DESCRIPTION

TNF beta also known as lymphotoxin alpha (LTA), is a member of the tumor necrosis factor family, and is a cytokine produced by lymphocytes. TNF beta is highly inducible, secreted, and forms heterotrimers with lymphotoxin-beta which anchor lymphotoxinalpha to the cell surface. TNF beta also mediates a large variety of inflammatory, immunostimulatory, and antiviral responses, is involved in the formation of secondary lymphoid organs during development and plays a role in apoptosis.

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	245.0	2.5	1.0
2	8	76.0	1.3	1.7
3	8	43.6	1.0	2.3

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	246.5	3.6	1.5
2	16	75.3	1.5	2.0
3	16	42.7	1.4	3.3

RECOVERY

The recovery of human/cynomolgus monkey TNF beta spiked to three different levels throughout the range of the assay in cell culture supernatant was evaluated.

Sample Type		Average% of Expected	Range (%)
Human cell culture supernatant	1:2	104	103-106
	1:4	104	104-105
Cynomolgus monkey cell culturesupernatant	1:2	97	94-103
	1:4	99	96-101

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 human cell culture supernatant was initially diluted 1:10. The cynomolgus monkey cell culture supernatant was initially diluted 1:2.)

		Human cell culture supernatant	Cynomolgus monkey cell culturesupernatant
1:2	Average% of Expected	100	100
	Range (%)	-	-
1:4	Average% of Expected	108	105
	Range (%)	106-110	101-108
1:8	Average% of Expected	114	104
	Range (%)	111-118	100-108
1:16	Average% of Expected	126	102
	Range (%)	122-131	101-103

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human/Cynomolgus Monkey TNF beta One-Step ELISA Kit produced at Sunlong Medical™.