Golimumab mAb-based ELISA Assay

Additional Information

Assay Principle

This Eagle Biosciences ELISA Assay Kit is based on Golimumab-specific mouse monoclonal antibody (catcher Ab, ImmunoGuide clone DWZ-1E4). Diluted standards and samples are incubated in the microtiter plate coated with IG-DWZ-1E4 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 Golimumab. 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 Golimumab in the sample or standard. Results of samples can be determined by using the standard curve. Binding of Golimumab to the solid phase, pre-coated with DWZ-1E4, is inhibited by recombinant human TNF-a in a concentration dependent manner. Therefore, the ImmunoGuide Golimumab ELISA (mAb-based) measures the free form of Golimumab.

1. Pipette 100µL of Assay Buffer into each of the wells to be used.
2. Pipette 50 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 10 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.

Specificity

There is no cross reaction with any other proteins present in native human serum. A screening test was performed with 48 different native human sera. All produced OD450/620 nm values less than the mean OD of standard D (6 ng/mL). In addition, binding of Golimumab is inhibited by recombinant human TNF-a in a concentration dependent manner. Therefore, the ImmunoGuide Golimumab ELISA (mAb-Based) measures the biologically active free form of Golimumab, i.e. not pre-occupied by TNF-a. No cross reaction was observed with sera spiked with the other therapeutic antibodies including Infliximab, Adalimumab, Etanercept, Rituximab, Trastuzumab, Tocilizumab, Omalizumab and Bevacizumab at concentrations up to 2 mg/mL.

Typical Standard Curve