Golimumab ELISA Assay Kit
The Golimumab ELISA Assay Kit is For Research Use Only
Size: 12×8 wells
Sensitivity: <2.5 ng/mL
Standard Range: 2.5-160 ng/ml
Incubation Time: 2.5 hours
Sample Type: Serum, Plasma, Cell Culture Supernatants
Sample Size: 100 µL
Alternative Name: Simponi
Golimumab (CNTO 148) is a human monoclonal antibody which is used as an immunosuppressive drug and marketed under the brand name Simponi. Golimumab targets tumor necrosis factor alpha (TNF-alpha), a proinflammatory molecule and hence is a TNF inhibitor. Profound reduction in C-reactive protein (CRP) levels, interleukin (IL)-6, intercellaular adhesion molecules (ICAM)-1, matrix metalloproteinase (MMP)-3, and vascular endothelial growth factor (VEGF) demonstrates golimumab as an effective modulator of inflammatory markers and bone metabolism.
Sample Preparation and Storage:
Blood is taken by venipuncture. Serum is separated after clotting by centrifugation. Plasma can be used, too. Lipaemic, hemolytic or contaminated samples should not be run. Repeated freezing and thawing should be avoided. If samples are to be used for several assays, initially aliquot samples and keep at – 20°C. For Cell Culture Supernatant – If necessary, centrifuge to remove debris prior to analysis. Samples can be stored at -20°C or -80⁰C. Avoid repeated freeze-thaw cycles.
Anti-Golimumab (Simponi) ELISA
Golimumab mAb-based ELISA Assay
Natalizumab (Tysabri) ELISA Kit
The method employs the quantitative sandwich enzyme immunoassay technique. Antibodies to Golimumab are pre-coated onto microwells. Samples and standards are pipetted into microwells and human Golimumab present in the sample are bound by the capture antibody. Then, a HRP (horseradish peroxidase) conjugated anti-Golimumab antibody is pipetted and incubated. After washing microwells in order to remove any non-specific binding, the ready to use substrate solution (TMB) is added to microwells and color develops proportionally to the amount of Golimumab in the sample. Color development is then stopped by addition of stop solution. Absorbance is measured at 450 nm.
1. It is strongly recommended that all Controls and Samples be run in duplicates or triplicates. A standard curve is required for each assay. All steps must be performed at 37°C.
2. Pipette out 50 μl of Assay Diluent in each well.
3. Add 100 μl of Standards or Samples into the respective wells.
4. Cover the plate and incubate for 60 minutes at 37°C
5. Aspirate and wash plate 4 times with Wash Buffer (1X) and blot residual buffer by firmly tapping plate upside down on absorbent paper. Wipe off any liquid from the bottom outside of the microtiter wells as any residue can interfere in the reading step.
6. Pipette without delay in the same order 100 μl of Anti-Golimumab: HRP Conjugate into each well.
7. Cover the plate and incubate for 60 minutes at 37°C
8. Aspirate and wash plate 4 times with Wash Buffer (1X) and blot residual buffer by firmly tapping plate upside down on absorbent paper. Wipe off any liquid from the bottom outside of the microtiter wells as any residue can interfere in the reading step.
9. Add 100 μl of TMB Substrate in each well.
10. Incubate the plate at 37°C for 30 minutes in dark. DO NOT SHAKE or else it may result in higher backgrounds and worse precision. Positive wells should turn bluish in color.
11. Pipette out 100 μl of Stop Solution. Wells should turn from blue to yellow in color.
12. Read the absorbance at 450 nm with a microplate reader.