DESCRIPTION

ENTPD1(Ectonucleoside triphosphate diphosphohydrolase 1), also known as CD39 or NTPDase 1, is a cell surface enzyme that plays a crucial role in regulating extracellular nucleotide levels and immune responses. ENTPD1 is highly expressed on regulatory T cells (Tregs), where it contributes to their suppressive functions by depleting extracellular ATP and generating adenosine. It is a noncovalent tetrameric protein, and detergent inhibition of enzymatic activity is caused by dissociation of ectoapyrase tetramers into monomers. Moreover, ENTPD1 is involved in the regulation of dendritic cell function and T cell activation. It modulates the local concentration of nucleotides and nucleosides, which in turn affects immune cell signaling and activation.

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	296.5	21.3	7.2
2	8	77.6	2.0	2.6
3	8	40.5	0.9	2.3

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	297.9	21.0	7.0
2	16	76.8	2.2	2.9
3	16	39.7	1.4	3.6

RECOVERY

The recovery of human CD39/ENTPD1 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Human serum	1:4	85	84-86
	1:8	93	92-95
Tissue lysate	1:2,000	100	98-102
	1:4,000	97	97-98

LINEARITY

To assess the linearity of the assay, human serum samples were spiked with high concentrations of human CD39/ENTPD1 and diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. Tissue lysate samples were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. ( The tissue lysate was initially diluted 1:250.)

		Human serum	Tissue lysate
1:2	Average% of Expected	114	100
	Range (%)	111-118	-
1:4	Average% of Expected	105	104
	Range (%)	103-107	103-105
1:8	Average% of Expected	109	108
	Range (%)	108-110	105-112
1:16	Average% of Expected	126	114
	Range (%)	126-127	111-118

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human CD39/ENTPD1 One-Step ELISA Kit produced at Sunlong Medical™.