Tocilizumab (Actemra) ELISA (mAb-based) Assay Kit

$1,395.00

The Eagle Biosicences Tocilizumab ELISA (mAb-based) Assay Kit is an enzyme immunoassay for the specific and precise measurement of free Tocilizumab in serum and plasma samples. This assay utilizes the specific monoclonal antibodies as the coating material on the microtiter plate so this assay specifically measures only Tocilizumab and no other TNF-α therapeutic.  The Eagle Biosciences Tocilizumab ELISA (mAb-based) Assay Kit is for research use only and not to be used in diagnostic procedures.

Tocilizumab (Actemra) ELISA (mAb-based) Assay Kit

For Research Use Only

Size: 1 x 96 wells
Sensitivity: 2 ng/mL
Dynamic Range: 6 – 200 ng/mL
Incubation Time: 2.5 hours
Sample Type: Serum, Plasma
Sample Size: 10 µL

Additional Information

Assay Background


The drug Tocilizumab (trade name Actemra®) is a humanized monoclonal antibody (mAb) that specifically targets both soluble interleukin-6 receptor (sIL-6R) and membrane bound interleukin-6 receptor (mIL-6R) with high affinity, thereby preventing pro-inflammatory effects of IL-6. The specificity of this testsystem is achieved by using a monoclonal antibody (clon IG-7C84) for the coating of the microtiter plate and also as a conjugate.
This Eagle Biosciences Tocilizumab ELISA (mAb-based) is developed for the specific measurement of Tocilizumab in sera, plasma and other biological fluids by the advantage of using a site-directed IG-7C84 mouse monoclonal antibody (mAb) specific for Tocilizumab only. Binding of Tocilizumab to the solid phase, pre-coated with IG-7C84, is inhibited by recombinant human interleukin-6 receptor alpha (rh-IL6Ra) in a concentration dependent manner. Therefore, this Tocilizumab ELISA (mAb-Based) measures the free form of Tocilizumab. The choice of specifically measuring the free form allows investigators to analyze the concentration-effect relationship. Due to the use of a mAb as catcher and also as detection reagent this test kit has an unmatched specificity.

Assay Principle


This ELISA is based on a Tocilizumab-specific monoclonal antibody (catcher Ab, ImmunoGuide clone IG-7C84). Diluted standards and diluted samples are incubated in the microtiter plate coated with IG-7C84 mAb. After incubation, the wells are washed. A horseradish peroxidase (HRP)-conjugated anti-Tocilizumab antibody is added and binds to the Tocilizumab. Following incubation, the wells are washed and the bound enzymatic activity is detected by addition of chromogen-substrate. The colour developed is proportional to the amount of Tocilizumab in the sample or standard. Results of samples can be determined by using the standard curve. Binding of Tocilizumab to the solid phase, pre-coated with IG-7C84, is inhibited by recombinant human interleukin-6 receptor-α (rh-IL6Rα) in a concentration dependent manner. Therefore, the ImmunoGuide Tocilizumab ELISA (mAb-Based) measures the free form of Tocilizumab.

Assay Procedure


  1. Pipette 100 µL of Assay Buffer into each of the wells to be used.
  2. Pipette 75 µL of each 1:10 Diluted Standard, and 1:1000 Diluted Samples into the respective wells of the microtiter plate.
  3. Cover the plate with adhesive seal. Shake plate carefully. Incubate 60 min at room temperature (RT, 20-25°C).
  4. 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.
  5. Pipette 100 μL of Enzyme Conjugate (HRP-anti Tocilizumab) into each well.
  6. Cover plate with adhesive seal. Shake plate carefully. Incubate 60 min at RT.
  7. 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.
  8. Pipette 100 µL of Ready-to-Use TMB Substrate Solution into each well.
  9. Incubate 10 min at RT. Avoid exposure to direct sunlight.
  10. 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.
  11. Measure optical density (OD) with a photometer at 450 nm (Reference at OD620 nm is optional) within 15 min after pipetting.

Typical Standard Curve


Manual

Product Manual


Publications

Citations


  • Inciarte-Mundo J, Ruiz-Esquide V, Hernández MV, Cañete JD, Cabrera-Villalba SR, Ramirez J, Yagüe J, Sanmarti R. Calprotectin more accurately discriminates the disease status of rheumatoid arthritis patients receiving tocilizumab than acute phase reactants. Rheumatology (Oxford). 2015 Aug 4. pii: kev251. [Epub ahead of print]
  • Kennedy GA, Varelias A, Vuckovic S, Le Texier L, Gartlan KH, Zhang P, Thomas G, Anderson L, Boyle G, Cloonan N, Leach J, Sturgeon E, Avery J, Olver SD, Lor M, Misra AK, Hutchins C, Morton AJ, Durrant ST, Subramoniapillai E, Butler JP, Curley CI, MacDonald KP, Tey SK, Hill GR. Addition of interleukin-6 inhibition with tocilizumab to standard graft-versus-host disease prophylaxis after allogeneic stem-cell transplantation: a phase 1/2 trial. Lancet Oncol. 2014;15(13):1451-9
  • Besada E. Potential patient benefit of a subcutaneous formulation of tocilizumab for the treatment of rheumatoid arthritis: a critical review. Patient Prefer Adherence. 2014;8:1051-9.
  • Ohta S, Tsuru T, Terao K, Mogi S, Suzaki M, Shono E, Ishida Y, Tarumi E, Imai M. Mechanism-based approach using a biomarker response to evaluate tocilizumab subcutaneous injection in patients with rheumatoid arthritis with an inadequate response to synthetic DMARDs (MATSURI study). J Clin Pharmacol. 2014;54(1):109-19.
  • Lee SJ, Park W, Park SH, Shim SC, Baek HJ, Yoo DH, Kim HA, Lee SK, Leee YJ, Park YE, Cha HS, Park JK, Lee EY, Lee EB, Song YW. Low baseline interleukin-17A levels are associated with better treatment response at 12 weeks to tocilizumab therapy in rheumatoid arthritis patients. J Immunol Res. 2015;2015:487230. doi: 10.1155/2015/487230. Epub 2015 Apr 2
  • Ogata A, Tanimura K, Sugimoto T, Inoue H, Urata Y, Matsubara T, Kondo M, Ueki Y, Iwahashi M, Tohma S, Ohta S, Saeki Y, Tanaka T; Musashi Study Investigators. Phase III study of the efficacy and safety of subcutaneous versus intravenous tocilizumab monotherapy in patients with rheumatoid arthritis. Arthritis Care Res (Hoboken). 2014;66(3):344-54.
  • Mori S, Ueki Y. Primary lack of efficacy of infliximab therapy for rheumatoid arthritis: pharmacokinetic characterization and assessment of switching to tocilizumab. Mod Rheumatol. 2011;21(6):628-36.
  • Zhang X, Peck R. Clinical pharmacology of tocilizumab for the treatment of patients with rheumatoid arthritis. Expert Rev Clin Pharmacol. 2011 Sep;4(5):539-58. doi: 10.1586/ecp.11.33.
  • Burmester GR, Rigby WF, van Vollenhoven RF, Kay J, Rubbert-Roth A, Kelman A, Dimonaco S, Mitchell N. Tocilizumab in early progressive rheumatoid arthritis: FUNCTION, a randomised controlled trial. Ann Rheum Dis. 2015 Oct 28. pii: annrheumdis-2015-207628. doi: 10.1136/annrheumdis-2015-207628. [Epub ahead of print]
  • Gabay C, Riek M, Hetland ML, Hauge EM, Pavelka K, Tomšič M, Canhao H, Chatzdionysiou K, Lukina G, Nordström DC, Lie E, Ancuta I, Hernández MV, van Riel PL, van Vollenhoven R, Kvien TK. Effectiveness of tocilizumab with and without synthetic disease-modifying antirheumatic drugs in rheumatoid arthritis: results from a European collaborative study. Ann Rheum Dis. 2015 Sep 15. pii: annrheumdis-2015-207760. doi: 10.1136/annrheumdis-2015-207760
  • Espinoza F, Jorgensen C, Pers YM. Efficacy of Tocilizumab in the treatment of Eosinophilic fasciitis: Report of one case. Joint Bone Spine. 2015 Jul 7. pii: S1297-319X(15)00101-3. doi: 10.1016/j.jbspin.2015.02.008. [Epub ahead of print]
  • Roddy JV, Haverkos BM, McBride A, Leininger KM, Jaglowski S, Penza S, Klisovic R, Blum W, Vasu S, Hofmeister CC, Benson DM, Andritsos LA, Devine SM, Efebera YA. Tocilizumab for steroid refractory acute graft-versus-host disease. Leuk Lymphoma. 2015 Jul 3:1-5. [Epub ahead of print]
  • Kim NH, Kim SK, Kim DS, Zhang D, Park JA, Yi H, Kim JS, Shin HC. Anti-proliferative action of IL-6R-targeted antibody tocilizumab for non-small cell lung cancer cells. Oncol Lett. 2015;9(5):2283-2288.
    Hammoudeh M, Al Awadhi A, Hasan EH, Akhlaghi M, Ahmadzadeh A, Sadeghi Abdollahi B Safety, Tolerability, and Efficacy of Tocilizumab in Rheumatoid Arthritis: An Open-Label Phase 4 Study in Patients from the Middle East. Int J Rheumatol. 2015;2015:975028. doi: 10.1155/2015/975028.
  • Yang L, Xing R, Li C, Liu Y, Sun L, Liu X, Wang Y. Active immunization with Tocilizumab mimotopes induces specific immune responses. BMC Biotechnol. 2015 Jun 2;15:46. doi: 10.1186/s12896-015-0161-9.
  • Burmester GR, Rubbert-Roth A, Cantagrel A, Hall S, Leszczynski P, Feldman D, Rangaraj MJ, Roane G, Ludivico C, Bao M, Rowell L, Davies C, Mysler EF. Efficacy and safety of subcutaneous tocilizumab versus intravenous tocilizumab in combination with traditional DMARDs in patients with RA at week 97 (SUMMACTA). Ann Rheum Dis. 2015 Jun 8. pii: annrheumdis-2015-207281. doi: 10.1136/annrheumdis-2015-207281.
  • Romão VC, Santos MJ, Polido-Pereira J, Duarte C, Nero P, Miguel C, Costa JA, Bernardes M, Pimentel-Santos FM, Barcelos F, Costa L, Melo Gomes JA, Pereira da Silva JA, Cunha Branco J, Canas da Silva J, Pereira da Silva JA, Fonseca JE, Canhão H. Comparative Effectiveness of Tocilizumab and TNF Inhibitors in Rheumatoid Arthritis Patients: Data from the Rheumatic Diseases Portuguese Register, Reuma.pt. Biomed Res Int. 2015;2015:279890. doi: 10.1155/2015/279890
  • Wang G, Mu R, Xu H. Management of rheumatoid arthritis in People’s Republic of China – focus on tocilizumab and patient considerations. Int J Gen Med. 2015;8:187-94.
  • Bensaoud N, Rostom S, Bahiri R, Hajjaj-Hassouni N. Efficacy of tocilizumab on MRI-determined bone oedema in rheumatoid arthritis. Clin Rheumatol. 2015;34(6):1031-7.
  • Narváez J, Díaz-Torné C, Magallares B, Hernández MV, Reina D, Corominas H, Sanmartí R, de la Serna AR, Llobet JM, Nolla JM. Comparative effectiveness of tocilizumab with either methotrexate or leflunomide in the treatment of rheumatoid arthritis. PLoS One. 2015 Apr 1;10(4):e0123392. doi: 10.1371/journal.pone.0123392. eCollection 2015