Binding assays cobalamins

Co] Cobalamin (known quantity) FIGURE 9.23 Competitive binding assay. 

Protein binding assays for cobalamin analysis were first suggested in the 1960s (Rothenberg 1961). The principle is based on two main steps ... 

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). 

Figure 26.4 Automatic measurement of cobalamin (Cbl) in human serum samples on platforms design of the methods. All three methods employ protein binding assays using intrinsic factor (IF) but differ in the detailed assay design. See the main text for details. The figure is published with permission from Clinical Chemistry Laboratory Medicine (Lildballe et al. 2009).

We chose the method developed by Abbott as an example of automatic protein binding assays for analysis of serum cobalamin. Minor variations occur for other automatic platforms. 

Measurement of serum cobalamin includes release of cobalamin from its binding proteins transcobalamin and haptocorrin, conversion of the various forms of cobalamin into one form of the vitamin, and quantification employing microbiological or protein binding assays. 

Standardized protein binding assays have been developed for measurement of total cobalamin in serum. Further studies are required to optimize assays of cobalamin in other matrixes than serum and in order to explore the use of measurement of sub-fractions of cobalamin such as holoTC. 

Radioisotope dilution assays are based on the principle of competition between radioactive labeled ( Co) vitamin B 2 and cobalamins extracted from matrices for binding sites on the intrinsic factor (a glycoprotein). Binding is in proportion to the concentration of the radioactive and nonradio active B 2 with the concentration of intrinsic factor as the limiting factor. Free cobalamins are separated from those bound on the intrinsic factor by absorption... 

In quantitative immunoassays, e.g., enzyme-linked immunosorbent assay and radioimmunoassay, a known amount of labeled vitamin is mixed with sample extract in which the vitamin content should be determined. Methods of labeling include radioisotopes (e.g., cobalamine), fluorescence, or luminescence markers (e.g., folate). The mixture is subjected to binding agent, equally forming complexes with both labeled and unlabeled vitamin. This complex is then isolated, and the amount of labeled vitamin present is measured. Sample vitamin concentration can be deduced from the ratio of labeled vitamin added to labeled vitamin measured after isolation. Advantages of immunoassays are short analysis time, and the possibility of automating them on clinical analyzers. 

Major vitamin Bi2-dependent metabolic processes include the formation of methionine from homocysteine, and the formation of succinyl coenzyme A from methylmalonyl coenzyme A. Thus, apart from directly determining vitamin B12 concentration in serum, elevated levels of both methylmalonic acid and homocysteine may indicate a vitamin B12 deficiency. Serum cobalamine concentration is often determined by automated immunoassays using an intrinsic factor as binding agent. These assays have mainly replaced the microbiological methods. Literature data about vitamin B12 concentration in serum varies. Values <110-150pmoll are considered to reflect deficiency, whereas values >150-200pmoll represents an adequate status. 

To our knowledge, all commercially available assays today employ intrinsic factor as the binding protein. The challenge with this is that the most commonly used source of intrinsic factor is the porcine stomach, a source that contains a substantial amount of haptocorrin in addition to intrinsic factor. Because of this, care is needed to remove haptocorrin prior to using porcine gastric intrinsic factor. It seems obvious that, in order to standardize assays for cobalamin across the analytical platforms, the use of recombinant intrinsic factor is relevant. Recombinant human intrinsic factor is available and has... 

Bayer Diagnostics 2008 Roche 2008). One way to do this is to couple the binding protein covalently to solid-phase particles in the form of paramagnetic beads. Another approach is to use biotinylated cobalamin as the competitive agent after binding to the protein, biotinylated cobalamin is precipitated onto streptavidin linked to solid-phase particles. In either case, the protein-cobalamin equilibrium may be disturbed in washing steps, and leakage from the solid phase may also be an issue to consider especially if these analytical principles are used in in-house assays. 

All three assays use extraction at high pH and include cyanide. All assays use chemiluminescence for the final detection and all three assays employ intrinsic factor as the binding protein. As outlined in Figure 26.4, the designs of the assays differ with regard to the washing procedures and whether the chemiluminescence is linked to cobalamin or to the binding protein. 

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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