The recoveryELISA RPE kit allows the simultaneous quantitative determination of free PCSK9, the PCSK9 neutralization rate and the available therapeutic antibody Evolocumab in human serum samples. This test should only be used for patients treated with Evolocumab 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:
- PCSK9 levels without and with additional Evolocumab against extinction (optical density)
- Evolocumab levels against PCSK9 recovery
RecoveryELISA is performed in a 96-well microplate. The wells of the microplate are pre-coated with a specific capture antibody against human PCSK9 that binds free PCSK9 from the patient sample. A simultaneous incubation of the PCSK9 calibrators (with and without the addition of Evolocumab), the samples and the detection conjugate (Evolocumab 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 PCSK9 and Evolocumab in the patient samples using the measured OD values. Determination of the therapeutic antibody is based on the principle that the presence of Evolocumab in patient samples leads to a systematic reduction in the recovery of PCSK9. 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:
PCSK9 Levels in samples without Evolocumab 40 – 400 ng / mL
PCSK9 Levels in samples with Evolocumab 40 – 350 ng / mL
Evolocumab level: 0.2 – 24 µg / mL
LINK TO THE INSTRUCTION
