DESCRIPTION

CD14 is a 50-55 kDa glycosylphosphatidylinositol-anchored glycoprotein. CD14 is preferentially expressed on monocytes and macrophages. CD14 acts as a co-receptor (along with TLR4 and MD-2) for bacterial liposaccharides (LPS). It plays a major role in the inflammatory response of monocytes to LPS. Soluble forms of CD14 (sCD14) have been detected in cell culture supernatants and in human serum and urine, either shedded from the cell surface or released from intracellular pools. Increased release of sCD14 from monocytes is observed after stimulation with various agents, and sCD14 may therefore be a marker for activation of monocytes/macrophages.

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	1,085.2	58.1	5.4
2	8	263.2	17.5	6.6
3	8	130.1	8.7	6.7

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	1,084.4	73.4	6.8
2	16	265.5	18.3	6.9
3	16	129.9	8.5	6.5

RECOVERY

The recovery of mouse CD14 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Mouse serum	1:800	103	96-114
	1:1,600	96	91-101
Cell culture supernatant	1:8	104	100-107
	1:16	103	99-109

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 mouse serum was initially diluted 1:100.)

		Mouse serum	Cell culture supernatant
1:2	Average% of Expected	100	100
	Range (%)	-	-
1:4	Average% of Expected	88	115
	Range (%)	88-89	110-120
1:8	Average% of Expected	84	111
	Range (%)	83-84	107-115
1:16	Average% of Expected	82	114
	Range (%)	80-85	98-129

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant mouse CD14 produced at Sunlong Medical™.