title: Characterization of a monoclonal antibody with specificity for holo-transcobalamin.
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.
  
  
  CONCLUSIONS
  The identified antibody possesses a unique specificity for holotranscobalamin and can be used to develop a direct assay for the quantification of holotranscobalamin.
authors.affiliation: Axis-Shield AS, POB 206 Økern, N-0510 Oslo, Norway. lars.orning@no.axis-shield.com
authors.forename: Lars
authors.initials: L
authors.lastname: Orning
authors.forename: Anne
authors.initials: A
authors.lastname: Rian
authors.forename: Andrew
authors.initials: A
authors.lastname: Campbell
authors.forename: Jeff
authors.initials: J
authors.lastname: Brady
authors.forename: Sergey N
authors.initials: SN
authors.lastname: Fedosov
authors.forename: Birgit
authors.initials: B
authors.lastname: Bramlage
authors.forename: Keith
authors.initials: K
authors.lastname: Thompson
authors.forename: Edward V
authors.initials: EV
authors.lastname: Quadros