Adalimumab (Humira®) ELISA Assay Kit


The Eagle Biosciences Adalimumab ELISA Assay Kit is an enzyme immunoassay for the quantitative determination of free Adalimumab in serum and plasma samples. This assay utilizes the target molecule (TNF-α) as the binding reagent on the microtiter plate so this assay will measure any other therapeutic that targets TNF-α including Entanercept, Infliximab, and Golimumab.  The Adalimumab ELISA Assay Kit is for research use only and not to be used in diagnostic procedures.

Adalimumab (Humira®) ELISA Assay Kit

For Research Use Only

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

Controls Included

Additional Information

Assay Background

The drug Adalimumab (trade name Humira®) is a recombinant human IgG1 monoclonal antibody specific for human tumor necrosis factor alpha (TNF-α). Adalimumab was created using phage display technology resulting in an antibody with human derived heavy and light chain variable regions and human IgG1-Κ constant regions. Adalimumab is produced by recombinant DNA technology in a mammalian cell expression system and it consists of 1330 amino acids and has a molecular weight of approximately 148 kilodaltons. Adalimumab binds specifically to (TNF-α)) and blocks its interaction with the p55 and p75 cell surface TNF receptors. Adalimumab also lyses surface TNF expressing cells in vitro in the presence of complement.

Identification of biomarkers for (non-)response and risk factors for adverse drug reactions that might be related to serum concentrations and maintaining the effective concentration of Adalimumab in order to potentially avoid some side effects with a reliable method might be beneficial.

Assay Principle

This Eagle Biosciences Adalimumab ELISA Assay Kit is based on sandwich type ELISA. Diluted standards and samples (serum or plasma) are incubated in the microtiter plate coated with recombinant human TNF-α (rh TNF-α). 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 Adalimumab pre-captured by the rhTNF-α on the surface of the wells. Following incubation, the wells are washed and the bound enzymatic activity is detected by addition of chromogen-substrate. The color developed is proportional to the amount of free Adalimumab in the sample or standard. Results of samples can be determined by using the standard curve.

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 1:50 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 human IgG mAb) into each well.
  6. Cover plate with adhesive seal. Shake plate carefully. Incubate 30 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 15 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 the Stop Solution.

Typical Standard Curve


Product Manual



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