DBC 25-Hydroxyvitamin D [25(OH)] Elisa

PRINCIPLE OF THE TEST 
This kit measures the total concentration of both 25- hydroxyvitamin D2 and 25-hydroxyvitamin D3 (25(OH)D). The results are expressed in ng/mL. DBC’s immunoassay of 25(OH)D is a sequential competitive assay that uses two incubations, with a total assay incubation time of less than two hours. During the first incubation, unlabelled 25(OH)D (present in the standards, controls, serum and plasma samples) is dissociated from binding proteins such as vitamin D binding protein and binds to the anti-25(OH)D antibody immobilized on the microplate wells. A washing step is performed next. During the next incubation, the complex of 25(OH)D-biotin conjugate and streptavidin-HRP conjugate competes with antibody-bound 25(OH)D for antibody binding sites. The washing and decanting procedures remove any unbound materials. The TMB substrate is added next which reacts with HRP to form a coloured product. The intensity of the colour is proportional to the amount of immobilized HRP. Stopping solution is added next which stops the colour development reaction. The optical density of each well is measured in a microplate reader. The absorbance values are inversely proportional to the concentration of 25(OH)D in the sample. A set of calibrators is used to plot a standard curve from which the concentrations of 25(OH)D in the samples and controls can be directly read.

CLINICAL APPLICATIONS 
Vitamin D concentration in blood should be measured regularly to ensure that satisfactory physiological levels are maintained year round (see references). Vitamin D is assimilated from food sources (both vitamin D2 and vitamin D3) or produced in the skin by sun exposure (vitamin D3). The body stores both vitamin D2 and vitamin D3 mainly in the form of 25-hydroxyvitamin D2 or 25-hydroxyvitamin D3 respectively. Therefore, the best approach to assess the physiological levels of vitamin D is to analyze the total concentration of both hydroxylated forms [25(OH)D].

SPECIMEN COLLECTION AND STORAGE 
A minimum of 0.05 mL of serum or plasma is required per duplicate determination. Appropriate sample collection is essential for the accurate determination of 25(OH)D. 
Serum: Collect 4–5 mL of blood into an appropriately labelled tube and allow it to clot. Centrifuge and carefully remove the serum layer. Store at 4°C for up to 24 hours or at -10°C or lower if the analyses are to be done at a later date. 
Plasma: Collect 4–5 mL of blood into EDTA plasma tubes. Store at 4°C for up to 24 hours or at -10°C or lower if the analyses are to be done at a later date.

CALCULATIONS 
Using immunoassay software, choose either a 4-parameter or 5-parameter curve fitting method for calculating results