DESCRIPTION

CD99 is a highly O-glycosylated type I transmembrane protein that is mainly expressed in cells of the hematopoietic system and at the intercellular borders of endothelial cells. CD99 is essential for transendothelial migration (TEM) of monocytes, neutrophils, and lymphocytes. It is frequently overexpressed in many types of tumors, particularly pediatric tumors including Ewing sarcoma and specific subtypes of leukemia. CD99 is also a novel marker of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) stem cells.

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	1,218.8	42.5	3.5
2	8	297.4	5.8	2.0
3	8	142.2	4.8	3.4

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	1,202.0	47.0	3.9
2	16	297.9	12.0	4.0
3	16	142.7	6.7	4.7

RECOVERY

The recovery of human CD99 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	91	86-96
	1:16	91	88-94
Urine	1:400	95	91-97
	1:800	97	96-97
Cell lysate	1:160	95	93-100
	1:320	94	92-96

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 urine was initially diluted 1:100. The cell lysate was initially diluted 1:80.)

		Cell culturesupernatant (SampleDiluent PT 4B1)	Urine (Sample DiluentPT 6-em)	Cell lysate (SampleDiluent PT 4B1)
1:2	Average% of Expected	100	100	100
	Range (%)	-	-	-
1:4	Average% of Expected	103	87	107
	Range (%)	100-107	85-90	101-113
1:8	Average% of Expected	121	83	100
	Range (%)	114-128	80-86	99-101
1:16	Average% of Expected	106	84	97
	Range (%)	104-108	79-88	94-100

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human CD99 produced at Sunlong Medical™.