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

Brady, Jeff

Bramlage, Birgit

Campbell, Andrew

Fedosov, Sergey N

Orning, Lars

Quadros, Edward V

Rian, Anne

Thompson, Keith

English

PubMed

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

NORA - Norwegian Open Research Archives

Characterization of a monoclonal antibody with specificity for holo-transcobalamin

Springer - Publisher Connector

Changes in Stokes radius on binding of vitamin B12 to human intrinsic factor and transcobalamins. Biochim Biophys Acta

Characterization of monoclonal antibodies to epitopes of human transcobalamin II.

Cobalamin binding proteins.

Depletion of serum holo-transcobalamin II; an early sign of negative vitamin B12 balance. Lab Invest

Epitope mapping using phage-displayed peptide libraries.

Fedosov SN: Quantification of holotranscobalamin, a marker of vitamin B12 deficiency. Clin Chem

Free modelling with Rosetta

Mayatepek E, Kölker S: Neurodegeneratiobn in methylmalonic aciduria involves inhibition of complex II and the tricarboxylic acid cycle, and synergistically acting excitotoxicity.

Measuring and interpreting holo-transcobalamin (holotranscobalamin II). Clin Chem

Modeling structurally variable regions in homologous proteins with rosetta. Proteins

OK: Holo-transcobalamin is an early marker 22 of changes in cobalamin homeostasis; a randomized placebo-controlled study. Clin Chem

Phage display method. In Real-time analysis of biomolecular interactions.

Prusoff WH: Relationship between the inhibition constant (KI) and the concentration of an inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol

Sali A: Protein structure predictions and structural genomics. Science

Seetharam B: Cobalamin (vitamin B12) binding, phylogeny, and synteny of human transcobalamin. Arch Biochem Biophys

SP: Transcobalamin II synthesized in the intestinal villi facilitates transfer of cobalamin to the portal blood.

Sundrehagen E, Orning L: Direct assay for cobalamin bound to transcobalamin (holotranscobalamin) in serum. Clin Chem

TE: Assembly of the intrinsic factor domains and oligomerization of the protein in the presence of cobalamin. Biochemistry

TE: Composite organization of the cobalamin binding and cubilin recognition sites of intrinsic factor. Biochemistry

TE: Conformational changes of transcobalamin induced by aquocobalamin binding: mechanism of substitution of the cobalt-coordinated group in the bound ligand.

TE: Mapping the functional domains of human transcobalamin using monoclonal antibodies.

The binding properties of the human receptor for the cellular uptake of vitamin B12. Biochem Biophys Res Commun

Update on cobalamin, folate, and homocysteine. Hematology (Am Soc Hematol Educ Program)

Vitamin B12 &quot;Cobalamin&quot;. In

file:///data/core-remote/dit/data/Springer-OA/pdf/078/aHR0cDovL2xpbmsuc3ByaW5nZXIuY29tLzEwLjExODYvMTc0My03MDc1LTMtMy5wZGY=.pdf

Characterization of a monoclonal antibody with specificity for holo-transcobalamin

Abstract

Similar works

Full text

Available Versions

NORA - Norwegian Open Research Archives

Springer - Publisher Connector