Omalizumab IgE ELISA Assay

Additional Information

Assay Background

Allergic asthma is a widespread illness that is initiated or aggravated by allergy-causing substances (allergens). Type E immunoglobulins (IgE) are formed in the organism as a reaction to the allergens, which set in motion the distribution of allergically acting transmitters such as histamine, leukotrines and bradykinins from the mast cells. In the immediate type I reaction, inhalation of the allergen leads directly to constriction of the airways.

In particularly severe cases of allergic bronchial asthma, where the therapeutic antibody Omalizumab (recombinant humanised antibody against IgE) is used, the IgE responsible for triggering the allergic reaction is neutralised by binding. Omalizumab acts as an anti-antibody that prevents the cascade allergic reactions and reduces the binding of IgE to the mast cells. The therapeutic goal of Omalizumab is the reduction of IgE levels in the organism.

The recoveryELISA Omalizumab Free IgE ELISA Assay Kit makes simultaneously determining the free IgE and the available therapeutic antibody possible where Omalizumab is used (Research Use Only).

Assay Priciple

RecoveryELISA (Enzyme-Linked Immunosorbent Assay) Omalizumab Free IgE ELISA Assay Kit is an immunological quantitative detection method based on a sandwich ELISA. In comparison to a classic sandwich ELISA, a two-dimensional calibration is carried out for recoveryELISA obtaining two analysis results within the same assay. The following calibrations are performed:

1. IgE levels without and with additional Omalizumab against extinction (optical density) 
2. Omalizumab levels against IgE recovery 

RecoveryELISA Omalizumab Free IgE ELISA Assay Kit is performed in a 96-well microplate. The wells of the microplate are pre-coated with a specific capture antibody against human IgE that binds free IgE from the patient sample. There occurs a simultaneous incubation of the IgE levels (with and without the addition of Omalizumab) of the samples and of the detection conjugate (peroxidase conjugate of an anti-IgE antibody). Incubation takes place over 16 to 22 h at 2 to 8°C. After washing, the colour substrate TMB (tetramethylbenzidine) is added to the wells. After incubation the enzymatic colour reaction is stopped using a sulphuric acid solution. A colour change from blue to yellow occurs that 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 to be performed, an evaluation procedure can be carried out to determine the concentrations of free IgE and Omalizumab in the patient samples using the measured OD values. Determination of the therapeutic antibody is based on the principle that the presence of Omalizumab in patient samples leads to a systematic reduction in the recovery of IgE. The evaluation is performed using non-linear regression (Marquardt-Levenberg algorithm), the Michaelis-Menten model from enzyme kinetics and the Langmuir isotherm from surface binding.

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