Rituximab mAb-based ELISA Assay


The Rituximab mAb-based ELISA Assay is developed for specific measurement of Rituximab in serum and plasma.  The kit is a solid phase enzyme-linked immunosorbent assay (ELISA) is based on rituximab-specific 9F9 monoclonal antibody (mAb).  The Rituximab (Rituxan®, Mabthera®) ELISA (mAb-based) Assay Kit  is for research use only and is not intended for diagnostic or therapeutic procedures.

Rituximab mAb-based ELISA Assay

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

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

Controls Included

Assay Background

The drug Rituximab (trade name Rituxan® and Mabthera®) is a genetically engineered chimeric murine/human monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes. The antibody is a glycosylated IgG1 kappa immunoglobulin containing murine light- and heavy-chain variable region sequences (Fab domain) and human constant region sequences (Fc domain). Rituximab is composed of 1,328 amino acids and has an approximate molecular weight of 144 kD. Rituximab has a high binding affinity for the CD20 antigen.

The specificity of this test system is achieved by using a monoclonal antibody (clon 9D5b) for the coating of the microtiter plate. This antibody is specific for Rituximab only (regardless whether Rituxan ® and Mabthera ®) and does not cross react with other CD20 catchers.

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. The usual precautions for venipuncture should be observed. It is important to preserve the chemical integrity of a blood specimen from the moment it is collected until it is assayed. Do not use grossly hemolytic, icteric or grossly lipemic specimens. Samples appearing turbid should be centrifuged before testing to remove any particulate material.

Related Products

Anti-Rituximab (Rituxan) ELISA Kit
Rituximab (Rituxan) ELISA Assay
Ustekinumab mAb-based ELISA

Additional Information

Assay Principle

This Eagle Biosciences Rituximab ELISA is based on Rituximab-specific mouse monoclonal antibody (catcher Ab, ImmunoGuide clone IG-9D5b). Diluted standards and samples are incubated in the microtiter plate coated with IG-9D5b 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 Rituximab. Following incubation, wells are washed and the bound enzymatic activity is detected by addition of chromogen-substrate. The colour developed is proportional to the amount of Rituximab in the sample or standard. Results of samples can be determined by using the standard curve. Binding of Rituximab to the solid phase, pre-coated with 9D5b, is inhibited by recombinant human CD20 protein. Therefore, the ImmunoGuide Rituximab ELISA (mAb-Based) measures the free form of Rituximab.

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 1:2000 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 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 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.



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Product Citations