DESCRIPTION

CD2 is a transmembrane glycoprotein of the immunoglobulin superfamily expressed on the surface of T cells, NK cells, thymocytes, and dendritic cells. The CD2 antigen was identified as one of the earliest T cell markers and was shown to be important for thymocyte development. CD2 stimuli were mandatory for Treg cell survival with reduced Bim expression. Moreover, its down-regulation may attenuate anti-tumor T-cell responses with implications for checkpoint immunotherapies.

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 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	733.2	41.7	5.7
2	8	183.5	8.7	4.8
3	8	88.9	4.3	4.9

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	715.4	44.5	6.2
2	16	180.9	11.6	6.4
3	16	88.0	4.8	5.5

RECOVERY

The recovery of mouse CD2 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Mouse serum	1:32	110	98-114
Tissue lysate	1:1,024	99	95-106

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 1:2. The tissue lysate was initially diluted 1:64.)

		Mouse serum	Tissue lysate
1:2	Average% of Expected	100	100
	Range (%)	-	-
1:4	Average% of Expected	114	101
	Range (%)	107-119	99-103
1:8	Average% of Expected	124	107
	Range (%)	123-125	105-108
1:16	Average% of Expected	125	106
	Range (%)	121-128	105-107

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant mouse CD2 produced at Sunlong Medical™.