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Human TWEAK / TNFSF12 ELISA Standard Curve
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Human TWEAK/TNFSF12 DuoSet ELISA Summary

Assay Type
Solid Phase Sandwich ELISA
96-well strip plate
Sample Volume Required
100 µL
Sufficient Materials
For fifteen 96-well plates*
Please see the product datasheet

* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.

This DuoSet ELISA Development kit contains the basic components required for the development of sandwich ELISAs to measure natural and recombinant human TWEAK / TNFSF12. The suggested diluent is suitable for the analysis of most cell culture supernate samples. Diluents for complex matrices, such as serum and plasma, should be evaluated prior to use in this DuoSet ELISA.

Product Features

  • Optimized capture and detection antibody pairings with recommended concentrations save lengthy development time
  • Development protocols are provided to guide further assay optimization
  • Assay can be customized to your specific needs
  • Economical alternative to complete kits

Kit Content

  • Capture Antibody
  • Detection Antibody
  • Recombinant Standard
  • Streptavidin conjugated to horseradish-peroxidase (Streptavidin-HRP)

Other Reagents Required

PBS: (Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2 - 7.4, 0.2 µm filtered

Wash Buffer: (Catalog # WA126), or equivalent

Reagent Diluent: 1% BSA in PBS, pH 7.2-7.4, 0.2 m filtered (R&D Systems Catalog # DY995). Quality of BSA is critical (see Technical Hints).

Blocking Buffer: 1% BSA in PBS, pH 7.2-7.4, 0.2 m filtered (R&D Systems Catalog # DY995). Quality of BSA is critical (see Technical Hints).

Substrate Solution: 1:1 mixture of Color Reagent A (H2O2) and Color Reagent B (Tetramethylbenzidine) (Catalog # DY999)

Stop Solution: 2 N H2SO4 (Catalog # DY994)

Microplates: R&D Systems (Catalog # DY990), or equivalent

Plate Sealers: ELISA Plate Sealers (Catalog # DY992), or equivalent


Data Example

Human TWEAK / TNFSF12 ELISA Standard Curve

Product Datasheets

Preparation and Storage

The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.

Background: TWEAK/TNFSF12

TWEAK is a type II transmembrane protein belonging to the TNF superfamily. The extracellular domains of human and mouse TWEAK share 89% amino acid sequence identity. A soluble form of TWEAK is generated from the membrane-associated molecule by proteolytic cleavage after Arg 93, suggesting that TWEAK may have long-range effects. TWEAK is expressed widely in many tissues and cells.

Long Name:
TNF-like and Weak Inducer of Apoptosis
Entrez Gene IDs:
8742 (Human); 21944 (Mouse); 360548 (Rat)
Alternate Names:
APO3 ligand; APO3LMGC20669; DR3LGTWEAKAPO3/DR3 ligand; MGC129581; TNF-related weak inducer of apoptosis; TNFSF12; tumor necrosis factor (ligand) superfamily, member 12; tumor necrosis factor ligand superfamily member 12; TWEAK

Assay Procedure


Plate Preparation

  1. Dilute the Capture Antibody (to the working concentration stated in the product datasheet ) in PBS without carrier protein. Immediately coat a 96-well microplate with 100 µL per well of the diluted Capture Antibody. Seal the plate and incubate overnight at room temperature.
  2. Aspirate each well and wash with Wash Buffer, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer (400 µL) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or by inverting the plate and blotting it against clean paper towels.
  3. Block each well of the microplate as recommended in the product datasheet. Incubate at room temperature for a minimum of 1 hour.

    Note: The recommended Reagent Diluent typically contains 1% BSA. Some DuoSet Development Kits require alternative blocking agents, or for plates to be blocked overnight with a higher percentage of BSA, please see the product datasheet for details.
  4. Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.


The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face and clothing protection when using this material.

Assay Procedure

  1. Add 100 µL of sample or standards in Reagent Diluent, or an appropriate diluent, per well. Cover with an adhesive strip and incubate 2 hours at room temperature.
  2. Repeat the aspiration/wash as in step 2 of Plate Preparation.
  3. Add 100 µL of the Detection Antibody, diluted in Reagent Diluent (as recommended in the product datasheet), to each well. Cover with a new adhesive strip and incubate 2 hours at room temperature.
  4. Repeat the aspiration/wash as in step 2 of Plate Preparation.
  5. Add 100 µL of the working dilution of Streptavidin-HRP to each well. Cover the plate and incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
  6. Repeat the aspiration/wash as in step 2.
  7. Add 100 µL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
  8. Add 50 µL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
  9. Determine the optical density of each well immediately, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.

Citations for Human TWEAK/TNFSF12 DuoSet ELISA

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

10 Citations: Showing 1 - 10
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  1. Soluble CD163 and TWEAK in early pregnancy gestational diabetes and later glucose intolerance
    Authors: J Dereke, J Nilsson, C Nilsson, H Strevens, M Landin-Ols, M Hillman
    PLoS ONE, 2019;14(5):e0216728.
    Species: Human
    Sample Types: Plasma
  2. Biofluid quantification of TWEAK/Fn14 axis in combination with a selected biomarker panel improves assessment of prostate cancer aggressiveness
    Authors: X Ruiz-Plaza, E Rodríguez-, M Alves, A Altuna-Coy, J Lozano-Bar, M Portero-Ot, JF García-Fon, S Martínez-G, J Segarra, MR Chacón
    J Transl Med, 2019;17(1):307.
    Species: Human
    Sample Types: Serum
  3. Synovial fluid levels of TWEAK and matrix metalloproteinase 1 in patients with osteoarthritis, and associations with disease severity
    Authors: IY Hwang, YS Youm, SD Cho, SW Choi, MH Bae, SJ Park, HW Kim
    J Orthop Surg (Hong Kong), 2018;26(1):2309499018760.
    Species: Human
    Sample Types: Synovial Fluid
  4. Effect of influenza vaccine on tumor necrosis factor-like weak inducer of apoptosis (TWEAK) in older adults
    Authors: M Keshtkar-J, M Ouyang, M Keshtkarja, S Almed, H Li, JD Walston, R Rios, SX Leng
    Vaccine, 2018;0(0):.
    Species: Human
    Sample Types: Serum
  5. Evaluation of interleukin-2, interleukin-8, and tumor necrosis factor-like weak inducer of apoptosis in hemodialysis and renal transplant patients and healthy controls
    Authors: Nasar Alwahaibi
    Saudi J Kidney Dis Transpl, 2016;27(6):1123-1128.
    Species: Human
    Sample Types: Serum
  6. TWEAK/Fn14 system and crescent formation in IgA nephropathy.
    Authors: Sasaki Y, Shimizu Y, Suzuki Y, Horikoshi S, Tomino Y
    BMC Nephrol, 2015;16(0):27.
    Species: Human
    Sample Types: Serum
  7. Reduced circulating sTWEAK levels are associated with metabolic syndrome in elderly individuals at high cardiovascular risk.
    Authors: Diaz-Lopez A, Bullo M, Chacon M, Estruch R, Vendrell J, Diez-Espino J, Fito M, Corella D, Salas-Salvado J
    Cardiovasc Diabetol, 2014;13(0):51.
    Species: Human
    Sample Types: Serum
  8. Serum sCD163 levels are associated with type 2 diabetes mellitus and are influenced by coffee and wine consumption: results of the study.
    Authors: Rojo-Martinez G, Maymo-Masip E, Rodriguez M, Solano E, Goday A, Soriguer F, Valdes S, Chaves F, Delgado E, Colomo N, Hernandez P, Vendrell J, Chacon M
    PLoS ONE, 2014;9(6):e101250.
    Species: Human
    Sample Types: Serum
  9. Lipocalin 2, the TNF-like receptor TWEAKR and its ligand TWEAK act downstream of NFAT1 to regulate breast cancer cell invasion.
    Authors: Gaudineau B, Fougere M, Guaddachi F, Lemoine F, de la Grange P, Jauliac S
    J Cell Sci, 2012;125(0):4475-86.
    Species: Human
    Sample Types: Cell Culture Supernates
  10. Tumour necrosis factor-like weak inducer of apoptosis (TWEAK), an important mediator of endothelial inflammation, is associated with the pathogenesis of Henoch-Schonlein purpura.
    Authors: Chen T, Guo ZP, Li MM, Li JY, Jiao XY, Zhang YH, Liu HJ
    Clin. Exp. Immunol., 2011;166(1):64-71.
    Species: Human
    Sample Types: Serum


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By Anonymous on 09/26/2016
Application: Sample Tested: Serum,EDTA Plasma

We run human serum and plasma samples at a 1:5 dilution in this assay. Due to large variation in human soluble TWEAK levels, a lot of samples need further dilution.