Human FGF-19 Quantikine ELISA Summary

Product Summary

The Quantikine Human FGF-19 Immunoassay is a 4.5 hour solid-phase ELISA designed to measure human FGF-19 in cell culture supernates, serum, and plasma. It contains E. coli-expressed recombinant human FGF-19 and has been shown to accurately quantitate the recombinant factor. Results obtained using natural human FGF-19 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 FGF-19.

Preparation and Storage

Background: FGF-19

The Fibroblast Growth Factor (FGF) family consists of at least 22 highly conserved proteins and are found in many species ranging from C. elegans and Drosophila to humans (1). FGFs are heparin-binding growth factors with a core 120 amino acid (aa) FGF domain that allows for a common tertiary structure. In general, FGFs are expressed during embryonic development and in restricted adult tissues. They act on cells of mesodermal and neuroectodermal origin to regulate diverse physiologic functions including angiogenesis, cell growth, pattern formation, metabolic regulation, cell migration, neurotrophic effects, and tissue repair (2). The activities of the FGF family are mediated by four receptors, FGF R1 through FGF R4 (2). The receptors are transmembrane proteins with a tyrosine kinase domain and are thought to act as classical receptor tyrosine kinases. FGF R5/FGF RL1 has also been described, but lacks the kinase domain and signaling capability (3). 

Human FGF-19 cDNA predicts a 251 aa precursor protein with a 22 aa signal peptide and a 229 aa secreted mature protein with no potential N-linked glycosylation sites (4, 5). It shares approximately 50% aa sequence identity with mouse and rat FGF-15 and 62% aa sequence identity with chicken FGF-19 (4-6). Unlike most FGFs, which bind to and activate more than one FGF receptor, FGF-19 appears to bind only FGF R4 (5). Receptor binding is capable of activating MAP Kinase and inducing the Prolactin promoter (7). FGF-19 has two putative disulfide bonds, one of which is conserved in the FGF family (8). Uniquely, it also has an extended loop structure that may affect heparan sulfate binding (8). 

The functional roles of FGF-19 continue to be elucidated and include developmental, metabolic, and tumorigenic activities. During chick embryogenesis, FGF-19 has been shown to act synergistically with Wnt-8c to initiate inner ear development (6). However, the mouse FGF-19 ortholog, FGF-15, is not required for this activity (9). FGF-19 is also expressed in the embryonic chicken eye where it may play a role in lens development (10). Questions regarding the functions of human FGF-19 have been addressed using ectopic expression. Transgenic over-expression of human FGF-19 in mice results in tumor formation, increased energy expenditure, decreased adiposity, and a resistance to weight gain in response to a high fat diet (11, 12). Similar affects have been observed in mice treated intravenously with recombinant FGF-19 (13).

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.