Aflibercept ELISA Assay Kit


The Aflibercept ELISA Assay Kit is an enzyme immunoassay for the measurement of free Aflibercept in serum and plasma samples. The Aflibercept ELISA Assay Kit is for research use only and not to be used in diagnostic procedures.

Aflibercept ELISA Assay Kit

The Aflibercept ELISA Assay Kit is 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
Alternative Names: Eyelea, Zaltrap

Assay Background

The drug Aflibercept (trade names Eylea ®, Zaltrap ®) is a recombinant fusion protein consisting of VEGF-binding portions from the extracellular domains of human VEGF receptors 1 and 2 fused to the Fc portion of human IgG1. Aflibercept has broad affinity for all ligands that bind to these receptors, including isoforms of VEGF-A, VEGF-B, and placental growth factors. Aflibercept demonstrated antitumour effects and antiangiogenic activity as a single agent and enhanced activity in combination with chemotherapy.

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 Aflibercept in order to potentially avoid some side effects with a reliable method might be beneficial.

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.

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Additional Information

Assay Principle

This Eagle Biosciences Aflibercept 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 vascular endothelial growth factor-A (rhVEGF-A). 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 Aflibercept pre-captured by the rhVEGF-A 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 Aflibercept 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 1:100 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).
    Remove adhesive seal. Aspirate or decant the incubation solution. Wash the plate 3 X 300 μL of Diluted Wash Buffer per well.
  4. 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.
    Remove adhesive seal. Aspirate or decant the incubation solution. Wash the plate 3 X 300 μL of Diluted Wash Buffer per well.
  7. 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

Aflibercept ELISA Assay Kit


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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