The recoveryELISA RTA is a diagnostic test kit (Research Use Only) for the simultaneous quantitative determination of free TNFα, the TNFα neutralization rate and the available therapeutic antibody Adalimumab in human serum samples. This test should only be used for patients treated with Adalimumab as mono biological therapy. It is a manual, non-automated kit for the determination of 7 samples.
The recoveryELISA (Enzyme-Linked Immunosorbent Assay) is an immunological quantitative detection method based on a sandwich ELISA. In contrast to a classic sandwich ELISA, a two-dimensional calibration is carried out for recoveryELISA.
The following calibrations are performed:
TNFα levels without and with additional Adalimumab against extinction (optical density)
Adalimumab levels against TNFα recovery
RecoveryELISA is performed in a 96-well microplate. The wells of the microplate are pre-coated with a specific capture antibody against human TNFα that binds free TNFα from the patient sample. A simultaneous incubation of the TNFα calibrators (with and without the addition of Adalimumab), the samples and the detection conjugate (Adalimumab peroxidase conjugate) is carried out. The incubation time is 16 to 22 hours at 2 to 8°C. After washing, the colour substrate TMB (tetramethylbenzidine) is added to the wells. The enzymatic colour reaction is stopped using a sulphuric acid solution. A colour change from blue to yellow is optically detected. Using a wavelength of 450 nm (reference value 620 nm), the optical density (OD) of the reaction product is measured with a suitable microplate reader.
With the aid of the two calibrations, an evaluation procedure can be carried out to determine the concentrations of free TNFα and Adalimumab in the patient samples using the measured OD values. Determination of the therapeutic antibody is based on the principle that the presence of Adalimumab in patient samples leads to a systematic reduction in the recovery of TNFα. The evaluation is performed using non-linear regression (Marquardt-Levenberg algorithm) of a logarithmic model from enzyme kinetics and the Langmuir isotherm from surface binding.
Measurement Range for undiluted samples:
TNFα Levels in samples without Adalimumab 4 – 400 ng / mL
TNFα Levels in samples with Adalimumab 4 – 160 ng / mL
Adalimumab level: 0.7 – 30 µg / mL
LINK TO THE INSTRUCTION
