DESCRIPTION

Tumor necrosis factor ligand superfamily member 4 (TNFSF4) is also named as OX40 ligand (OX40L), TXGP1 and CD252. TNFSF4 (OX40L or CD134L) is the only known ligand of TNFRSF4 (OX40 or CD134). It is a type II transmembrane protein that contains the conserved tumor necrosis factor (TNF) homology domain that enables trimerization. TNFSF4 is a co-stimulatory checkpoint protein expressed by several types of immune and non-immune cells, and was shown in the past to enhance the anti-neoplastic activity of T cells. TNFSF4 has long been known to be expressed on antigen presenting cells and to be inducible in T cells. Expression of TNFSF4 by monocytes was shown to promote T follicular helper cell polarization and pathogenesis in human lupus.

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	409.8	12.4	3.0
2	8	113.5	4.8	4.2
3	8	56.0	2.4	4.3

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	423.3	21.1	5.0
2	16	113.9	4.3	3.8
3	16	57.0	2.3	4.1

RECOVERY

The recovery of human OX40L/TNFSF4 spiked to three different levels throughout the range of the assay in human serum was evaluated.

Sample Type		Average% of Expected	Range (%)
Human serum	1:2	79	70-90
	1:4	83	75-93

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 serum
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	104
	Range (%)	103-105
1:8	Average% of Expected	118
	Range (%)	117-119
1:16	Average% of Expected	89
	Range (%)	83-95

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human OX40L/TNFSF4 One-Step ELISA Kit produced at Sunlong Medical™.