Human Free IGFBP-1 Quantikine ELISA Kit Summary

Product Summary

The Quantikine Human Free IGFBP-1 Immunoassay is a 4.5 hour solid-phase ELISA designed to measure human free IGFBP-1 in cell culture supernates, cell lysates, serum, plasma, urine, and human milk. It contains NS0-expressed recombinant human IGFBP-1 and has been shown to accurately quantitate the recombinant factor. Results obtained using natural human IGFBP-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 naturally occurring human free IGFBP-1.

Preparation and Storage

Background: IGFBP-1

The superfamily of insulin-like growth factor (IGF) binding proteins include the six high-affinity IGF binding proteins (IGFBP) and at least four additional low-affinity binding proteins referred to as IGFBP related proteins (IGFBP-rP). All IGFBP superfamily members are cysteine-rich proteins with conserved cysteine residues, which are clustered in the amino- and carboxy-terminal thirds of the molecule. IGFBPs modulate the biological activities of IGF proteins. Some IGFBPs may also have intrinsic bioactivity that is independent of their ability to bind IGF proteins. Post-translational modifications of IGFBP, including glycosylation, phosphorylation and proteolysis, have been shown to modify the affinities of the binding proteins to IGF. ALS (Acid Labile Subunit) is a liver-derived protein that exists in a ternary complex with Insulin-like Growth Factor (IGF)-binding Protein-3 (IGFBP-3) or IGFBP-5, and either IGF-I or IGF-II. ALS increases the half-life of IGF/IGFBP complexes in circulation.

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.