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The quantitative detection of Rat EEA1 antigen is based on the ELISA (Enzyme-Linked Immunosorbent Assay) technique. The microplate wells are pre-coated with an antibody specific to Rat EEA1. After incubation, the corresponding antigen in the sample or standards will bind to the immobilized antibody. After washing the microplate wells to remove all unbound sample material, horseradish peroxidase (HRP) labeled antibody conjugate is added, which binds to the antigen, forming antibody-antigen-enzyme-labeled antibody complex upon incubation. In a second wash step, all unbound material is removed. The immune complex formed by the bound conjugate is visualized by adding Tetramethylbenzidine (TMB) substrate, which gives a blue color reaction in wells containing the complex. A stop solution containing acid terminates the reaction, producing a yellow end-point coloration proportional to the amount of specific antigen present in the sample. Absorbance is measured at 450 nm using an ELISA microwell plate reader. Analysis is completed by comparing the OD values of samples to the standard curve. |
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