Human CEACAM-1/CD66a DuoSet ELISA Summary

Product Summary

The Quantikine Human FGF-21 Immunoassay is a 4.5 hour solid-phase ELISA designed to measure human FGF-21 in cell culture supernates, serum, and plasma. It contains E. coli-expressed recombinant human FGF-21 and has been shown to accurately quantitate the recombinant factor. Results obtained using natural human FGF-21 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-21.

Preparation and Storage

Background: FGF-21

Fibroblast growth factor 21 (FGF-21) is a member of the FGF gene family, which contains 22 mammalian members. Based on its structure, it is further classified as a member of the FGF-19 subfamily, which also includes FGF-19 and FGF-23 (1-4). FGF family members contain a 120 amino acid (aa) core FGF domain that exhibits a beta -trefoil structure. FGF-19 subfamily members, unlike other FGFs, lack one strand of the beta -trefoil and bind poorly to extracellular matrix molecules such as heparin (3). They are consequently more diffusible than other FGFs and are considered endocrine rather than paracrine (1-4). All three subfamily members impact some aspect of metabolism; all three are induced by a nuclear receptor heterodimer that includes RXR (retinoid X receptor), and all three bind FGF receptors (FGF R) indirectly through co-receptors of the klotho family (5-9). FGF-21 binds to beta -Klotho via its C-terminal sequence. This binding, along with amino acids at the N-terminus, is required for signaling through FGF R (7, 8). FGF-21 is selective for FGF R1 isoform 1c, with varying reports of using isoforms 2c or 3c (10-12). Presence of the required klotho and FGF R family members determines tissue specificity of FGF-19 subfamily members, and thus concentrates FGF-21 activity within adipose tissue (3, 9-11). Mature human FGF-21 shares 81% aa sequence identity with mouse and rat FGF-21. 

FGF-21 is produced by hepatocytes in response to free fatty acid (FFA) stimulation of a PPAR alpha /RXR dimeric complex (4, 13-15). This situation occurs during starvation, diabetic ketosis, or following the ingestion of a high-fat/low-carbohydrate or ketogenic diet (5, 14-16). Upon FGF-21 secretion, white adipose tissue is induced to release FFAs from triglyceride stores. Once FFAs reach the hepatocytes, they are oxidized and reduced to acetyl-CoA (16). The acetyl-CoA is recombined into 4-carbon ketone bodies (acetoacetate and beta -hydroxybutyrate), released, and transported to peripheral tissues for energy generation (5, 15, 16). FGF-21 production is also induced upon differentiation of human or mouse fibroblasts to adipocytes (17, 18). In adipose tissue, FGF-21 induces glucose uptake by signaling in synergy with PPAR gamma to increase production of the glucose transporter, GLUT1 (10, 12, 19). 

FGF-21 production follows a circadian pattern in mice (20). It diffuses across the blood-brain barrier and this may facilitate induction of a state of torpor, or decreased activity, in response to increased FGF-21 (16, 21). These characteristics appear to induce a hibernation-like state during fasting and short days in winter (22). In diet-induced obese mice and mouse models of diabetes such as db/db and ob/ob, administration or transgenic overexpression of FGF-21 restores circulating glucose and triglyceride values to near normal and increases insulin sensitivity (5, 6, 14, 23, 24). In some of these states and in human obesity and type II diabetes, FGF-21 is already elevated prior to treatment, suggesting that resistance to FGF-21 is possible (17, 25, 26). Although FGF-21 administration corrects obesity in mice, it is unclear whether the same benefit would be seen in humans (2, 3, 17, 26-28).

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.