Human IGF-I/IGF-1 Quantikine ELISA Kit Summary

Product Summary

The Quantikine^® Human IGF-I/IGF-1 Immunoassay is a 4.5 hour solid-phase ELISA designed to measure human IGF-1 in cell culture supernates, serum, and plasma. It contains E. coli-expressed recombinant human IGF-1 and has antibodies raised againt the recombinant protein. Results obtained for naturally occurring human IGF-1 showed linear curves that were parallel to the standard curves obtained using the Quantikine^® kit standards. These results indicate that this kit can be used to determine relative mass values for natural human IGF-1.

Preparation and Storage

Background: IGF-I/IGF-1

Insulin-like growth factor 1 (IGF-1, also known as somatomedin C) is a 7.6 kDa, 70 amino acid (aa) polypeptide with three internal disulfide bonds. The sequence of human IGF-1 is identical to that of bovine and porcine IGF-1, and is 70% identical to human IGF2. IGF-1 is a single-chain molecule with about 50% identity to the sequences of the A- and B-chains of human insulin. 

 IGF-1 is a growth hormone that plays a role in a variety of biological events. It is produced primarily by hepatocytes, serving an endocrine function. It is also produced by many other cells, where it may act in an autocrine or paracrine manner. It binds two tyrosine kinase receptors, the IGF-1 receptor (IGF1R) and the insulin receptor, to initiate downstream events like the AKT and PI3K signal transduction pathways. This triggers cell proliferation and protects cells from apoptosis. IGF-1 also interacts with seven IGF-binding proteins (IGFBP-1 through IGFBP7), influencing IGF-1 binding to IGF1R and increasing IGF-1 half-life to closely regulate IGF-1 signaling. For example, IGFBP-3 binds over 90% of the total IGF in serum in a complex of IGF, IGFBP, and an acid-labile subunit. This ternary complex greatly stabilizes IGF in the circulation, changing the half-life from minutes to hours. Proteases also facilitate IGF-1R binding by cleaving IGFBPs to modify their affinity for IGF or completely eliminate the IGFBP. The interactions of IGF, IGFBP, IGFBP proteases, and IGF receptors are referred to as the IGF axis. 

 The IGF axis affects many primary physiological and pathological processes, including development, growth, metabolic regulation, tumorigenesis, atherosclerosis, and angiogenesis. Its ability to inhibit apoptosis and stimulate cell growth and proliferation plays a significant role in prenatal development, growth to adulthood, and metabolic control. Serum levels of IGF-1 have been reported to increase from birth to puberty, followed by a slow decline through adulthood. IGF-1 also induces amino acid uptake, protein synthesis, and glucose utilization. In the brain, IGF-1 acts as a neurotrophic factor to promote neurogenesis and neuronal survival. Exercise increases levels of IGF-1 in blood serum, indicating it plays a role as a key mediator of exercise-induced neurogenesis.

Assay Procedure

Refer to the product datasheet for the complete assay procedure.

Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.

1.     Prepare all reagents, standard dilutions, and samples as directed in the product insert.

2.     Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

3.     Add 50 μL of Assay Diluent to each well.

4.     Add 50 μL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours.

5.     Aspirate each well and wash, repeating the process 4 times for a total of 5 washes.

6.     Add 100 μL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 2 hours.

7.     Aspirate and wash 5 times.

8.     Add 100 μL Substrate Solution to each well.

9.     Add 100 μL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.