DESCRIPTION

Tumor necrosis factor (TNF) is a multifunctional cytokine that plays a key role in regulating inflammation, immune functions, host defense, and apoptosis. TNF signals through two distinct cell surface receptors, TNFR1 (TNFRSF1A, CD120a) and TNFR2 (TNFRSF1B, CD120b). TNFR1, which contains a death domain (DD) within its intracytoplasmic region, is thought to be the key receptor for TNF signaling. This receptor can be released to the extracellular space by two mechanisms, ectodomain shedding and constitutive release of full-length 55-kDa TNFR1. Soluble TNFR1 (sTNFR1) could function as TNF-binding protein, inhibiting TNF bioactivity.

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	2,077.5	35.2	1.7
2	8	583.7	9.2	1.6
3	8	265.9	15.4	5.8

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	2,246.0	177.0	7.9
2	16	562.1	33.2	5.9
3	16	281.2	19.6	7.0

RECOVERY

The recovery of mouse TNFR1/CD120a spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Mouse serum	1:8	86	82-90
Cell culture supernatant	1:2	115	107-119
	1:4	82	79-87

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.

		Mouse serum	Cell culture supernatant
1:2	Average% of Expected	100	100
	Range (%)	-	-
1:4	Average% of Expected	126	80
	Range (%)	123-129	76-84
1:8	Average% of Expected	128	72
	Range (%)	127-129	71-72
1:16	Average% of Expected	118	-
	Range (%)	104-132	-

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Mouse TNFR1/CD120a One-Step ELISA Kit produced at Sunlong Medical™.