DESCRIPTION

A glycosylphosphatidyl-inositol-(GPI)-anchored glycoprotein, carcinoembryonic antigen (CEA)-related cell adhesion molecule 8 (CEACAM8), also known as CD66b, NCA-95 and CD67, was identified in human neutrophils. It is solely expressed by granulocytes, of which 95% are polymorphonuclear neutrophils (PMN). CEACAM8 is cell-adhesion proteins on neutrophils that belong to the human carcinoembryonic antigen (CEA) family. CEA family members are believed to function as intercellular adhesion molecules in vitro. CEACAM8 has been shown to adhere to the closely related molecule CEACAM6 (NCA-50/90 and CD66c), suggesting a role in the interaction between granulocytes or between granulocytes and epithelial cells expressing CEACAM6 in vivo.

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	1,636.4	27.3	1.7
2	8	412.0	10.7	2.6
3	8	211.2	4.6	2.2

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	1,629.4	38.2	2.3
2	16	412.1	8.1	2.0
3	16	209.7	4.1	2.0

RECOVERY

The recovery of human CEACAM8/CD66b spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Human plasma	1:4	95	91-98
	1:8	109	104-117
Cell culture supernatant	1:2	115	105-123
	1:4	111	106-120

LINEARITY

To assess the linearity of the assay, cell culture supernatant samples were spiked with high concentrations of human CEACAM8/CD66b and diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. Human plasma samples were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay.

		Human plasma	Cell culture supernatant
1:2	Average% of Expected	100	107
	Range (%)	-	102-112
1:4	Average% of Expected	107	101
	Range (%)	106-108	99-104
1:8	Average% of Expected	104	102
	Range (%)	96-112	100-105
1:16	Average% of Expected	95	106
	Range (%)	93-97	103-110

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human CEACAM8/CD66b One-Step ELISA Kit produced at Sunlong Medical™.