Human TNF-alpha (Tumor Necrosis Factor Alpha) QuickTest ELISA Kit

Mfr.No.

QT-EH0302

Description

This kit was based on sandwich ELISA method. The experiment lasted 120 minutes. Capture antibody was conjugated to an affinity tag that was recognized by a specific antibody coated on the QuickTest plate. Add the Cap/Det Ab working solution into each well, then add the standards and pilot samples into individual wells. If the sample contains TNF-alpha, a capture antibody-TNF-alpha-biotin-detection antibody complex was formed. After incubation, unbound conjugates were removed by wash buffer. HRP-Streptavidin was added. After washing, TMB substrates were added to visualize HRP enzymatic reaction. TMB was catalyzed by HRP to produce a blue color product that turned yellow after adding a stop solution. Read the O.D. absorbance at 450nm in a microplate reader. The concentration of TNF-alpha in the sample was calculated by drawing a standard curve. The concentration of the target substance is proportional to the OD450 value.

Alternative Names

Tumor necrosis factor ELISA Kit, Cachectin ELISA Kit, TNF-alpha ELISA Kit, Tumor necrosis factor ligand superfamily member 2 ELISA Kit, TNF-a ELISA Kit, Tumor necrosis factor, membrane form ELISA Kit, N-terminal fragment ELISA Kit, NTF ELISA Kit, Intracellular domain 1 ELISA Kit, ICD1 ELISA Kit, Intracellular domain 2 ELISA Kit, ICD2 ELISA Kit, C-domain 1 ELISA Kit, C-domain 2 ELISA Kit, Tumor necrosis factor, soluble form ELISA Kit, TNF ELISA Kit, TNFA ELISA Kit, TNFSF2 ELISA Kit

Reactivity

Human

Detection Method

Double Antibody

Detection Range

15.625-1000pg/ml

Sample Type

Cell lysates, Tissue lysate, Other biological fluids, Serum, Plasma, Cell culture supernatants

Assay Type

Sandwich

Assay Duration

120 minutes

Kit Components

Kit Components	Item	Size(48T)	Size(96T)	Storage Condition for Opened Kit
E001	ELISA Microplate(Dismountable)	8x6	8x12	Put the rest strips into a sealed foil bag with the desiccant. Stored for 1 month at 2-8°C; Stored for 6 month at -20°C
E002	Lyophilized Standard	1vial	2vial	Put the rest standards into a desiccant bag. Stored for 1 month at 2-8°C; Stored for 6 month at -20°C
E054	Cap/Det Ab (Ready to use, blue)	3ml	6ml	2-8°C (Avoid Direct Light)
E053	HRP-Streptavidin (Ready to use, orange)	5ml	10ml
E024	TMB Substrate	5ml	10ml
E039	Sample Dilution Buffer	20ml	20ml	2-8°C
E026	Stop Solution	5ml	5ml
E038	Wash Buffer(25X)	15ml	30ml
E006	Plate Sealer	3 pieces	5 pieces
E007	Product Description	1 copy	1 copy

Recovery

Add a certain amount of TNF-alpha into the sample. Calculate the recovery by comparing the measured value with the expected amount of TNF-alpha in the sample.

Sample Type	Recovery Range(%)	Average(%)
serum(n=10)	92-103	95
EDTA plasma(n=10)	92-99	93
Heparin plasma(n=10)	86-99	94

Linearity

Dilute the sample with a certain amount of TNF-alpha at 1:2, 1:4 and 1:8 to get the recovery range.

Sample Type	1:2	1:4	1:8
serum(n=10)	81-93%	82-95%	86-95%
EDTA plasma(n=10)	86-103%	84-100%	83-94%
Heparin plasma(n=10)	85-96%	80-95%	81-99%

Sensitivity

9.375pg/ml

Standard Curve

This product is detected by QC department and meets performance required in the manual. (Laboratory Humidity: 20%-60%; Temperature: 18°C -25°C; Equilibrate TMB substrate to 37°C before staining. After adding into the ELISA wells, incubate for 15min at 37°C in dark.)
Due to different assay environments and operations, assay data below and standard curve are provided for reference. Experimenters should establish standard curve according to their own assay.

STD.(pg/ml)	OD-1	OD-2	Average
0	0.142	0.149	0.145
15.625	0.187	0.197	0.191
31.25	0.246	0.258	0.251
62.5	0.302	0.317	0.308
125	0.422	0.444	0.431
250	0.746	0.784	0.761
500	1.256	1.32	1.281
1000	2.05	2.154	2.092

Assay Procedure Summary

Step 1:
Take out the required plate wells, add 50ul Cap/Det Ab into each well, then add 50ul Standard or Sample into individual well. (When adding standard or sample, the disposable tip lightly touches the liquid level. Change the disposable tips for different samples and standards.) Gently tap the plate for 10s to ensure thorough mixing then static incubate for 60 minutes at 37°C.

Washing:
Wash the plate twice without immersion.

Step 2:
Add 100ul HRP-Streptavidin (orange) into each well, seal the plate and static incubate for 30 minutes at 37°C.

Washing:
Wash the plate five times without immersion.

Step 3:
Add 90ul TMB substrate solution, seal the plate and static incubate for 10-20 minutes at 37°C. (Accurate TMB visualization control is required.)

Step 4:
Add 50ul stop solution. Read at 450nm immediately and calculate.

Stability

Perform the stability test for the sealed kit at 37°C and 2-8°C and get relevant data.

ELISA kit(n=5)	37°C for 1 month	2-8°C for 6 months
Average(%)	80	95-100

Notes

Specifically binds with TNF-alpha , no obvious cross reaction with other analogues.

UniProt ID	P01375
Usage Notes	Microplate reader (wavelength: 450nm) 37°C incubator (CO2 incubator for cell culture is not recommenced.) Automated plate washer or multi-channel pipette/5ml pipettor (for manual washing purpose) Precision single (0.5-10uL, 5-50uL, 20-200uL, 200-1000uL) and multi-channel pipette with disposable tips(Calibration is required before use.) Sterile tubes and Eppendorf tubes with disposable tips Absorbent paper and loading slot Deionized or distilled water

Precision

Intra-assay Precision: samples with low, medium and high concentration are tested 20 times on the same plate.
Inter-assay Precision: samples with low, medium and high concentration are tested 20 times on three different plates.

Item	Intra-assay Precision			Inter-assay Precision
Sample	1	2	3	1	2	3
n	20	20	20	20	20	20
Mean (pg/ml)	31.68	118.4	485	32.21	125.2	494
Standard deviation	1.76	7.09	28.71	1.78	7.32	27.12
CV(%)	5.56	5.99	5.92	5.53	5.85	5.49

Storage	2-8°C(Sealed), Don't cryopreserve.

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SKU	Supplier No.	Your price	Quantity
BHE10810147	QT-EH0302	$280		Add to Cart

Human TNF-alpha (Tumor Necrosis Factor Alpha) QuickTest ELISA Kit

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