DBC Vasoactive Intestinal Polypeptide (VIP) Elisa

PRINCIPLE OF THE TEST 
The VIP ELISA is a two-step competitive immunoassay. In the first incubation step, competition occurs between VIP present in calibrators, controls, specimen samples and a biotin-labelled antigen (biotin conjugate) for a limited number of anti-VIP antibody binding sites on the microplate wells. 
Excess and unbound materials are removed by a washing step. In the second incubation step, streptavidin-HRP conjugate is added, which binds specifically to any bound biotin conjugate. After a washing step that removes unbound materials, the TMB substrate (enzyme substrate) is added which reacts with HRP to form a blue-coloured product that is inversely proportional to the amount of VIP present. Following an incubation, the enzymatic reaction is terminated by the addition of the stopping solution, converting the colour from blue to yellow. The absorbance is measured on a microplate reader at 450 nm. A set of calibrators is used to plot a calibrator curve from which the amount of VIP in specimen samples and controls can be directly read.

Specimen Collection & Storage 
Follow the specimen collection procedure steps in the order provided below to avoid any delays that could potentially affect the stability of specimen samples. K2 and K3 EDTA collection tubes are suitable for plasma sample collection. 
Approximately 0.2 mL of K2 or K3 EDTA plasma is required per duplicate determination. 
1. Prior to sample collection, place a K2 or K3 EDTA plasma collection tube into a container of ice for at least 10 minutes. 
2. Collect 4–5 mL of venous blood into an appropriately labelled precooled EDTA plasma collection tube. 
3. Mix the tube by inverting several times. 
4. Place the collection tube into a container of ice to keep cool prior to centrifugation. 
5. Centrifuge the tube at 2000x g for 10 minutes and carefully transfer the plasma into a new storage tube or container. 
6. If the sample will not be tested immediately, the plasma should be aliquoted and stored frozen at ≤ -20°C for up to 3 months. Repeated freezing and thawing should be avoided. 
Consider all human specimens as possible biohazardous materials and take appropriate precautions when handling. 6.2 Specimen Pre-Treatment Specimen pre-treatment is not required.
Specimen pre-treatment is not required.

CALCULATIONS 
1. Calculate the mean optical density for each calibrator, control and specimen sample duplicate. 
2. Use a 4-parameter or 5-parameter curve fit with immunoassay software to generate a calibrator curve. 
3. The immunoassay software will calculate the concentrations of the controls and specimen samples using the mean optical density values and the calibrator curve. 
4. If a sample reads more than 800 pg/mL and needs to be diluted and retested, then dilute with Assay Buffer not more than 1:5. The result obtained must be multiplied by the dilution factor.