DESCRIPTION

CXCL4, alternatively known as platelet factor 4 (PF-4), is the oldest member of the chemokine family. CXCL4 is a chemokine produced by activated platelets and immune cells involved in pathological conditions such as cancer, infections and inflammatory diseases like systemic sclerosis (SSc), rheumatoid arthritis (RA) and psoriatic arthritis (PsA), among others. CXCL4 is present in the α-granules of platelets in amounts that constitute up to 2% platelet protein mass. CXCL4 plays a determinant role in distinct physiological processes such as in hematopoiesis, angiogenesis, coagulation and modulation of immune responses. For instance, CXCL4 can prevent monocyte apoptosis and promote cell survival, induces the production of TNF and reactive oxygen species (ROS) and promotes monocyte differentiation into a unique macrophage-like phenotype.

PRINCIPLE OF THE ASSAY

Sandwich ELISA structure (Detection antibody labeled with biotin) A capture antibody is pre-coated onto the bottom of wells which binds to analyte of interest. A detection antibody labeled with biotin also binds to the analyte. Streptavidin-HRP binds to the biotin. TMB acts as the HRP substrate and the solution color will change from colorless to blue. A stop solution containing sulfuric acid turns 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 20 times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in 24 separate assays to assess inter-assay precision.

Intra-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	20	874.5	84.3	9.6
2	20	207.1	13.7	6.6
3	20	53.5	4.4	8.3

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	24	1,010.2	100.2	9.9
2	24	231.5	19.8	8.6
3	24	50.9	3.7	7.3

RECOVERY

The recovery of mouse CXCL4/PF4 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Mouse serum	1:80,000	96	84-120
	1:160,000	90	82-108
Cell culture supernatant	1:40	100	84-116
	1:80	101	89-116

LINEARITY

To assess the linearity of the assay, mouse serum and cell culture supernatant was diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. (The mouse serum samples were initially diluted 1:10,000, the cell culture supernatant samples were initially diluted 1:5)

		Mouse serum	Cell culture supernatant
1:2	Average% of Expected	100	100
	Range (%)	-	-
1:4	Average% of Expected	94	92
	Range (%)	91-100	89-96
1:8	Average% of Expected	95	100
	Range (%)	90-100	79-115
1:16	Average% of Expected	91	100
	Range (%)	84-102	89-112

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Mouse CXCL4/PF4 Sandwich ELISA Kit produced at Sunlong Medical™.