Vitamin B12 Food ELISA Kit


The Vitamin B12 Food ELISA 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.

SKU: B1206-K01 Categories: , ,

Vitamin B12 Food ELISA Kit

The Vitamin B12 Food ELISA Kit is 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
Controls Included

Assay 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 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).

Related Products

Vitamin B12 Microtiter Plate Assay
25-OH Vitamin D ELISA Assay Kit

Additional Information

Assay Principle

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.

Assay Procedure

  1. 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.
  2. 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).
  3. 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.
  4. Pipet 100 µL of substrate solution into each well.
  5. Allow the reaction to develop in the dark (e.g. cupboard or drawer; the chromogen is lightsen­si­tive) for 20 minutes at room tem­perature.
  6. 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.
  7. After thorough mixing, measure absorbance at 450 nm (reference wavelength 620 nm), using an ELISA reader. The colour is stable for 30 minutes.

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