Etanercept mAb-based ELISA Assay


The Etanercept mAb-based ELISA Assay is an enzyme immunoassay for the specific and precise measurement of free Etanercept in serum and plasma samples. The Etanercept ELISA (maB-based) Assay Kit is for research use only and not to be used in diagnostic procedures.

Etanercept mAb-based ELISA Assay

The Etanercept mAb-based ELISA Assay is For Research Use Only

Size: 1 x 96 wells
Sensitivity: 5 ng/mL
Dynamic Range: 6 – 200 ng/mL
Incubation Time: 2 hours
Sample Type: Serum, Plasma
Sample Size: 10 µL
Alternative Names: Enbrel Assay

Assay Principle

This Etanercept ELISA is based on Etanercept-specific mouse monoclonal antibody (catcher Ab, ImmunoGuide clone 5A1). Diluted standards and samples are incubated in the microtiter plate coated with IG-5A1 mAb. After incubation, the wells are washed. A horseradish peroxidase (HRP)-conjugated anti-human IgG monoclonal antibody is added and binds to the Fc part of Etanercept. Following incubation, wells are washed and the bound enzymatic activity is detected by addition of chromogen-substrate. The color developed is proportional to the amount of Etanercept in the sample or standard. Results of samples can be determined by using the standard curve. Binding of Etanercept to the solid phase, pre-coated with 5A1, is inhibited by recombinant human TNF-alpha in a concentration dependent manner. Therefore, the ImmunoGuide Etanercept ELISA (mAb-based) measures the free form of Etanercept. Pipette 100µL of Assay Buffer into each of the wells to be used.

The kit is shipped at ambient temperature and should be stored at 2-8°C. Keep away from heat or direct sun light. The microtiter strips are stable up to the expiry date of the kit in the broken, but tightly closed bag when stored at 2–8°C.

Related Products

Anti-Etanercept (Enbrel) ELISA Kit
Etarnacept (Enbrel) ELISA Assay
Bevacizumab (Avastin®) (mAb-based) ELISA

Additional Information

Assay Background

The drug Etanercept (trade name Enbrel®) is a dimeric fusion protein consisting of the extracellular ligand-binding portion of the human 75 kilodalton (p75) tumor necrosis factor receptor (TNFR) linked to the Fc portion of human IgG1. Etanercept binds specifically to human tumor necrosis factor alpha (TNF-alpha) and blocks its interaction with cell surface TNF receptors. Serum concentration of Etanercept might be related to predict some clinical outcome during maintenance therapy. The specificity of this testsystem is achieved by using a monoclonal antibody named “5A1 mAb” for the coating of the microtiter plate. This antibody is specific for Etanercept only and does not cross react with other TNF-α catchers.

Assay Procedure

  1. Pipette 50 µL of each 1:10 Diluted Standard, and 1:50 Diluted Samples into the respective wells of the microtiter plate.
  2. Cover the plate with adhesive seal. Shake plate carefully. Incubate 60 min at room temperature (RT, 20-25°C).
  3. Remove adhesive seal. Aspirate or decant the incubation solution. Wash the plate 3 X 300 μL of Diluted Wash Buffer per well. Remove excess solution by tapping the inverted plate on a paper towel.
  4. Pipette 100 μL of Enzyme Conjugate (HRP-anti human IgG mAb) into each well.
  5. Cover plate with adhesive seal. Shake plate carefully. Incubate 30 min at RT.
  6. Remove adhesive seal. Aspirate or decant the incubation solution. Wash the plate 3 X 300 μL of Diluted Wash Buffer per well. Remove excess solution by tapping the inverted plate on a paper towel.
  7. Pipette 100 µL of Ready-to-Use TMB Substrate Solution into each well.
  8. Incubate 10 min at RT. Avoid exposure to direct sunlight.
  9. Stop the substrate reaction by adding 100 µL of Stop Solution into each well. Briefly mix contents by gently shaking the plate. Color changes from blue to yellow.
  10. Measure optical density (OD) with a photometer at 450 nm (Reference at OD620 nm is optional) within 15 min after pipetting the Stop Solution.

Typical Standard Curve


Product Manual


Please note: All documents above are for reference use only and should not be used in place of the documents included with this physical product. If digital copies are needed, please contact us.



  • Cheong CU, Chang CP, Chao CM, Cheng BC, Yang CZ, Chio CC. Etanercept attenuates traumatic brain injury in rats by reducing brain TNF- α contents and by stimulating newly formed neurogenesis. Mediators Inflamm. 2013;2013:620837. doi: 10.1155/2013/620837.
  • Jung YS, Park W, Na K. Temperature-modulated noncovalent interaction controllable complex for the long-term delivery of etanercept to treat rheumatoid arthritis. J Control Release. 2013 Oct 28;171(2):143-51.
  • Erdemli Ö, Özen S, Keskin D, Usanmaz A, Batu ED, Atilla B, Tezcaner A. In vitro evaluation of effects of sustained anti-TNF release from MPEG-PCL-MPEG and PCL microspheres on human rheumatoid arthritis synoviocytes. J Biomater Appl. 2014 Oct;29(4):524-42.
  • Chen DY, Chen YM. Response to: ‘Towards optimal cut-off trough levels of adalimumab and etanercept for a good therapeutic response in rheumatoid arthritis. Results of the INMUNOREMAR study’ by Sanmarti et al. Ann Rheum Dis. 2015;74(8):e43. doi: 10.1136/annrheumdis-2015-207543.
  • Jani M, Chinoy H, Warren RB, Griffiths CE, Plant D, Fu B, Morgan AW, Wilson AG, Isaacs JD, Hyrich K, Barton A; Biologics in Rheumatoid Arthritis Genetics and Genomics Study Syndicate Collaborators. Clinical utility of random anti-tumor necrosis factor drug-level testing and measurement of antidrug antibodies on the long-term treatment response in rheumatoid arthritis. Arthritis Rheumatol. 2015 May;67(8):2011-9.
  • Kneepkens EL, Krieckaert CL, van der Kleij D, Nurmohamed MT, van der Horst-Bruinsma IE, Rispens T, Wolbink GJ. Lower etanercept levels are associated with high disease activity in ankylosing spondylitis patients at 24 weeks of follow-up. Ann Rheum Dis. 2015 Oct;74(10):1825-9.
  • Chen DY, Chen YM, Tsai WC, Tseng JC, Chen YH, Hsieh CW, Hung WT, Lan JL. Significant associations of antidrug antibody levels with serum drug trough levels and therapeutic response of adalimumab and etanercept treatment in rheumatoid arthritis. Ann Rheum Dis. 2015 Mar;74(3):e16. doi: 10.1136/annrheumdis-2013-203893.
  • Garcês S, Antunes M, Benito-Garcia E, da Silva JC, Aarden L, Demengeot J. A preliminary algorithm introducing immunogenicity assessment in the management of patients with RA receiving tumour necrosis factor inhibitor therapies. Ann Rheum Dis. 2014 Jun;73(6):1138-43.
  • Mahil SK, Arkir Z, Richards G, Lewis CM, Barker JN, Smith CH. Predicting treatment response in psoriasis using serum levels of adalimumab and etanercept: a single-centre, cohort study. Br J Dermatol. 2013 Aug;169(2):306-13.
  • Sivamani RK, Goodarzi H, Garcia MS, Raychaudhuri SP, Wehrli LN, Ono Y, Maverakis E. Biologic therapies in the treatment of psoriasis: a comprehensive evidence-based basic science and clinical review and a practical guide to tuberculosis monitoring. Clin Rev Allergy Immunol. 2013;44(2):121-40.
  • Hutmacher MM, Nestorov I, Ludden T, Zitnik R, Banfield C. Modeling the exposure-response relationship of etanercept in the treatment of patients with chronic moderate to severe plaque psoriasis. J Clin Pharmacol. 2007;47(2):238-48.
  • Yim DS, Zhou H, Buckwalter M, Nestorov I, Peck CC, Lee H. Population pharmacokinetic analysis and simulation of the time-concentration profile of etanercept in pediatric patients with juvenile rheumatoid arthritis. J Clin Pharmacol. 2005 Mar;45(3):246-56.
  • Andrick BJ, Schwab AI, Cauley B, O’Donnell LA, Meng WS. Predicting Hemagglutinin MHC-II Ligand Analogues in Anti-TNFα Biologics: Implications for Immunogenicity of Pharmaceutical Proteins. PLoS One. 2015 Aug 13;10(8):e0135451. doi: 10.1371/journal.pone.0135451
  • Emery P, Vencovský J, Sylwestrzak A, Leszczyński P, Porawska W, Baranauskaite A, Tseluyko V, Zhdan VM, Stasiuk B, Milasiene R, Barrera Rodriguez AA, Cheong SY, Ghil J. A phase III randomised, double-blind, parallel-group study comparing SB4 with etanercept reference product in patients with active rheumatoid arthritis despite methotrexate therapy. Ann Rheum Dis. 2015 Jul 6. pii: annrheumdis-2015-207588. doi: 10.1136/annrheumdis-2015-207588.
  • Arstikyte I, Kapleryte G, Butrimiene I, Venalis A. Influence of Immunogenicity on the Efficacy of Long-Term Treatment with TNF α Blockers in Rheumatoid Arthritis and Spondyloarthritis Patients. Biomed Res Int. 2015;2015:604872. doi: 10.1155/2015/604872.
  • Baldo BA. Chimeric fusion proteins used for therapy: indications, mechanisms, and safety. Drug Saf. 2015;38(5):455-79.
  • Deehan M, Garcês S, Kramer D, Baker MP, Rat D, Roettger Y, Kromminga A Managing unwanted immunogenicity of biologicals. Autoimmun Rev. 2015;14(7):569-74.
  • Senabre-Gallego JM, Santos-Ramírez C, Santos-Soler G, Salas-Heredia E, Sánchez-Barrioluengo M, Barber X, Rosas J; AIRE-MB group. Long-term safety and efficacy of etanercept in the treatment of ankylosing spondylitis. Patient Prefer Adherence. 2013;7:961-72.
  • Hunt L, Emery P. Etanercept in the treatment of rheumatoid arthritis. Expert Opin Biol Ther. 2013;13(10):1441-50.
  • Jamnitski A, Bartelds GM, Nurmohamed MT, van Schouwenburg PA, van Schaardenburg D, Stapel SO, Dijkmans BA, Aarden L, Wolbink GJ. The presence or absence of antibodies to infliximab or adalimumab determines the outcome of switching to etanercept. Ann Rheum Dis. 2011;70(2):284-8.

Product Citations