25-OH-Vitamin-D ELISA

Features

• RUO
• standards 1-6: 0-120 ng/ml
• limit of detection 1.6 ng/ml
• Material of animal origin used in the preparation of the kit have been obtained from certified healthy animals but these materials should be handled as potentially infectious

Research topic

Bone and cartilage metabolism

Summary

The 25-OH Vitamin D ELISA is designed for the serological determination of the Vitamin D concentration in the human organism. Types of Vitamin D that are differentiated are Vitamin D2 (ergocalciferol) that is contained in plant food (mushrooms, avocado) and Vitamin D3 (cholecalciferol) that is produced from 7-dehydrocholesterol in the skin under ultra-violet irradiation or found in animal food or products (sea fish, egg yolk, butter) [1, 2, 3, 4]. These two forms of Vitamin D, which are not yet biologically active, are bound by a protein called VDBP (Vitamin D binding protein) in the bloodstream, then metabolised in the liver and converted into 25-OH Vitamin D3 (calcidiol) and subsequently to the biological active form 1,25(OH)2 Vitamin D3 (calcitriol) in the kidney [1]. In contrast to other commercially available tests, the ELISA uses a newly designed monoclonal antibody which is equally specific for both forms of the vitamin. This is necessary because sometimes Vitamin D2 instead of D3 is used in therapy [5, 6, 7]. The new ELISA test kit is designed for the in vitro determination of 25-OH Vitamin D in human serum or plasma samples. In the first analysis step, the calibrators and samples are diluted with biotin-labelled 25-OH Vitamin D and added to microplate wells coated with monoclonal anti-25-OH Vitamin D antibodies. During the incubation an unknown amount of 25-OH Vitamin D in the sample and a known amount of biotin-labelled 25-OH Vitamin D compete for the antibody binding sites in the microplate wells plate. Unbound 25-OH Vitamin D is removed by washing. For the detection of bound biotin-labelled 25-OH Vitamin D, a second incubation is performed using peroxidase-labelled streptavidin. In a third incubation using the peroxidase substrate tetramethylbenzidine (TMB) the bound peroxidase promotes a colour reaction. The colour intensity is inversely proportional to the 25-OH Vitamin D concentration.