Anti-Adalimumab (Humira) ELISA Assay Kit

$640.00

The Eagle Biosciences Anti-Adalimumab (Humira) ELISA is used as an analytical tool for quantitative determination of Anti-Adalimumab in serum, plasma and cell culture supernatant.

Anti-Adalimumab (Humira) ELISA Assay Kit

For Research Use Only

Size: 12×8 wells
Sensitivity: <1.56 ng/mL
Standard Range: 1.56-100 ng/ml
Incubation Time: 2.5 hours
Sample Type: Serum, Plasma, Cell Culture Supernatants
Sample Size: 100 µL

Additional Information

Assay Background


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 levels of TNF are also found in psoriasis (Ps) plaques. Anti-Drug Antibodies (ADA) may induce unwanted side effects in biopharmaceuticals. Hence, ADA has been subjected to increase in scrutiny by the regulatory authorities using immunogenicity safety studies. ADA has been observed in pre-clinical and clinical studies, resulting in significant changes in toxicology, pharmacokinetics and efficacy. These effects result from the generation of drug-induced (neutralizing) autoantibodies against Adalimumab and can be responsible for allergic reaction, or even anaphylactic shock. This ELISA kit detects antibodies for Anti-Adalimumab and may be used for monitoring immunogenicity.

Assay Principle


The method employs the quantitative sandwich enzyme immunoassay technique. Adalimumab is pre-coated onto microwells. Samples and standards are pipetted into microwells and antibodies to Adalimumab present in the sample are bound by the capture antibody. Then, a HRP (horseradish peroxidase) conjugated Adalimumab is pipetted and incubated. After washing microwells in order to remove any nonspecific binding, the ready to use substrate solution (TMB) is added to microwells and color develops proportionally to the amount of Anti-Adalimumab in the sample. Color development is then stopped by addition of stop solution. Absorbance is measured at 450 nm.

Assay Procedure


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 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.

Manual

Product Manual