Vitamin B12 ELISA Assay kit:
For Research Use Only
Size: 1×96 wells
Sensitivity: 0.3 ng/mL
Dynamic Range: 0.4 – 40 ng/mL
Incubation Time: 1.5 hours
Sample Type: Food Products, Cell Culture, Tissue
Sample Size: 3 – 10 g
Product Developed and Manufactured in Germany
Product Support in the USA
The Eagle Biosciences Vitamin B12 ELISA Assay kit is intended for the quantitative determination of Vitamin B12 in food products by enzyme linked immunoassay (ELISA). The Eagle Biosciences Vitamin B12 ELISA Assay kit is for research use only and not to be used in diagnostic procedures.
The Eagle Biosciences 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 labelled 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.
- Pipet 50 µL standards or prepared samples in duplicate into the appropriate wells of the microtiter plate. Immediately add 50 µL vitamin B12-peroxidase conjugate into each well.
- Cover the microtiter plate with a plastic foil and incubate for 60 minutes at room temperature on a microtiter plate shaker (or 90 minutes without shaker).
- Wash the plate three times as follows: Discard the contents of the wells (dump or aspirate). Pipet 300 µL of diluted washing solution into each well. After the third repetition empty the wells again and remove residual liquid by striking the plate against a paper towel. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbencies.
- Pipet 100 µL of substrate solution into each well.
- Allow the reaction to develop in the dark (e.g. cupboard or drawer; the chromogen is lightsensitive) for 20 minutes at room temperature.
- Stop enzyme reaction by adding 100 µL of stop solution (0.5 M H2SO4) into each well. The blue colour will turn yellow upon addition.
- After thorough mixing, measure absorbance at 450 nm (reference wavelength 620 nm), using an ELISA reader. The colour is stable for 30 minutes.
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 six fold 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 (1), but also HPLC and thin-layer chromatography, all of which are associated with a high amount of time and instrumentation. With the present Eagle Biosciences Vitamin B12 ELISA Assay test kit it is possible, to determine vitamin B12 quantitatively in vitaminated food (2) in a significantly faster way (2.5 to 4 hours inclusive sample pre-treatment) compared with a conventional microbiological assay (24 to 48 hours).
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