Antibody to Certolizumab Pegol ELISA Assay Kit

$895.00

The Eagle Biosciences Certolizumab pegol Antibodies ELISA Assay Kit is an enzyme immunoassay for the semi-quantitative determination of  free antibodies to Vedolizumab in serum and plasma. The Certolizumab pegol Antibodies ELISA Assay Kit is for research use only and is not intended for diagnostic or therapeutic procedures.

Antibody to Certolizumab Pegol ELISA Assay Kit

For Research Use Only

Size: 1×96 wells
Sensitivity: Cut-Off
Incubation Time: 2.75 hours
Sample Type: Serum, Plasma
Sample Size: 10 µL

Additional Information

Assay Background

The drug 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 (PEG). The Fab’ fragment is manufactured in E. coli and is subsequently subjected to purification and conjugation to PEG2MAL40K, to generate Certolizumab pegol.

The Eagle Biosciences Certolizumab pegol Antibodies ELISA Assay Kit has been designed for the measurement of free antibodies against this drug. It does not detect such antibodies which already are bound to the drug.

Assay Principle

This anti-drug antibody(ies) (ADA) kit is a bridging type ELISA for the determination of free antibodies against the drug Certolizumab pegol in serum and plasma samples. During the first incubation period, ADA in serum or plasma samples are captured by the drug coated on the microtiter wells. After washing away the unbound components from samples, a biotinylated drug conjugate is added and then incubated. ADA, if present in sample, will make a bridge, with its identical Fab arms, between the drug coated on the well and the other drug molecule labelled with biotin. Following incubation, wells are washed, and the horseradish peroxidase (HRP)-conjugated streptavidin is added and binds to the biotinylated Certolizumab pegol. Following incubation, wells are washed, and the bound enzymatic activity is detected by addition of tetramethylbenzidine (TMB) chromogen-substrate. Finally, the reaction is terminated with stop solution. The positive reaction is expected to be related to the presence of ADA in the sample.

Assay Procedure

  1. Pipette 100 µL of Assay Buffer into each of the wells to be used.
  2. Pipette 50 µL of each Ready-to-Use Negative Control, Ready-to-Use Positive Control, and 1:10 Diluted Samples (as described in section 10.2) into the respective wells of the microtiter plate. Bubble formation during the pipetting of standards and samples must be avoided.
  3. Cover the plate with adhesive seal. Shake plate carefully by tapping several times. Incubate the plate on bench top for 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 Biotinylated CZP into each well.
  6. Cover the plate with adhesive seal. Shake plate carefully by tapping several times. Incubate the plate on bench top for 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 enzyme conjugate (HRP-Streptavidin) into each well.
  9. Cover the plate with adhesive seal. Shake plate carefully by tapping several times. Incubate the plate on bench top for 30 min at RT.
  10. 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.
  11. Pippette 100 µL of Ready-To-Use TMB Substrate Solution into each well
  12. Incubate 15 min at RT. Avoid exposure to direct sunlight.
  13. Stop the substrate reaction by adding 100 µL of Stop Solution into each well. Color changes from blue to yellow. Breifly mix contents by gently shaking the plate
  14. Measure optical density (OD) with a phtometer at 450 nm (reference OD620 nm is optional) within 15 min after pipetting the Stop Solution.

Manual

Product Manual