Human TNF alpha ELISA Kit (HUFI00262)- High Sensitivity

	Save Time	Pre-coated 96 well plate
	Quick Start	Kit includes all necessary reagents
	Publication Ready	Reproducible and reliable results

Product Name:	Human TNF alpha ELISA Kit
SKU:	HUFI00262
Size:	96 Assays
Synonyms:	TNFalpha, Tumor Necrosis Factor Alpha, TNF-alpha, DIF, TNF-alpha, TNFA, TNFSF2
Detection Method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human TNF-alpha concentrations in serum plasma and other biological fluids.
Sensitivity:	9.375pg/ml
Range:	15.625-1000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

TNF-Alpha Background

TNF Alpha is involved in both physiological and pathological processes within the body. It acts as a potent pro-inflammatory cytokine, initiating and amplifying inflammatory responses. Upon activation, TNF Alpha stimulates the production of other cytokines, chemokines, and adhesion molecules, leading to recruitment and activation of immune cells at the site of inflammation. It also promotes the migration of immune cells across the endothelium, facilitating their infiltration into tissues.

TNF-Alpha Strcuture

The TNF Alpha gene, also known as TNF or TNFA, encodes the protein tumor necrosis factor alpha (TNF Alpha). The TNF Alpha gene is located on chromosome 6p21.3 in humans. It consists of four exons and three introns.The TNF Alpha protein is a cytokine that is primarily produced as a transmembrane protein but can also be processed into a soluble form. It belongs to the tumor necrosis factor superfamily and is involved in various biological processes, particularly immune responses and inflammation.

The expression of the TNF Alpha gene is tightly regulated and can be induced by various stimuli, including microbial pathogens, pro-inflammatory cytokines, and cellular stress. Once produced, TNF Alpha acts as a pleiotropic cytokine, exerting its effects through binding to two receptors, TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2), present on the surface of target cells.The binding of TNF Alpha to its receptors initiates a signaling cascade that leads to the activation of various downstream pathways, such as NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and MAPK (mitogen-activated protein kinase) pathways. These pathways regulate gene expression and modulate immune responses, inflammation, cell proliferation, and apoptosis.

Component	Size (96T)	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2 vials	4°C / -20°C
Biotin-labeled Antibody (Concentrated)	120 µL	4°C (Protect from light)
HRP-Streptavidin Conjugate (SABC)	120 µL
TMB Substrate	10 mL
Sample/Standard Dilution Buffer	20 mL	4°C
Antibody Dilution Buffer	10 mL
SABC Dilution Buffer	10 mL
Stop Solution	10 mL
Wash Buffer (25X)	30 mL
Plate Sealer	5 pieces

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer reservoir

Recovery:

Matrices listed below were spiked with certain levels of Human TNF-alpha, and the recovery rates were calculated by comparing the measured value to the expected amount of Human TNF-alpha in samples.

Matrix	Recovery Range (%)	Average (%)
Serum (n=5)	85-105	93
EDTA Plasma (n=5)	91-105	96
UFH Plasma (n=5)	88-105	94

Linearity:

The linearity of the kit was assayed by testing samples spiked with an appropriate concentration of Human TNF-alpha and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
Serum (n=5)	85-103%	87-104%	88-100%
EDTA Plasma (n=5)	83-101%	83-99%	89-100%
UFH Plasma (n=5)	80-97%	82-97%	81-100%

Precision (CV%):

Intra-Assay: CV < 8%
Inter-Assay: CV < 10%

UniProt:	P01375
NCBI GenInfo Identifier:	135934
NCBI Gene ID:	7124
Molecular Weight:	25,644 Da
Protein Family:	Tumor necrosis factor

Note: Protocols are specific to each batch/lot. For the exact instructions, please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Steps	Protocol
Step 1:	Set standard, test sample, and control (zero) wells on the pre-coated plate respectively, and record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample, and control (zero) wells.
Step 2:	Aliquot 0.1 ml standard solutions into the standard wells.
Step 3:	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
Step 4:	Add 0.1 ml of properly diluted sample (Human serum, plasma, tissue homogenates, and other biological fluids) into test sample wells.
Step 5:	Seal the plate with a cover and incubate at 37°C for 90 minutes.
Step 6:	Remove the cover and discard the plate content. Clap the plate on absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate 2 times.
Step 7:	Add 0.1 ml of Biotin-detection antibody working solution into the above wells (standard, test sample, and zero wells). Add the solution at the bottom of each well without touching the side wall.
Step 8:	Seal the plate with a cover and incubate at 37°C for 60 minutes.
Step 9:	Remove the cover, and wash the plate 3 times with Wash buffer. Let the wash buffer rest in wells for 1 min between each wash.
Step 10:	Add 0.1 ml of SABC working solution into each well, cover the plate, and incubate at 37°C for 30 minutes.
Step 11:	Remove the cover and wash the plate 5 times with Wash buffer. Let the wash buffer stay in the wells for 1-2 min each time.
Step 12:	Add 90 µl of TMB substrate into each well, cover the plate, and incubate at 37°C in the dark for 10-20 minutes. (The optimal time should be determined by the end user.) The shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions); the other wells show no obvious color.
Step 13:	Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
Step 14:	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum:	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma:	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 minutes at 1000 × g within 30 minutes of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid:	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 minutes at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant:	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 minutes at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates:	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20°C.
Tissue homogenates:	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 minutes at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates:	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20°C.
Breast Milk:	Collect milk samples and centrifuge at 10,000 x g for 60 minutes at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

What is the TNF-Alpha ELISA Kit used for?

The measurements can help researchers and healthcare professionals understand the role of TNF Alpha in various physiological and pathological processes, monitor disease progression, evaluate therapeutic interventions, and assess the inflammatory status of individuals.

What are the advantages of using the TNF-Alpha ELISA Kit?

The TNF-Alpha ELISA Kit offers several advantages, including high sensitivity, accuracy, and reproducibility. It provides a user-friendly and reliable method to quantify TNF-Alpha levels in biological specimens, allowing for precise measurements and robust data analysis.

What sample types are compatible with TNF-Alpha ELISA Kit?

The TNF-Alpha ELISA Kit is compatible with various sample types, including serum, plasma, cell lysates, and tissue homogenates. It provides flexibility in sample selection, allowing researchers to analyze TNF-Alpha levels in different biological matrices.

What are the storage requirements with TNF-Alpha ELISA Kit?

The TNF-alpha ELISA Kit components should be stored according to the instructions provided in the kit manual. Generally, it is recommended to store the kit components at the recommended temperature to ensure their stability and optimal performance.

What should I do if my assay results are not optimal?

If you encounter any issues or have suboptimal assay results, we recommend contacting our dedicated support team for assistance. They will be available to provide troubleshooting guidance, answer your questions, and ensure you achieve the best possible results with the TNF-alpha ELISA Kit.

Kontomanolis et al.	Cytokine Plasma Levels in Breast Cancer Patients, Before and After Surgery	Oncology and Immunology 2024	PubMed ID: 38386538
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Yusuf et al.	Surface modification of silver nanoparticle (AgNP) by liposomal encapsulation mitigates AgNP-induced inflammation	Nanotechnology and Pharmaceutical Sciences 2019	PubMed ID: 31493545

Human TNF alpha ELISA Kit (HUFI00262)

Description