Enzyymimmunoassay for the quantitative determination of allergen-specific immunoglobulin E (IgE) in human serum or plasma
The calibrators are pipetted into the wells of the first microstrips, the samples are pipetted into the remaining wells, each sample in plural, depending on the number of allergens to be determined. During the first incubation, the anti-IgE antibodies in the wells bind to the IgE of the samples (both allergen-specific and non-specific IgE). A first wash is performed to avoid any interference with other immunoglobulins, e.g. Allergen-specific IgG, which are possibly in the serum. The next step is to add anti-IgE biotene conjugate to the wells that have been incubated with the calibrator, while the various allergen biotine conjugates are pipetted into the corresponding sample wells. During this second incubation, the anti-IgE-biotine conjugate binds to the IgE of the calibrators tied to the Wells to form a “sandwich” of solid phase-╟ anti-IgE: IgE: Anti-IgE biotin.
The biotinylated allergens bind to the specific IgE, which were bound during the first incubation and form an immune complex of solid phase ╟Anti-IgE: IgE: allergen biotin.
After washing, the streptavidin-peroxidase-conjugate is pipetted into all Wells. During incubation, both the anti-IgE biotin, remaining in the calibrator wells, and also the allergen biotin, located in the sample wells, are bound by the streptavidin-peroxidase conjugate. In the last washing step, unresponsive antibodies are removed. Finally, incubation with the chromogen substrate (TMB/H2O2) allows detection of the streptavidine peroxidase conjugate, which is bound to the solid stage immune complex. The resulting coloring correlates with the IgE concentration of the calibrator wells and with the concentration of the specific IgE in the sample wells. The optical densities (O.D.s) are read with a microtiter plate reader. Sample O.D.s are a measure of the specific IgE concentration in the samples. A concentration determination in units or classes is made by interpolating the sample O.D.s on the oak curve.
Content and components:
Specific IgE basic-kit in 3 sizes for 96/480/960 tests
IgE microtiter plate:
Coated with anti-IgE. A set for 96 tests includes a microtiter plate with 12 stripes x 8 wells. Each well is coated with monoclonal human anti-IgE antibodies. Store the unused strips at 2-8 °C in the closeable plastic bag together with the desiccant.
HRP streptaidine conjugate:
Each vial contains 13 ml of streptavidin-peroxidase conjugate in red colored buffer, pH 5.5, with 0.001% proclin 300. Ready to use.
Specific IgE incubation buffer:
Each vial contains 8 ml TRIS buffer, pH 8.4, with 0.005% Proclin 300. Ready to use.
Each vial contains 13 ml of a stabilized and ready-to-use mixture of 3.3′, 5.5′ thetramethylbenzidine (TMB) and hydrogen peroxide (H2O2). Ready to use.
Each vial contains 13 ml of 0.3 M of sulphuric acid. Ready to use.
Washing buffer concentrate 10x:
Each vial contains 100 ml of a 10x concentrated washing liquid. To prepare the working liquid, mix the contents of the vial with 900 ml of distilled water.
Calibrators for specific IgE:
Each vial with calibrator liquid contains 1.3 ml of Human-IgE in horse serum with 0.01 percent proclin 300. IgE concentrations specified above have been calibrated against the international standard of WHO 2nd IRP 75/502. Store at temperatures between 2-8 °C. Ready to use.
Specific IgE control serum:
Each vial contains 1.3 ml of Human-IgE in horse serum and 0.01 percent proclin 300. The permitted range for the specific IgE control serum can be found in the certificate of analysis enclosed in this kit. This control serum is only to be used as a calibration control with the Nexgen Four Allergy test. Ready to use.
Anti-IgE biotin conjugate:
Each vial contains 10 ml of Human-anti-IgE, conjugated with biotin in TRIS buffer liquid pH 8.1 as well as 0.04 percent proclin 300 and Bronidox 0.02%. Ready to use.