DESCRIPTION

Insulin-like growth factor binding protein 3, IGFBP3, is an N-linked glycosylated, phosphorylated, secretory protein with known antiproliferative and pro-apoptotic functions. IGFBP3 belongs to a family of high affinity insulin-like growth factor (IGF) binding proteins, which function to sequester extracellular IGF-1, preventing IGF-1 activation of the insulin-like growth factor receptor, IGF-1R. IGFBP3 is highly expressed in the endometrium and at the maternal-fetal interface in multiple species, including humans. In a mouse embryo coculture system, IGFBP3 was found to be secretedby endometrial cells that promote embryonic developmentby enhancing the effect of IGF.

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	2,599.6	60.7	2.3
2	8	581.0	11.5	2.0
3	8	285.7	5.6	2.0

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	2,616.5	72.4	2.8
2	16	579.9	17.6	3.0
3	16	285.3	7.8	2.7

RECOVERY

The recovery of mouse IGFBP3 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Mouse serum	1:4,000	96	89-104
	1:8,000	97	89-106
Cell culture supernatant	1:2	113	111-114
	1:4	109	102-117

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 mouse serum was initially diluted 1:500. The cell culture supernatant was initially diluted 1:16.)

		Mouse serum	Cell culture supernatant
1:2	Average% of Expected	100	100
	Range (%)	-	-
1:4	Average% of Expected	96	100
	Range (%)	96-97	94-105
1:8	Average% of Expected	99	105
	Range (%)	98-100	95-115
1:16	Average% of Expected	105	108
	Range (%)	101-108	93-123

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant mouse IGFBP3 produced at Sunlong Medical™.