Adalimumab ELISA Assay
The Adalimumab ELISA Assay is For Research Use Only
Size: 12×8 wells
Sensitivity: 4.8 ng/mL
Standard Range: 5-320 ng/ml
Incubation Time: 2.5 hours
Sample Type: Serum, Plasma, Cell Culture Supernatants
Sample Size: 100 µL
Alternative Name: Humira
Assay Background for Adalimumab Assay
Adalimumab is a recombinant human IgG1 monoclonal antibody specific for human tumor necrosis factor alpha (TNF-α). Adalimumab is produced by recombinant DNA technology in a mammalian cell expression system and is purified by a process that includes specific viral inactivation and removal steps. Adalimumab binds specifically to (TNF-α) and blocks its interaction with the p55 and p75 cell surface TNF receptors. TNF is a naturally occurring cytokine that is involved in normal inflammatory and immune responses. Elevated levels of TNF are found in the synovial fluid of rheumatoid arthritis, including juvenile idiopathic arthritis, psoriatic arthritis, and ankylosing spondylitis patients and play an important role in both the pathologic inflammation and the joint destruction that are hallmarks of these diseases. Increased TNF levels are also found in psoriasis (Ps) plaques.
Sample Preparation and Storage for Adalimumab ELISA:
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 samples can be stored at -20°C or -80⁰C. Avoid repeated freeze-thaw cycles.
Anti-Adalimumab (Humira) ELISA
TNF-alpha Neutralization Rate Adalimumab ELISA Assay
Bevacuzimab (Avastin) Assay Kit
The method employs the quantitative sandwich enzyme immunoassay technique. Antibodies to Adalimumab are pre-coated onto microwells. Samples and standards are pipetted into microwells and human Adalimumab present in the sample are bound by the capture antibody. Then, a HRP (horseradish peroxidase) conjugated anti-Adalimumab 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 Adalimumab 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 100 μl of Standards or Samples into the respective wells.
3. Cover the plate and incubate for 60 minutes at 37°C
4. 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.
5. Pipette without delay in the same order 100 μl of Anti-Adalimumab: HRP Conjugate into each well.
6. Cover the plate and incubate for 60 minutes at 37°C
7. 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.
8. Add 100 μl of TMB Substrate in each well.
9. 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.
10. Pipette out 100 μl of Stop Solution. Wells should turn from blue to yellow in color.
11. Read the absorbance at 450 nm with a microplate reader.
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