DRG 25-OH Vitamin D (total) Elisa

PRINCIPLE OF THE TEST
The DRG 25-OH Vitamin D (total) ELISA is a solid phase enzyme-linked immunosorbent assay (ELISA) based on the principle of competitive binding. The microtiter wells are coated with a monoclonal (mouse) antibody directed towards a unique antigenic site of the 25-OH Vitamin D (25-OH D) molecule. The patient sample is incubated together with Release Reagent in the wells to dissociate endogenous 25-OH D from Vitamin D binding protein (VDBP). Released 25-OH D then binds to the coated antibody of the well. After a washing step, biotin-labeled 25-OH D (Enzyme Conjugate) and peroxidase-labeled streptavidin (Enzyme Complex) are added. Added Biotin-25-OH D competes with endogenous 25-OH D for the binding to the coated antibody. Bound Biotin − 25-OH Vitamin D is then detected by Streptavidin-HRP. After incubation, unbound components are washed off. The amount of bound biotin-streptavidin complex is inversely proportional to the concentration of 25-OH Vitamin D in the sample. Subsequently, substrate solution is added and the color development is stopped after a defined time. The intensity of the color formed is inversely proportional to the 25-OH D concentration in the sample. The absorbance is measured at 450 nm with a microtiter plate reader.

Specimen Collection
Serum: Collect blood by venipuncture (e.g. Sarstedt Monovette for serum), allow to clot, and separate serum by centrifugation at room temperature. Do not centrifuge before complete clotting has occurred. Patients receiving anticoagulant therapy may require increased clotting time.

Plasma: Whole blood should be collected into centrifuge tubes containing anti-coagulant (e.g. Sarstedt Monovette with the appropriate plasma preparation) and centrifuged immediately after collection.

QUALITY CONTROL
Good laboratory practice requires that controls be run with each calibration curve. A statistically significant number of controls should be assayed to establish mean values and acceptable ranges to assure proper performance. 
It is recommended to use control samples according to state and federal regulations. The use of control samples is advised to assure the day to day validity of results. Use controls at both normal and pathological levels. 
The controls and the corresponding results of the QC-Laboratory are stated in the QC certificate added to the kit. The values and ranges stated on the QC sheet always refer to the current kit lot and should be used for direct comparison of the results. It is also recommended to make use of national or international Quality Assessment programs in order to ensure the accuracy of the results. 
Employ appropriate statistical methods for analysing control values and trends. If the results of the assay do not fit to the established acceptable ranges of control materials patient results should be considered invalid.
In this case, please check the following technical areas: Pipetting and timing devices; photometer, expiration dates of reagents, storage and incubation conditions, aspiration and washing methods.
After checking the above mentioned items without finding any error contact your distributor or DRG directly.