Total Infliximab ELISA Assay

Additional Information

Assay Principle

The Eagle Biosciences Total Antibody to Infliximab ELISA Assay Kit serves for the determination of total antibodies (free and Infliximab-bound) against Infliximab in serum and plasma samples. During sample preparation, the Infliximab-bound anti-Infliximab antibodies are dissociated from the complex in order to acquire free anti-Infliximab antibodies. During the incubation period, antibodies to Infliximab (ATI) are replaced and bridged in the well with its Fab arms, between the Infliximab coated on the well and the conjugate Infliximab labelled with peroxidase. After washing away the components 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 related with the presence of ATI 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 Enzyme Conjugate (HRP-Infliximab) into each well.
3. Pipette 40 µL of dissociation buffer-mixed (as described in section 10.2) Negative Control, Positive Control and Samples into the respective wells of the microtiter ELISA plate.
4. Cover the plate with adhesive seal. Shake plate carefully. Incubate 60 min at room temperature (RT) (20-25°C).
5. 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.
6. Pipette 100 µL of Ready-to-Use TMB Substrate Solution into each well.
7. Incubate 15 min at RT. Avoid exposure to direct sunlight.
8. 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.
9. Measure optical density (OD) with a photometer at 450 nm (Reference at OD620 nm is optional) within 15 min after pipetting the Stop Solution.