DBC Adiponectin ELISA

PRINCIPLE OF THE TEST 
The principle of the adiponectin ELISA is a two-step sandwich enzyme immunoassay. The assay makes use of two highly specific monoclonal antibodies: A monoclonal antibody specifi c for adiponectin is immobilized onto the microplate and another monoclonal antibody specific for a different epitope of adiponectin is conjugated to biotin. During the first step, adiponectin present in the samples and standards is bound to the immobilized antibody and to the biotinylated antibody, thus forming a sandwich complex. Unbound biotinylated antibody is removed by a washing. In the second step, streptavidin-HRP is added, which binds specifi cally to bound biotinylated antibody. Unbound streptavidin-HRP is removed by washing. Next, the enzyme substrate (TMB) is added. The colour intensity of the enzymatic reaction is directly proportional to the concentration of adiponectin. The enzymatic reaction is terminated by the addition of stopping solution. The absorbance is measured on a microplate reader at 450 nm. The concentration of adiponectin in samples and controls can be calculated from of a plot of the standard curve, either graphically or by using immunoassay software.

CLINICAL APPLICATIONS 
Adiponectin is a hormone that modulates glucose regulation and fatty acid oxidation. It is secreted from adipose tissue and placenta into the bloodstream and represents 0.01% of all plasma protein. Adiponectin increases insulin sensitivity and decreases plasma glucose by increasing tissue fat oxidation. Adiponectin concentrations correlate negatively with glucose, insulin and triglycerides (TG) concentrations, liver fat content and body mass index and positively with high density lipoprotein cholesterol levels, hepatic insulin sensitivity and insulin stimulated glucose disposal. Adiponectin levels decrease in patients with type 2 diabetes and in patients with coronary heart disease. Adiponectin could be used as a marker for: 
• Energy metabolism and body weight regulation. 
• Metabolic syndrome. 
• Type 2 diabetes. 
• Coronary artery disease. 
• Atherosclerosis

SPECIMEN COLLECTION AND STORAGE 
Approximately 0.05 mL of serum or plasma is required per duplicate determination. Serum: Collect 2–3 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 2–3 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 
1. Calculate the mean optical density of each calibrator duplicate. 
2. Plot a calibrator curve with the mean optical densities on the Y-axis and the calibrator concentrations on the X-axis. If immunoassay software is used, choose a 4-parameter or 5-parameter curve fi tting method. 
3. Calculate the mean optical density of each unknown duplicate. 
4. Read the values of the serum and plasma samples directly off the calibrator curve.
5. The measured concentration of samples calculated from the standard curve must be multiplied by the dilution factor of 1000. Example: 15 ng/mL (from standard curve) x 1000 (dilution factor for serum and plasma samples) = 15 µg/mL. 
6. If a sample reads more than calibrator F then dilute the original 1000x diluted sample with working dilution buffer at a dilution of no more than 1:8. The result obtained must be multiplied by the dilution factor.