DESCRIPTION

CD45, also known as protein tyrosine phosphatase, receptor type C, is a type I transmembrane protein expressed on the surface of all haematopoietic cells with the exception of erythrocytes and platelets. CD45 is a pan-haematopoietic cell marker and has been shown to be essential for Tand B-cell activation and signalling.

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	538.7	11.1	2.1
2	8	136.5	5.6	4.1
3	8	69.5	3.4	4.9

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	536.3	11.3	2.1
2	16	134.6	6.7	5.0
3	16	67.9	3.2	4.7

RECOVERY

The recovery of mouse CD45 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Cell culture supernatant	1:8	97	92-100
	1:16	99	97-103
Lysate	1:160	85	74-95

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 1:1,000.)

		Cell culture supernatant	Lysate
1:2	Average% of Expected	100	100
	Range (%)	-	-
1:4	Average% of Expected	110	107
	Range (%)	108-112	97-118
1:8	Average% of Expected	118	109
	Range (%)	113-122	103-115
1:16	Average% of Expected	119	112
	Range (%)	113-125	105-119

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant mouse CD45 produced at Sunlong Medical™.