DBC Leptin Elisa

PRINCIPLE OF THE TEST 
The principle of the following enzyme immunoassay test follows a typical two-step capture or ‘sandwich’ type assay. The assay makes use of two highly specific monoclonal antibodies: A monoclonal antibody specific for leptin is immobilized onto the microplate and another monoclonal antibody specific for a different epitope of leptin is conjugated to biotin. During the first step, leptin present in the samples and standards is bound to the immobilized antibody and to the biotinylated antibody, thus forming a sandwich complex. Excess and unbound biotinylated antibody is removed by a washing step. In the second step, streptavidin-HRP is added, which binds specifically to any bound biotinylated antibody. Again, unbound streptavidin-HRP is removed by a washing step. Next, the enzyme substrate is added (TMB), forming a blue coloured product that is directly proportional to the amount of leptin present. The enzymatic reaction is terminated by the addition of the stopping solution, converting the blue colour to a yellow colour. The absorbance is measured on a microtiter plate reader at 450 nm. A set of standards is used to plot a standard curve from which the amount of leptin in patient samples and controls can be directly read.

CLINICAL APPLICATIONS 
Human Leptin is a 16 kDa, 146 amino acid residue, nonglycosylated polypeptide. Leptin is encoded by the OB gene. Its major source is the adipose tissue, and its circulating concentrations indirectly reflect body fat stores. Plasma or serum concentrations of leptin are increased in obese humans and strongly correlate with the degree of adiposity as expressed by percentage of body fat or body mass index. The recently discovered hormone leptin contributes to the regulation of energy balance by informing the brain of the amount of adipose tissue in the body. The brain may then make the appropriate adjustments in either energy intake or expenditure. Many areas of leptin physiology remain to be investigated. The roles of leptin in metabolism, insulin sensitivity, as a potential therapeutic modality for weight loss as well as involvement in endocrine function are active areas of research. While the future for leptin as a therapeutic agent is not clear, its involvement in many areas of physiology undoubtedly makes this a new hormone which requires extensive study in the future to understand its physiology.

SPECIMEN COLLECTION AND STORAGE 
Approximately 0.1 mL of serum is required per duplicate determination. 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. Consider all human specimens as possible biohazardous materials and take appropriate precautions when handling.

CALCULATIONS 
1. Calculate the mean optical density of each calibrator duplicate. 
2. Draw a calibrator curve on semi-log paper with the mean optical densities on the Y-axis and the calibrator concentrations on the X-axis. If immunoassay software is being used, a 4-parameter or 5-parameter curve is recommended. 
3. Calculate the mean optical density of each unknown duplicate. 
4. Read the values of the unknowns directly off the calibrator curve. 
5. If a sample reads more than 100 ng/mL then dilute it with assay buffer at a dilution of no more than 1:8. The result obtained should be multiplied by the dilution factor.