DESCRIPTION

Leptin is a product of the obese (ob) gene and, following synthesis and secretion from fat cells in white adipose tissue, binds to and activates its cognate receptor, the leptin receptor (LEP-R). Still, smaller quantities have been detected in other body tissues, including the brown adipose tissue (BAT), placenta, fetal tissue, stomach, muscles, bone marrow, teeth, and brain. Leptin circulates in the blood in both free and protein-bound forms, where the free form of leptin is the biologically active form. Leptin can enter the central nervous system (CNS) (in the area of the choroid plexus) by receptor-mediated transport. Leptin secretion is higher in subcutaneous than in visceral adipose tissue.

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	972.2	19.0	2.0
2	8	228.6	2.6	1.2
3	8	101.2	4.6	4.5

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	976.0	26.7	2.7
2	16	234.0	13.2	5.6
3	16	101.2	3.7	3.6

RECOVERY

The recovery of human Leptin spiked to three different levels throughout the range of the assay was evaluated.

Sample Type		Average% of Expected	Range (%)
Human plasma	1:80	101	82-116
Cell culture supernatant	1:2	109	98-124

LINEARITY

To assess the linearity of the assay, cell culture supernatant samples were spiked with high concenthumanions of human Leptin 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 1:5. )

		Human plasma	Cell culture supernatant
1:2	Average% of Expected	100	96
	Range (%)	-	95-96
1:4	Average% of Expected	110	97
	Range (%)	109-110	87-108
1:8	Average% of Expected	119	98
	Range (%)	117-122	90-105
1:16	Average% of Expected	107	101
	Range (%)	98-117	99-103

CALIBRATION

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