DBC Resistin 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 resistin is immobilized onto the microplate and another monoclonal antibody specific for a different epitope of resistin is conjugated to biotin. During the first step, resistin 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 resistin 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 resistin in patient samples and controls can be directly read.

CLINICAL APPLICATIONS 
Resistin is a 12.5 kDa protein containing 108 amino acids. It is synthesised as a pre-peptide and its hydrophobic signal peptide is cleaved before secretion. Resistin circulates in human blood as a dimeric protein consisting of two 92 amino acid polypeptides that are linked by a disulfide bridge. Resistin belongs to the resistin-like molecule (RELM) hormone family. 
The RELM family comprises RELM-α, RELM-β, RELM-γ and resistin. RELM-β is related to resistin and is expressed in the colon. In rodents, resistin is produced by adipose tissue and is a significant regulator of glucose metabolism and insulin sensitivity. Hyperresistinemia in rodents causes insulin resistance and predisposition to type 2 diabetes. In humans, resistin is produced by the macrophages, which stimulates the macrophage secretion of pro-inflammatrory cytokines. Some studies have shown a correlation between increased serum resistin levels and atherosclerosis. Another study shows an increase of resistin levels in mice with atherosclerotic lesions. Many studies have tried to translate the mouse data to humans by answering the question whether levels of resistin are increased in human obesity, insulin resistance, and/ or type 2 diabetes. Some groups failed to identify changes in resistin levels with obesity, insulin resistance, or type 2 diabetes while other studies that used diverse populations and different assays, have found significant relationships with one or more of these conditions.

SPECIMEN COLLECTION AND STORAGE 
Approximately 0.1 mL of serum is required per duplicate determination. Collect 2–5 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. 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 fitting method. 
3. Calculate the mean optical density of each unknown duplicate. 
4. Read the values of the serum samples directly off the calibrator curve.