VITAMIN B12 ASSAY

摘要

A competitive binding assay for vitamin B12 in a test sample gives a steep dose-response curve in the clinically significant concentration regions, yielding great accuracy. The assay uses immobilized vitamin B12 to compete with the vitamin B12 in the test sample for a binding partner. The quantity of the binding partner bound to the immobilized vitamin B12 can be measured and is inversely proportional to the concentration of free vitamin B12 in the test sample. The binding partner is typically biotinylated intrinsic factor prepared so that substantially all the molecules of intrinsic factor are biotinylated and the binding activity of the intrinsic factor for vitamin B12 is not diminished. The bound biotinylated intrinsic factor can then be determined by reacting it with avidin coupled to a reporter group. The reporter group is typically an enzyme such as horseradish peroxidase, but can be another detectable label. The immobilized vitamin B12 is typically bound to a solid support such as diazotized amine-derivatized beads, and can be bound through a protein linker. Other binding partners such as anti-vitamin B12 antibody are also usable. The binding partner can be labeled directly with the reporter group. The method is adaptable to the assay of serum, and methods of inactivating endogenous vitamin B12-binding proteins in serum are described.