Anti-Infliximab ELISA Assay Kit
The Anti-Infliximab ELISA Assay Kit is For Research Use Only
Size: 12×8 wells
Sensitivity: <10 ng/mL
Standard Range: 10-640 ng/ml
Incubation Time: 2.25 hours
Sample Type: Serum, Plasma, Cell Culture Supernatants
Sample Size: 100 µL
Alternative Name: Remicade
Infliximab is a monoclonal antibody which has high specificity for TNFα, and does not neutralize TNF beta. Infliximab causes programmed cell death of TNFα-expressing activated T lymphocytes, an important cell type mediating inflammation; hence it is generally assumed that resolution of activated T cells by Infliximab explains its efficacy in Crohn’s disease. 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 infliximab and can be responsible for allergic reaction, or even anaphylactic shock. This ELISA kit detects antibodies for Anti-Infliximab and may be used for monitoring immunogenicity.
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.
Infliximab ELISA Assay Kit
TNF-alpha Neutralization Rate Infliximab ELISA
Total Infliximab ELISA Assay
The method employs the quantitative sandwich enzyme immunoassay technique. Infliximab is pre-coated onto microwells. Samples and standards are pipetted into microwells and antibodies to Infliximab present in the sample are bound by the capture antibody. Then, a HRP (horseradish peroxidase) conjugated Infliximab 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 Anti-Infliximab 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 Infliximab: 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 15 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.