DESCRIPTION

RBP4 (retinol-binding protein 4) belongs to the lipocalin family and is the specific carrier for retinol (Vitamin A) in the blood. It is mainly synthesized in the liver and circulates in the bloodstream bound to retinol in a complex with transthyretin. RBP4 acts as a drug target for ophthalmology research and reduction in serum RBP4 level inhibits lipofuscin without inhibiting the visual cycle. Urinary RBP4 could act as a marker for glomerular disease. And RBP4 also was identified as an adipokine that elevated in some insulin-resistant states. Measurement of serum RBP4 could be used to assess the risk of insulin resistance, type 2 diabetes, obesity, and cardiovascular disease.

PRINCIPLE OF THE ASSAY

Sandwich ELISA structure (Detection antibody labeled with HRP) A capture antibody is pre-coated onto the bottom of wells which binds to analyte of interest. A detection antibody labeled with HRP also binds to the analyte. TMB acts as the HRP substrate and the solution color will change from colorless to blue. A stop solution containing sulfuric acid turns 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 20 times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in 24 separate assays to assess inter-assay precision.

Intra-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	20	12.3	0.65	5.3
2	20	18.4	0.75	4.0
3	20	40.8	0.92	2.3

Inter-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	24	12.6	0.25	2.0
2	24	18.0	0.60	3.3
3	24	37.9	1.68	4.4

RECOVERY

The recovery of human RBP4 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Human serum	1:8,000	89	79-94
	1:16,000	86	74-99
Cell culture supernatant	1:2	78	74-80
	1:4	77	72-81
Urine	1:4	91	78-98
	1:8	86	73-104
Saliva	1:2	103	90-119
	1:4	99	86-118

LINEARITY

To assess the linearity of the assay, samples were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. (The human serum was initially diluted 1:1000, cell culture supernatant was initially diluted 1:32)

		Human serum(Sample Diluent PT 4)	Cell culture supernatant(Sample Diluent PT 1-ef)	Urine (SampleDiluent PT 4)	Saliva (SampleDiluent PT 1-ef)
1:2	Average% ofExpected	100	100	100	100
	Range (%)	-	-	-	-
1:4	Average% ofExpected	115	108	116	117
	Range (%)	106-120	107-109	112-122	110-120
1:8	Average% ofExpected	107	101	100	98
	Range (%)	101-114	98-105	74-113	73-111
1:16	Average% ofExpected	98
	Range (%)	82-119

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human RBP4 produced at Sunlong Medical™.