DESCRIPTION

The hormone leptin, secreted predominantly from adipose tissue, plays a crucial role in the regulation of numerous neuroendocrine functions, from energy homeostasis to reproduction. Leptin acts through the leptin receptor (LEPR, also known as OB-R), a single-transmembrane-domain receptor of the cytokine receptor family. On ligand binding, LEPR mediates signaling through JAK2/STAT3. LEPR plays an important role in the regulation of glucose homeostasis via feeding and adiposity.

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 (ng/mL)	SD	CV%
1	8	15.27	0.37	2.42
2	8	3.84	0.08	2.08
3	8	1.08	0.03	2.78

Inter-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	16	15.36	0.35	2.28
2	16	3.92	0.19	4.85
3	16	1.08	0.03	2.78

RECOVERY

The recovery of human LEPR spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Human plasma	1:16	114	110-118
	1:32	111	95-120
Cell culture supernatant	1:2	122	116-125
	1:4	120	119-121

LINEARITY

To assess the linearity of the assay, cell culture supernatant samples were spiked with high concenthumanions of human LEPR and diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. Human plasma samples were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. (The human plasma was initially diluted 1:4.)

		Human plasma	Cell culture supernatant
1:2	Average% of Expected	100	114
	Range (%)	-	108-121
1:4	Average% of Expected	114	114
	Range (%)	113-115	107-120
1:8	Average% of Expected	122	119
	Range (%)	116-128	115-123
1:16	Average% of Expected	116	119
	Range (%)	115-117	114-123

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human LEPR produced at Sunlong Medical™.