Enzymimmunoassay for the quantitative determination of allergen-specific immunoglobulin G/G4 (IgG/IgG4) in human serum or plasma
Die ALLERgen-Kits basieren auf dem µ-capture Enzymimmunassay, wobei dieselben Festphasen-Mikrowells, die mit humanen anti-IgG(4)-Antikörpern beschichtet sind, für die Analyse der verschiedenen Allergene und zur Erstellung der Eichkurve verwendet werden.
In the ALLERgenA spec. IgG(4) kit for the detection of IgG(4) antibodies that are specific for certain allergens, two types of enzyme-linked immunosorbent assay are used. Sandwich type analysis is used in microwells for standards. Indirect solid-phase enzyme immunodetection is used in microwells used for blood serum samples. The surface of the microwells is coated with streptavidin. The kit uses two monoclonal antibodies that are specific for different epitopes of immunoglobulins IgG(4). One of these antibodies is conjugated to biotin esters; the other is conjugated to horseradish peroxidase (HRP). Binding of IgG(4) molecules contained in standards with anti-IgG(4) biotin conjugate and simultaneous immobilization in the solid phase by streptavidin molecules take place during the first incubation . At the same stage, the binding of biotinylated allergens with IgG(4) molecules takes place in the microwells filled with sample serum, which are specific for these allergens, and immobilizes them in the solid phase by streptavidin. The excess anti-IgG(4) biotin is removed from the microwells of the standards and the excess of biotinylated allergens is removed from the microwells of the samples by washing. During the second incubation, anti-IgG(4) peroxidase conjugate (MAB2) binds to IgG(4) molecules in the microwells of the standards with the calibrators and to IgG(4) molecules of the tested samples in the microwells. Excess anti-IgG(4) peroxidase conjugate is removed by decanting and washing the microwells. The color level changes during the incubation with TMB. The degree of color is related to the amount of bound anti-IgG(4) peroxidase conjugate. Based on calibration curves with an optical density value, measured at 450 nm and 405 nm, the concentration of specific IgG(4) in the tested samples is defined after the optical density measurement of the solution in the microwells.
Content and components:
Specific IgG(4) basic-kit in 3 sizes for 96/480/960 tests
Coated with anti-IgG(4). A set for 96 tests includes a microtiter plate with 12 stripes x 8 wells. Each well is coated with monoclonal human anti-IgG(4) antibodies. Store the unused strips at 2-8 °C in the closeable plastic bag together with the desiccant.
Each vial contains 5 ml monoclonal anti-IgG (4) antibody solution conjugated with biotin in red colored buffer, pH 5.5, with 0.001% Proclin 300. Ready to use.
Each vial contains 22 ml of anti-IgG (4) monoclonal antibodies conjugated with HRP in red colored buffer, pH 5.5, with 0.001% Proclin 300. Ready to use.
specific IgG(4) incubation buffer:
Each vial contains 16 ml TRIS buffer, pH 8.4, with 0.005% Proclin 300. Ready to use.
Each vial contains 14 ml of a stabilized, ready-to-use mixture of 3,3 ’, 5,5’ tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2). Ready to use.
Each vial contains 50 ml of 0.3 M sulfuric 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 für specific IgG(4)
Each vial with calibrator liquid contains 0.5 ml of Human-IgG(4) in horse serum with 0.01 percent ProClin 300. IgG(4) concentrations specified above have been calibrated against the international standard of WHO Immunglobulin G, A und M, Human Serum, NIBSC-Code: 67/086 Store at temperatures between 2-8 °C. Ready to use.
specific IgG(4) control serum:
Each vial contains 0.5 ml of Human-IgG(4) in horse serum and 0.01 percent ProClin 300. The permitted sector for the specific IgG(4) control serum can be found in the certificate of analysis enclosed in this kit. Ready to use.