Etanercept (Enbrel®) Antibodies ELISA Assay Kit


The Eagle Biosciences Antibody to Etanercept is an enzyme immunoassay for the semi-quantitative determination of free antibodies to Etanercept in serum and plasma samples. The Antibody to Etanercept ELISA Assay Kit is for research use only and not to be used in diagnostic procedures.

Etanercept (Enbrel®) Antibodies ELISA Assay Kit

For Research Use Only

Size: 1 x 96 wells
Sensitivity: Cut-off
Incubation Time: 2.5 hours
Sample Type:  Serum, Plasma
Sample Size: 10 µL

Additional Information

Assay Background

The drug Etanercept (trade name Enbrel®) is a dimeric fusion protein consisting of the extracellular ligand-binding portion of the human 75 kilodalton (p75) tumor necrosis factor receptor (TNFR) linked to the Fc portion of human IgG1. One of the major concerns, despite of its wide usage, is the potential development of anti-drug antibodies (ADA) which in turn may interfere with the drug efficacy as mainly judged by observing the relapse of signs and symptoms of disease and necessitate dose-escalation or potentially ending up the treatment.

The ImmunoGuide Antibody to Etanercept ELISA Kit can be used for the measurement of free antibodies against this drug. It does not detect such antibodies which already are bound to the drug.

Assay Principle

This Eagle Biosciences ImmunoGuide anti-drug antibody(ies) (ADA) kit is a bridging type ELISA for the determination of free antibodies against the drug Etanercept in serum and plasma samples. During the first incubation period, ADA in serum or plasma samples are captured by the drug coated on the microtiter wells. After washing away the unbound components from samples, a peroxidase-labelled drug conjugate is added and then incubated. ADA, if present in sample, will make a bridge, with its identical Fab arms, between the drug coated on the well and the other drug molecule labelled with peroxidase. After a second washing step, the bound enzymatic activity is detected by addition of tetramethylbenzidine (TMB) chromogen-substrate. Finally, the reaction is terminated with stop solution. The positive reaction is expected to be related to the presence of ADA in the sample.

Assay Procedure

  1. Pipette 100 µL of Assay Buffer into each of the wells to be used.
  2. Pipette 50 µL of each Ready-to-Use Negative Control, Ready-to-Use Positive Control, and 1:10 Diluted Samples (as described in section 10.2) 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-drug) into each well.
  6. Cover plate with adhesive seal. Shake plate carefully. Incubate 60 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 15 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.


Product Manual



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