Certolizumab Pegol mAb-based ELISA

Additional Information

Assay Background

Certolizumab pegol (trade name Cimzia®) is a tumor necrosis factor alpha (TNFα) blocker and binds to human TNFα with a KD of 90pM. Certolizumab pegol is a recombinant, humanized antibody Fab’ fragment, with specificity for TNFα, conjugated to an approximately 40kDa polyethylene glycol (PEG2MAL40K). The Fab’ fragment is manufactured in E. coli and is subsequently subjected to purification and conjugation to PEG2MAL40K, to generate Certolizumab pegol. Steady-state concentrations range from 0.5 to 90 µg/mL for a fixed dose of 400 mg of certolizumab pegol.  The ImmunoGuide Certolizumab pegol ELISA  (mAb-based) kit can be efficiently used for measuring free Certolizumab pegol levels in serum and plasma.

Assay Procedure

1. Pipette 100 μL of Biotinylated TNF into each well.
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-Streptavidin) 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