DESCRIPTION

CD44, a transmembrane cell surface glycoprotein, is normally expressed in all human cells. It is primarily involved in regulating cell adhesion, migration, angiogenesis, proliferation, and inflammation. It may take part in the process of tumorigenesis as CD44 and its variants have a potential role in the development of malignant tumors. CD44 has gained significant attention because of its usefulness as a stem cell marker and has surfaced as a budding therapeutic target, imposing a greater understanding of CD44 in various cancers. Additionally, Serum CD44 concentration correlated with tumor burden and metastasis of tumors.

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	3,137.1	120.5	3.8
2	8	791.1	33.3	4.2
3	8	352.5	23.9	6.8

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	3,177.9	111.6	3.5
2	16	785.3	33.0	4.2
3	16	365.3	26.3	7.2

RECOVERY

The recovery of human CD44 spiked to three different levels throughout the range of the assay in human serum was evaluated.

Sample Type		Average% of Expected	Range (%)
Human serum	1:800	106	100-111
	1:1,600	108	99-118

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 human serum was initially diluted 1:200.)

		Human serum
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	99
	Range (%)	97-100
1:8	Average% of Expected	96
	Range (%)	90-103
1:16	Average% of Expected	97
	Range (%)	95-100

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human CD44 produced at Sunlong Medical™.