Canine TNFa (Tumor Necrosis Factor Alpha) ELISA Kit-EnkiLife

KEY FEATURES

Full Name	Tumor Necrosis Factor Alpha
Synonym	DIF; TNF-A; TNFSF2; Cachectin; Tumor Necrosis Factor Ligand Superfamily Member 2
Assay Type	Sandwich
Reactivity	Canine
Range	15.6-1000pg/mL
Sensitivity	6.1pg/mL
Sample Type	Serum,plasma and other biological fluids
Sample Volume	100μL
Detection Wavelength	OD450
Transportation Temperature	2-8°C
Specificity	The kit detected Canine TNFa in the samples and no significant cross-species reactions were found
Microplate	96-wells plate breakable into 12 x 8 wells strip

TEST PRINCIPLE

This ELISA kit uses the Sandwich-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Canine TNFa. Standards or samples are added to the micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Canine TNFa and Avidin-Horseradish Peroxidase (HRP) conjugate are added successively to each micro plate well and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Canine TNFa, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by the addition of stop solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Canine TNFa. You can calculate the concentration of Canine TNFa in the samples by comparing the OD of the samples to the standard curve.

ELISA KIT COMPONENTS

Upon receipt, unpack promptly and store as recommended in the instructions.

Components	Specifications	Storage and Notes
Micro Plate	96T: 8 wells×12 strips 48T: 8 wells×6 strips	Unopened: -20°C, 12 months Unused: Put it back in the aluminum foil bag and seal it, store it at -20°C.
Reference Standard	96T: 2 vials 48T: 1 vial	Unopened: -20°C, 12 months Please use freshly dissolved standards for each experiment. Discard any unused standards after dissolution.
Biotinylated Detection Ab Concentrate (100×)	96T: 120μL×1 vial 48T: 60μL×1 vial	Unopened: -20°C, 12 months Unused: Please seal the concentrate and store it at -20°C, and discard the working solution.
HRP Conjugate Concentrate (100×)	96T: 120μL×1 vial 48T: 60μL×1 vial	Unopened: -20°C(Protect from light), 12 months Unused: Please seal the concentrate and store it at -20°C, and discard the working solution.
Biotinylated Detection Ab Diluent	14mL×1	2-8℃, 12 months
HRP Conjugate Diluent	14mL×1	2-8℃, 12 months
Reference Standard & Sample Diluent	20mL×1	2-8℃, 12 months
Washing Buffer Concentrate (25×)	30mL×1	2-8℃, 12 months
Substrate Reagent(TMB)	10mL×1	2-8°C(Protect from light),12 months
Stop Solution	7mL×1	2-8°C/Room temperature

ASSAY PROCEDURES

TYPICAL DATA

Canine TNFa ELISA Standard Curve

Typical data are for reference only and curves should be replotted for each experiment. The Logistics function is recommended for fitting.

PRECISION

Intra-Assay Precision (Precision within an assay): Three samples of known concentration were tested twenty times on one plate to assess intra-assay precision.

Inter-Assay Precision (Precision between assays): Three samples of known concentration were tested in twenty separate assays to assess inter-assay precision. Assays were performed by at least three technicians using two lots of components.

	Intra-assay Precision			Inter-assay Precision
Sample	1	2	3	1	2	3
n	20	20	20	20	20	20
Mean (pg/mL)	51.52	128.38	377.95	51.07	134.3	408.87
Standard deviation	3.3	6.65	18.94	3.2	6.98	16.27
CV(%)	6.4	5.18	5.01	6.27	5.2	3.98

RECOVERY

The recovery of Canine TNFa spiked to three different levels in samples throughout the range of the assay in various matrices was evaluated.

Sample Type	Range (%)	Average Recovery (%)
Serum(n=8)	89-100	94
EDTA plasma (n=8)	86-100	93
Cell culture media (n=8)	89-100	95

LINEARITY

To assess the linearity of the assay, samples containing and/or spiked with high concentrations of Canine TNFa in various matrices were diluted with the Reference Standard & Sample Diluent to produce samples with values within the dynamic range of the assay.