Calibrators, cobalamins

As mentioned above, the pool of cobalamins in a serum sample consists of several variants. The methyl- and 5 -adenosyl-cobalamins are Kght-sensitive and are converted into aquo-cobalamin when exposed to light. Aquo-cobala-min readily forms complexes with the side chain of the histidine amino add the aquo-group is replaced from the cobalt atom by the histidine side chain (Pratt 1972) and the cobalamin is associated with the denatured protein pellet. Addition of cyanide e.g. 20nmol/L KCN Raven et al. 1968) prevents this phenomenon as the cyanide ion rapidly displaces other Kgands on the cobalt atom (X on Figure 26.2). Therefore, cyanide is added during protein dena-turation to prevent precipitation of cobalamin with the protein debris. Simultaneously, addition of cyanide converts all cobalamins into cyanocobalamin. This ensures that the sample contains the same form of the vitamin as the form present in the calibrator of the assay. 

For most analytical processes, a measured signal of the sample is compared with the signals of calibrators in order to calculate the concentration present in the sample. An ideal calibrator contains a known concentration of the analyte and is by all means comparable to the sample to be analysed. As serum contains various protein bound cobalamins and cobalamin analogues in a very complex matrix, it is not possible to use an ideal calibrator when analysing cobalamin in serum. A suitable approximation is a solution of cyanocobalamin prepared as described in Box 26.1. For the final dilution of the calibrator, a buffer comparable to the one used for dilution of the samples to be analysed should be employed. 

The stock solution is to be used for preparation of calibrators of cyanocobalamin. A similar principle can be used for other forms of cobalamin. 

To prepare your calibrators, dilute accordingly. If the final concentration is below lOpmol/L, use of a buffer including cobalamin-free albumin (O.lmg/mL) is recommended. 

Labelled Cobalamin or Labelled Binding Protein. The first protein binding assays for cobalamin employed radiolabelled cobalamin and measured the amount of radioactive cobalamin bound when this label was added alone or in combination with calibrators or the sample to be tested (Rothenberg 1961). The amount of bound label decreased as the concentration of cobalamin in the cahbrator or sample increased. As an alternative to radioactive labels, the commercial companies have developed derivatives of cobalamin still able to bind to the binding protein but at the same time coupled to a property such as fluorescence (Abbott 2007 Bayer Diagnostics 2008). Another approach is to in-solubilize cobalamin and measure the amount of labelled binding protein not trapped on the in-solubilized cobalamin (Roche, 2008). 

Accuracy indicates the ability to measure the true value. The use of a set of calibrators with a traceable concentration is important to ensure accuracy. For cobalamin in serum, calibrators can be prepared from stock solutions of cyanocobalamin where the concentration is determined by spectrophotometry. 

In 2002, Sato and co-workers replaced IF with vitamin Bi2-targeting Lactobacillus helveticus B-1 in the vitamin Bn assay by CL method due to its cost and non-availability (Sato et al. 2002). Lactobacillus helveticus B-1 is assumed to have a vitamin Bn targeting (or binding) site on its cells and binds vitamin Bn instantly and quantitatively. This reaction is specific to complete vitamin Bn compounds, cobalamins, and was used for a vitamin Bn assay method by CL. The calibration graph was linear from 0.1 to 10 ng/mL. 

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