Hormone

Insulin ELISA - rat

rat insulin ELISA For the quantitative determination of insulin in rat serum and
Catalog Number: 
M046042
Qty/Size: 
96 wells

rat insulin ELISA from MD Bioproducts

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For the quantitative determination of insulin in rat serum and plasma samples

Overview

Overview: 

Rat insulin is a pancreatic hormone whose molecular weight is about 6000 Da. It is a protein composed of two polypeptide chains, a shorter A-chain of twenty-one residues and a longer B-chain of thirty. The two chains are connected by two disulphide (-S-S-) linkages, while a third such linkage forms an intra-chain precursor called pro-insulin , in which the future A- and B-chains are linked end to end by a peptide strand, C-peptide, before being joined by their –S-S-bonds. It is found in the ß-cell granules in the pancreatic Islets of Langerhans. Specific proteases act on pro-insulin to release the C-peptide and insulin within the granule. On stimulation the C-peptide and insulin are released into the bloodstream in approximately equimolar amounts.

 

Rat insulin differs from most other species in that it has two forms that are products of non-allelic genes. Translation of the two insulin mRNAs results in the synthesis of two preproinsulins differing by 7 amino acids. Processing of these peptides involves removal of the pre region and formation of proinsulins differing in 4 of 86 amino acids. The proinsulins are cleaved to mature insulins 1 and 2 which have identical A chains but differ by 2 amino acids in the B chain (positions 9 and 29). They are found roughly in the proportion 60% insulin 1 and 40% insulin 2 in the pancreas.

 

Several factors can effect the release of insulin. One of the main regulators of insulin release is the amount of glucose in the blood. A rise in blood glucose stimulates the release of insulin while a fall in blood glucose sup- presses its secretion. Amino acids also stimulate insulin-release to allow their uptake into muscle cells. Insulin is considered to be an anabolic hormone in that it promotes the synthesis of protein, lipid and glycogen and it inhibits the degradation of these compounds. The key target tissues of insulin are liver, muscle and adipose tissue. It promotes cell growth in many different cell types and is an absolute requirement for normal growth in all immature animals. Insulin exerts its effect through a receptor complex comprising two a sub-units of molecular weight 135 kDa and two ß sub-units of molecular weight 90 kDa. It is also well known for its involve- ment in diabetes, where insulin deficiency results in aberrant blood glucose homeostasis.

 

Rat insulin can be measured in the range 0.156-10 ng/mL. Each kit contains materials sufficient for 96 determi- nations permitting the construction of one standard curve and the assay of 40 unknowns in duplicate.

Data/Specifications

Data/Specifications: 

Species: rat

Sample Type:  serum and plasma 

Sample Size: 50 uL

Standard Curve Range:  0 - 10 ng/mL

Sample Size:  0.093 ng/mL

Assay Length:   2.5 hours

Specificity: Pig insulin (31%), Bovine Insulin (76%), Human Insulin (46%)

 

Literature/Support

Literature/Support: 

Product Insert:

rat Insulin ELISA Insert (PDF)

 

Articles/Troublshooting:

ELISA Troubleshooting Guide

ELISA Data Reduction Guide

 

 

 

 

How It Works

How It Works: 

The technology uses two high affinity monoclonal antibodies in an immunometric assay system. This assay is based on a two-step procedure. In the first step the standards and samples are incubated in streptavidin coated wells with biotin labelled monoclonal antibody (capture antibody). During a 1-hour incubation period with continuous agitation the capture antibody - antigen complex is developed and immobilized on the reactive surface of wells. After incubation wells are washed repeatedly.

 

In the second step the horseradish peroxidase (HRP) labelled monoclonal antibody (signal antibody) is added. It binds to an epitope of the insulin molecule different from that recognized by the capture-antibody, developing the formation of a capture antibody - antigen - signal antibody complex, also referred to as a ‘sandwich’. After the one-hour-incubation period with continuous agitation the reaction mixture is washed repeatedly. After the addition of a ready-to-use tetramethyl-benzidine (TMB) peroxide substrate the signal is measured in an ELISA photometer at 450 nm and 405 nm wavelength (620 nm as reference wavelength is recommended). The concentration of antigen is directly proportional to the optical density measured in the wells. The unknown concentration of rat insulin in samples is read off a calibration curve constructed by plotting binding values against a series of calibrators containing known amount of rat insulin.

 

rat insulin ELISA