Vitamin B12 ELISA

Enzyme Immunoassay for the quantitative determination of Vitamin B12 in food.

Background: Vitamin B12 as a trace element belongs to the biologically important chelate formers. The basic unit consists of a corrin ring with cobalt as a central atom. Cobalt is sixfold coordinated by four nitrogen atoms, one cyanide and a dimethylbenzimidazol group. Vitamin B12 forms a stable complex, which is absorbed in the lower part of the small intestine, with the so-called intrinsic factor present in the gastric juice. A lack of vitamin B12 can lead among other things to pernicious anemia. This disease is not generated by an insufficient supply of vitamin B12, but by the absence of intrinsic factor. A pernicious anemia can be treated by a high dosage of vitamin B12.
The existing detection procedures are mainly microbiological methods, but also HPLC and thinlayer chromatography, all of which are associated with a high amount of time and instrumentation. With the present test kit it is possible, to determine vitamin B12 quantitatively in vitaminated food in a significantly faster way (2.5 to 4 hours inclusive sample pretreatment) compared with a conventional microbiological assay (24 to 48 hours).

Description: The Vitamin B12 quantitative test is based on the principle of the enzyme linked immunosorbent assay. An antibody directed against vitamin B12 is bound on the surface of a microtiter plate. Vitamin B12 containing samples or standards and a vitamin B12-peroxidase conjugate are given into the wells of the microtiter plate. Enzyme labeled and free vitamin B12 compete for the antibody binding sites. After one hour incubation at room temperature, the wells are washed with diluted washing solution to remove unbound material. A substrate solution is added and incubated for 20 minutes, resulting in the development of a blue colour. The colour development is inhibited by the addition of a stop solution, and the colour turns yellow. The yellow colour is measured photometrically at 450 nm. The concentration of vitamin B12 is indirectly proportional to the colour intensity of the test sample.

Product Features:
- The kit contains reagents for 96 determinations;
- Microtiter plate consisting of 12 strips with 8 breakable wells;
- Vitamin B12 Standards: 0, 0.4, 1, 4, 10, 40 ng/mL;
- ELISA reader at 450 nm;
- Standard range: 0.4 - 40 ng/ml;
- Sensitivity analytical: 0.3 ng/mL.