifs with no direct sequence similarity to transcobalamin. A comparison of the motifs with a de novo generated three-dimensional model of transcobalamin identified two structures in the N-terminal part of transcobalamin that resembled the motif. Using this antibody an ELISA based prototype assay was developed and compared to the only available commercial assay for measuring holotranscobalamin, HoloTC RIA.

Conclusion

The identified antibody possesses a unique specificity for holotranscobalamin and can be used to develop a direct assay for the quantification of holotranscobalamin.