DESCRIPTION

The chemokine CXCL16, a member of the ELR-negative CXC subfamily, signals exclusively through its receptor CXCR6, a classic G protein-coupled receptor. Beyond its broad expression on dendritic cells, monocytes, macrophages, and B cells, the CXCR6/CXCL16 axis has emerged as a critical player in autoimmune pathogenesis. Its clinical relevance is highlighted by its strong association with disease severity, activity, and prognosis in a spectrum of disorders, including multiple sclerosis, autoimmune hepatitis, rheumatoid arthritis, Crohn's disease, and psoriasis.

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	576.4	15.6	2.7
2	8	139.2	3.1	2.2
3	8	70.5	1.3	1.8

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	569.8	18.9	3.3
2	16	140.9	4.0	2.8
3	16	70.4	1.6	2.3

RECOVERY

The recovery of mouse CXCL16 spiked to three different levels throughout the range of the assay was evaluated.

Sample Type		Average% of Expected	Range (%)
Mouse serum	1:32	91	87-98
	1:64	87	78-94
Cell culture supernatant	1:128	96	71-120
	1:256	86	77-91

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 1:4. The cell culture supernatant was initially diluted 1:2.)

		Mouse serum	Cell culture supernatant
1:2	Average% of Expected	100	100
	Range (%)	-	-
1:4	Average% of Expected	103	92
	Range (%)	100-106	90-94
1:8	Average% of Expected	95	85
	Range (%)	91-100	83-86
1:16	Average% of Expected	99	85
	Range (%)	99-100	85-86

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant mouse CXCL16 produced at Sunlong Medical™.