Enzymatic extraction of cobalamin from monoclonal antibody captured haptocorrin and transcobalamin.

Abstract:

OBJECTIVES:Current extraction methods for cobalamins from serum influence the molecular characteristics of the vitamin. Therefore, an extraction procedure that leaves the cobalamins unchanged is needed. DESIGN AND METHODS:Monoclonal antibodies towards transcobalamin (TC) and haptocorrin (HC) (in house) linked to magnetic microspheres were used for precipitation of the proteins. The cobalamins were subsequently released by degradation of TC or HC with subtilisin Carlsberg (EC.3.4.21.14). RESULTS:Recovery for the extraction of (57)Co-cyanocobalamin ((57)Co-hydroxycobalamin) was higher than 99 (93)% and 92 (93)% from TC and HC respectively (n=10 (3)). No change in retention time and recovery was observed for (57)Co-hydroxycobalamin analyzed with HPLC before and after extraction with subtilisin Carlsberg. Endogenous cobalamin in serum was extracted from TC and HC and measured with routine methods. Enzymatic extracted cobalamin constituted 118-187% of that measured in serum with routine methods (n=16). CONCLUSION:We present a method that allows extraction of cobalamins from their binding proteins without modifying the vitamin. The procedure may be useful especially for extractions aiming at characterizing the cobalamins bound to individual cobalamin binding proteins.

journal_name

Clin Biochem

journal_title

Clinical biochemistry

authors

Hardlei TF,Morkbak AL,Nexo E

doi

10.1016/j.clinbiochem.2007.09.008

subject

Has Abstract

pub_date

2007-12-01 00:00:00

pages

1392-7

issue

18

eissn

0009-9120

issn

1873-2933

pii

S0009-9120(07)00365-7

journal_volume

40

pub_type

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