Total IgE ELISA

The Total IgE ELISA is based on a solid phase enzyme-linked immunosorbent assay (ELISA). The assay system utilizes one monoclonal anti-IgE antibody for solid phase (microtiter wells) immobilization and goat anti-IgE antibody in the antibody-enzyme (horseradish peroxidase) conjugate solution. The serum sample is added to the IgE antibody coated microtiter wells and incubated with the Zero Buffer at room temperature for 30 minutes. If human IgE is present in the sample, it will combine with the antibody on the well. The well is then washed to remove any residual sample, and IgE antibody labeled with horseradish peroxidase (conjugate) is added. The conjugate will bind immunologically to the IgE on the well, resulting in the IgE molecules being sandwiched between the solid phase and enzyme-linked antibodies. After incubation at room temperature for 30 minutes, the wells are washed with water to remove unbound-labeled antibodies. A solution of TMB reagent is added and incubated at room temperature for 20 minutes, resulting in the development of a blue color. The color development is stopped with the addition of Stop Solution, and the color is changed to yellow and measured spectrophotometrically at 450 nm. The concentration of IgE is directly proportional to the color intensity of the sample.