DESCRIPTION

CD69 is a type II C-type lectin receptor that serves as an early activation marker for lymphocytes. It is rapidly expressed on the surface of T cells, B cells, and natural killer (NK) cells following activation. CD69 plays a crucial role in lymphocyte proliferation and functions as a signal-transmitting receptor, influencing cell cycle progression and cytokine production. It also regulates the differentiation of T helper (Th) subsets, including Th1, Th2, Th17, and regulatory T cells (Tregs), by modulating intracellular signaling pathways and cytokine secretion.

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	544.9	22.3	4.1
2	8	137.0	5.1	3.7
3	8	64.5	2.8	4.3

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	546.9	17.2	3.1
2	16	138.0	7.1	5.1
3	16	64.9	2.5	3.9

RECOVERY

The recovery of human CD69 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Human plasma	1:4	105	98-114
	1:8	103	97-111
Cell lysate	1:16	110	102-118

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 cell lysate was initially diluted 1:32.)

		Human plasma(Sample Diluent PT 3-ef)	Cell lysate(Sample Diluent PT 4B1)
1:2	Average% of Expected	100	100
	Range (%)	-	-
1:4	Average% of Expected	106	89
	Range (%)	104-109	79-100
1:8	Average% of Expected	107	99
	Range (%)	94-119	94-105
1:16	Average% of Expected	78	92
	Range (%)	71-85	78-106

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human CD69 produced at Sunlong Medical™.