Human Proinsulin ELISA Kit
Proinsulin is a polypeptide of 86 amino acids made in the beta cells of the pancreas and is the precursor molecule for insulin, most proinsulin is converted to insulin and C-peptide secreted into the blood. Increased circulating levels of proinsulin are found in patients with insulinomas, hypoglycemia and hyperinsulinema.
PRINCIPLE OF THE ASSAY
This assay is a quantitative sandwich ELISA. The immunoplate is pre-coated with a monoclonal antibody specific for human proinsulin. Standards and samples are pipetted into the wells and any human proinsulin present is bound by the immobilized antibody. After washing away any unbound substances, a biotin labelled monoclonal antibody specific for human proinsulin is added to the wells. After wash step to remove any unbound reagents, streptavidin-HRP conjugate (STP-HRP) is added. After the last wash step, an HRP substrate solution is added and colour develops in proportion to the amount of human proinsulin bound initially. The assay is stopped and the optical density of the wells determined using a microplate reader. Since the increases in absorbance are directly proportional to the amount of captured human proinsulin, the unknown sample concentration can be interpolated from a reference curve included in each assay.
A. Typical representation of standard curve
The following standard curve is provided for demonstration only. A standard curve should be generated for each assay.
The lowest insulin level that can be measured by this assay is 31.2 pg/ml.
Intra-assay Precision (Precision within an assay) C.V. < 10%.
Inter-assay Precision (Precision between assays) C.V. <10%.
The recovery of the assay was determined by adding various amounts insulin to a sample. The measured concentration of the spiked sample in the assay was compared to the expected concentration. The average recovery was 89.9%.
E. Linearity: 98-113%