DESCRIPTION

CLEC9A (C-type lectin domain family 9 member A), also known as DNGR1, CD370, is a type II membrane protein that contains Ctype lectin domain. The C-type lectin domain family members share a common protein fold and have diverse functions, such as cell adhesion, cell-cell signaling, glycoprotein turnover, and roles in inflammation and immune response. Mouse Clec9A is primarily restricted to dendritic cells (DCs) and selectively expressed in mouse CD8+ conventional DCs (cDCs) and plasmacytoid DCs (pDCs), whereas human CLEC9A is also expressed on human DC subtypes. CLEC9A functions as an activating receptor that recruits Syk kinase and can induce the production of proinflammatory cytokines.

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	13.68	0.37	2.70
2	8	3.35	0.14	4.18
3	8	1.67	0.07	4.19

Inter-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	16	13.44	0.47	3.50
2	16	3.23	0.18	5.57
3	16	1.62	0.09	5.56

RECOVERY

The recovery of human CLEC9A spiked to three different levels throughout the range of the assay in human serum was evaluated.

Sample Type		Average% of Expected	Range (%)
Human serum	1:2	77	71-88
	1:4	82	79-88

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.

		Human serum
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	105
	Range (%)	98-111
1:8	Average% of Expected	109
	Range (%)	100-125
1:16	Average% of Expected	113
	Range (%)	105-127

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human CLEC9A produced at Sunlong Medical™.