DESCRIPTION

CD18, also known as integrin beta-2, is a transmembrane protein that forms heterodimers with CD11a, CD11b, CD11c, CD11d (PMID: 1968349). CD11a/CD18 (LFA-1) is expressed by all leukocytes, while CD11b/CD18 (Mac-1) and CD11c/CD18 (p150,95) are normally restricted to expression on monocytes, macrophages, polymorphonuclear lymphocytes (PMNs), and natural killer cells. CD18 plays an important role in mediating cell adhesion during immune response and defects of CD18 cause leukocyte adhesion deficiency.

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 (ng/mL)	SD	CV%
1	8	4.98	0.03	0.60
2	8	1.28	0.01	0.78
3	8	0.64	0.01	1.56

Inter-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	16	4.96	0.05	1.01
2	16	1.28	0.01	0.78
3	16	0.64	0.01	1.56

RECOVERY

The recovery of human CD18 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Human plasma	1:8	90	86-92
	1:16	94	93-95
Cell culture supernatant	1:2	95	93-97
	1:4	102	100-103

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.

		Human plasma	Cell culture supernatant
1:2	Average% of Expected	100	100
	Range (%)	-	-
1:4	Average% of Expected	104	90
	Range (%)	103-105	83-97
1:8	Average% of Expected	116	97
	Range (%)	114-118	93-101
1:16	Average% of Expected	118	91
	Range (%)	117-119	85-98

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human CD18 produced at Sunlong Medical™.