DESCRIPTION

CD5 antigen (CD5, also known as Ly-1, Ly-A, Ly-12, and Lyt-1) is a pan-T cell marker and a negative regulator of TCR and BCR signaling, which increases expression during T-cell development in the thymus. It is associated with the B cell receptor (BCR) complex and is a substrate for BCR-induced tyrosine kinase activity. In addition to membrane-bound CD5, the circulating soluble form of CD5 (sCD5) has been shown in sera resulting from proteolytic cleavage following lymphocyte activation. The expression level of sCD5 is associated with the development of autoimmune and inflammatory disorders.

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 20 times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in 24 separate assays to assess inter-assay precision.

Intra-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	20	670.0	36.3	5.4
2	20	165.7	8.7	5.3
3	20	40.1	2.7	6.6

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	24	743.3	36.7	4.9
2	24	170.6	10.1	5.9
3	24	40.5	1.8	4.4

RECOVERY

The recovery of human CD5 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	110	98-129
	1:8	109	89-127
Cell lysate	1:3,200	89	86-91
	1:6,400	90	85-96

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 1:400.)

		Human serum	Cell lysate
1:2	Average% of Expected	100	100
	Range (%)	-	-
1:4	Average% of Expected	100	93
	Range (%)	99-101	90-96
1:8	Average% of Expected	88	94
	Range (%)	83-94	93-95
1:16	Average% of Expected	-	88
	Range (%)	-	87-89

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human CD5 produced at Sunlong Medical™.