Trastuzumab (Herceptin®) (mAb-based) ELISA Assay Kit

$1,190.00

The Eagle Biosciences Trastuzumab (mAb-based) ELISA Assay Kit is an enzyme immunoassay for the specific and precise determination of free Trastuzumab in serum and plasma. The Trastuzumab (mAb-based) ELISA Assay Kit is for research use only and not to be used in diagnostic procedures.

Trastuzumab (Herceptin®) (mAb-based) 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 Trastuzumab (trade names Herceptin® or Herclon®) is a recombinant DNA-derived humanized monoclonal antibody that selectively targets the extracellular domain of the human epidermal growth factor receptor 2 protein (HER2). The antibody is an IgG1 kappa that contains human framework regions with the complementarity-determining regions of a murine anti-p185 HER2 antibody that binds to HER2. Trastuzumab blood concentrations throughout the dosing interval expected to be remaining above those considered necessary for anticancer activity. Furthermore, in a separate analysis, patients with the lowest Trastuzumab serum trough concentrations had the highest rate of disease progression and shortest overall survival. This Eagle Biosciences ImmunoGuide Trastuzumab ELISA (mAb-based) is developed for the specific measurement of Trastuzumabin serum, plasma,and other biological fluids by the advantage of using a site-directed VME-5A6b mouse monoclonal antibody (mAb) specific for Trastuzumab only.

Assay Priniciple


This Eagle Biosciences ELISA Assay Kit is based on Trastuzumab-specific mouse monoclonal antibody (catcher Ab, ImmunoGuide clone VME-5A6b). Diluted standards and samples are incubated in the microtiter plate coated with IG-VME-5A6b 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 Trastuzumab. Following incubation, wells are washed and the bound enzymatic activity is detected by addition of chromogen-substrate. The color developed is proportional to the amount of Trastuzumab in the sample or standard. Results of samples can be determined by using the standard curve. Binding of Trastuzumab to the solid phase, pre-coated with VME-5A6b, is inhibited by recombinant human HER2 protein in a concentration dependent manner. Therefore, the ImmunoGuide Trastuzumab ELISA (mAb-Based) measures the free form of Trastuzumab.

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 1:1000 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.
  11. Briefly mix contents by gently shaking the plate. Color changes from blue to yellow.
  12. 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 any other proteins present in native human serum. A screening test was performed with 36 different native human sera. All produced OD450/620 nm values less than the mean OD of standard D (6 ng/mL). No cross reaction was observed with sera spiked with the other therapeutic antibodies including Infliximab, Adalimumab, Etanercept, Rituximab, Tocilizumab, Omalizumab and Bevacizumab at concentrations up to 2mg/mL. All produced mean OD450/620 nm values less than the mean OD of standard D (6 ng/mL). In addition, binding of Trastuzumab is inhibited by recombinant human HER2 in a concentration dependent manner. Therefore, the ImmunoGuide Trastuzumab ELISA (mAb-based) measures the biologically active free form of Trastuzumab, i.e. not pre-occupied by HER2.

Manual

Product Manual


Publications

References


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