DESCRIPTION

Fibroblast growth factor 21 (FGF21) is a peptide hormone that is expressed across several tissues, predominantly in liver and adipocyte tissues, and, to a lower extent, in the pancreas, skeletal muscle, heart, kidneys and testes. FGF21 plays a role in regulating adaptation to various metabolic abnormalities. In the liver, FGF21 plays an important role in the regulation of fatty acid oxidation both in the fasted state and in mice consuming a high-fat, low-carbohydrate ketogenic diet. FGF21 also regulates fatty acid metabolism in mice consuming a diet that promotes hepatic lipotoxicity.

PRINCIPLE OF THE ASSAY

An anti-tag antibody is pre-coated onto the bottom of wells. After adding antigen or samples, Tagged-Capture antibody and HRP-conjugated detection antibody, a sandwich complex is formed in the solution. TMB acts as a HRP substrate, and the solution color will change from colorless to blue. A stop solution containing sulfuric acid turns the solution yellow. The color intensity is proportional to the quantity of bound protein, which is measurable at 450 nm with the correction wavelength set at 630 nm.

PRECISION

Intra-assay Precision (Precision within an assay) Three samples of known concentration were tested 8 times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in 16 separate assays to assess inter-assay precision.

Intra-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	8	236.8	6.4	2.7
2	8	60.0	3.2	5.3
3	8	30.2	1.4	4.6

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	239.6	6.3	2.6
2	16	61.1	2.8	4.6
3	16	29.8	1.3	4.4

RECOVERY

The recovery of human/cynomolgus monkey FGF-21 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Human plasma	1:2	92	86-96
	1:4	113	102-119
Cynomolgus monkey serum	1:4	109	99-114
	1:8	111	101-117
Cell culture supernatant	1:2	121	109-135
	1:4	106	103-110

LINEARITY

To assess the linearity of the assay, cell culture supernatant samples were spiked with high concentrations of human FGF-21 and diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. Human plasma and cynomolgus monkey serum samples were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay.

		Human plasma(Sample Diluent PT 3)	Cynomolgus monkey serum(Sample Diluent PT 5)	Cell culture supernatant(Sample Diluent PT 4B1)
1:2	Average% ofExpected	100	100	94
	Range (%)	-	-	93-95
1:4	Average% ofExpected	109	98	97
	Range (%)	100-118	96-100	97-98
1:8	Average% ofExpected	109	98	111
	Range (%)	97-120	98-99	109-113
1:16	Average% ofExpected	84	96	115
	Range (%)	83-84	85-106	114-116

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human/cynomolgus monkey FGF-21 produced at Sunlong Medical™.