DESCRIPTION

CD27 (also known as TNFRSF7) is a lymphocyte-specific member of the TNF/NGF-R family, which is found in NK, T, and B cell populations. CD27 is a T and B cell co-stimulatory molecule. Its activity is governed by the transient availability of its TNF-like ligand CD70 on lymphocytes and dendritic cells. This interaction may have a key role in controlling the size and function of antigen-primed lymphocyte populations. CD27 is also required for the effective generation and long-term maintenance of antigen-specific CD4+ and CD8+ T cell immunity.

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	465.2	28.5	6.1
2	8	114.2	2.8	2.5
3	8	33.6	0.9	2.7

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	457.7	33.5	7.3
2	16	114.7	3.0	2.6
3	16	34.5	1.2	3.5

RECOVERY

The recovery of mouse CD27 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Mouse serum	1:80	104	98-114
Tissue lysate	1:1,280	98	86-112

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 1:5. The tissue lysate was initially diluted 1:80.)

		Mouse serum	Tissue lysate
1:2	Average% of Expected	100	100
	Range (%)	-	-
1:4	Average% of Expected	109	99
	Range (%)	108-110	94-104
1:8	Average% of Expected	112	111
	Range (%)	109-114	104-115
1:16	Average% of Expected	107	108
	Range (%)	93-120	106-111

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant mouse CD27 produced at Sunlong Medical™.