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Immunoassays | Prototype

Erenna® Human (MP-based) Insulin Immunoassay Prototype Assay (Cat# 03-0041-AA)

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Introduction

The Erenna® Human, MP-based Insulin Immunoassay Evaluation Reagent Kit uses a quantitative fluorescent sandwich immunoassay technique to measure insulin in human serum samples. A capture antibody specific for insulin has been pre-coated onto paramagnetic microparticles (MP). The user pipettes MP, standards, and samples into uncoated microplate wells. During incubation, the insulin present in the sample binds to the capture antibody on the coated MP. Unbound molecules are washed away during the subsequent buffer exchange and wash steps. Fluor-labeled detection antibody is added to each well and incubated. This detection antibody recognizes and binds to insulin that has been captured onto the MP. During the following wash step the MPs are transferred to a clean plate. Elution buffer is then added and incubated. The elution buffer dissociates the bound protein sandwich from the MP surface, releasing the labeled antibodies. These antibodies are separated during transfer to a final microplate. The plate is loaded into the Erenna System where the labeled molecules are detected and counted. The number of fluor-labeled detection antibodies counted is directly proportional to the amount of insulin present in the sample when captured. The amount of insulin in unknown samples is interpolated from a standard curve.

Assay Performance


TABLE 1. Analytical sensitivity of the Erenna® Human (MP-based) Insulin Immunoassay Prototype Assay1
Lower Limit of Detection 0.1 pg/mL
Lower Limit of Quantification2 0.3 pg/mL
Upper Limit of Quantification 70 pg/mL
Low-end CV% Range 9 - 10%
Low-end CV% Average 10%
Recommended Sample Volume 100 μL
Minimum Sample Volume Required3 1 μL

1 See product insert for updated values
2 LLoQ = 20% CV and ± 20% recovery
3 based upon median [Insulin (human, MP-based)] in a healthy reference


FIGURE 1. [Insulin] in EDTA plasma from 4 healthy donors, with median and interquartile range. The Erenna® Human, MP-based Insulin Immunoassay Evaluation Reagent Kit reliably quantifies insulin in healthy subjects, who have a median [insulin] of 342 pg/mL that is well above the detection limit of 0.1 pg/mL.

FIGURE 2. Erenna® Human, MP-based Insulin Immunoassay Evaluation Reagent Kit low-end standard curve signal (left) and curve fit (right).

Representative data shown for demonstration purposes only. Individual results may vary depending upon samples tested and protocol used.

Biology and Disease

Insulin is an important metabolic hormone produced by islet cells within the pancreas whose primary function is to regulate blood sugar levels in the body. When blood glucose level is high (ie. after food ingestion), insulin increases glucose uptake and forces liver cells to convert glucose to glycogen for storage. Insulin is also known to promote glycolysis, fatty acid synthesis and amino acid uptake. Patients who do not produce sufficient insulin (Type 1) or become resistant to insulin (Type 2) develop diabetes mellitus and suffer from hyperglycemia, which can lead to complications such as blindness, cardiovascular disease, and kidney damage. Many insulin analogs, which mimic either normal or rapid-acting (ie. lispro) insulin functions have been developed as treatment for diabetes.


TABLE 2. Additional UniProtKB/Swiss-Prot Information*
Protein Name: Insulin
UniProtKB/Swiss-Prot ID: P01308
Protein-protein interaction database: P01308
Alternative Names:
Gene Names: INS
Synonym Gene Names:
Function: Insulin decreases blood glucose concentration. It increases cell permeability to monosaccharides, amino acids and fatty acids. It accelerates glycolysis, the pentose phosphate cycle, and glycogen synthesis in liver.
Biological Processes: carbohydrate metabolism, glucose homeostasis, fatty acid homeostasis
Molecular Function: hormone activity, insulin receptor binding

* Source Info from www.uniprot.org.


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