Bevacizumab (Avastin®) ELISA Assay Kit

$895.00

The Eagle Biosciences Bevacizumab (Avastin®) ELISA Assay Kit is an enzyme immunoassay for the precise analytical determination of free Bevacizumab in serum and plasma samples.  The Bevacizumab (Avastin®) ELISA Assay Kit is for research use only and not to be used in diagnostic procedures.

Bevacizumab (Avastin®) ELISA Assay Kit

For Research Use Only

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

Additional Information

Assay Background

The drug Bevacizumab (trade name Avastin®)is a recombinant human IgG1:k monoclonal antibody specific for all human vascular endothelial growth factor-A (VEGF-A) isoforms and it has been approved by the FDA as a first-line treatment for metastatic colorectal cancer in combination with chemotherapy. Furthermore, VEGF is implicated in intraocular neovascularization associated with diabetic retinopathy and age-related macular degeneration.

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

Assay Principle

This Eagle Biosciences Bevacizumab 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 Bevacizumab 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 colour developed is proportional to the amount of Bevacizumab 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

Specificity:

There is no cross reaction with native serum immunoglobulins. Thirty seven native human sera were screened and all produced OD450/620 nm lower than 0.112. Other therapeutic antibodies (Omalizumab, Golimumab, Infliximab, Trastuzumab, Rituximab, Etanercept, Adalimumab and Tocilizumab) are also tested at the concentrations up to 400 μg/mL and observed that there are no cross reactions (OD 450/620nm values were less than 0.112). In addition, interference of Ranibizumab, binds to the same antigen VEGF-A, was tested at 25 ng/mL (nearly ten times of the serum Cmax of Ranibizumab) and no measurable inhibition was observed.

Manual

Product Manual


Publications

Citations

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