DESCRIPTION

Type I collagen, the major structural component of connective tissues such as skin, tendon and bone, is the most abundant and widely expressed collagen in humans. Type I collagen is a heterotrimer comprising one alpha 2(I) and two alpha 1(I) chains which are encoded by the unlinked loci COL1A2 and COL1A1 respectively. Type I collagen has a molecular mass of about 250300 kDa, while the alpha 2(I) chain has a molecular weight of about 100-140 kDa. Mutations in COL1A2 gene are associated with osteogenesis imperfecta, Ehlers-Danlos syndrome, idiopathic osteoporosis, and atypical Marfan syndrome.

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	10.08	0.20	1.98
2	8	2.74	0.14	5.11
3	8	1.35	0.04	2.96

Inter-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	16	9.75	0.44	4.51
2	16	2.62	0.19	7.25
3	16	1.30	0.06	4.62

RECOVERY

The recovery of human Collagen Type I spiked to three different levels throughout the range of the assay in human plasma was evaluated.

Sample Type		Average% of Expected	Range (%)
Human plasma	1:4	90	84-93
	1:8	94	88-98

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 plasma were initially diluted 1:2.)

		Human plasma
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	107
	Range (%)	103-111
1:8	Average% of Expected	110
	Range (%)	101-120
1:16	Average% of Expected	100
	Range (%)	95-105

CALIBRATION

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