Biotin ELISA Kit
For Research Use Only
Size: 1×96 wells
Sensitivity: 0.5 ng/mL
Dynamic Range: 1.0 – 25 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
Biotin serves as the prosthetic group of enzymes, which catalyze carboxylations in the organism. For this purpose, biotin is bound via its carboxy group to lysin residues of carboxylases, and the transfer of carbon dioxide takes place after its attachment to a nitrogen atom of biotin, forming the so-called active carbon dioxide.
The awareness of the population for a good health and its interest in healthy nutrition has increased significantly during the last years. After the content of vitamins in his nourishment has gained importance for the consumer, food has partially been vitaminized by the manufacturer. When there exists a lack of biotin, seborrhoea, dermatitis, anorexia, muscle pain, tiredness and nervous disorders can appear. As biotin is synthesized by the human intestinal flora, deficiency symptoms are rare, appear however after excessive ingestion of raw egg white, which can be explained by its content of biotin-binding avidin.
The Eagle Biosciences Biotin (Vitamin H) quantitative test is based on the principle of the enzyme linked immunosorbent assay. Avidin, which shows a high affinity to biotin, is bound on the surface of a microtiter plate. Biotin containing samples or standards and a biotin-alkaline phosphatase conjugate are given into the wells of the microtiter plate. Enzyme labelled and free biotin compete for the 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 30 minutes, resulting in the development of a yellow colour. The colour development is inhibited by the addition of a stop solution. The yellow colour is measured photometrically at 405 nm. The concentration of biotin is indirectly proportional to the colour intensity of the Eagle Biosciences Biotin ELISA Assay test sample.
- Pipet 50 µL standards or prepared samples in duplicate into the appropriate wells of the microtiter plate. Immediately add 100 µL biotin-AP 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 light-sensitive) for 30 minutes at room temperature.
- Stop enzyme reaction by adding 100 µL of stop solution (1 M NaOH) into each well. The yellow colour will darken upon addition.
- After thorough mixing, measure absorbance at 405 nm (reference wavelength 620 nm), using an ELISA reader. The colour is stable for 30 minutes.