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Principle |
The qualitative detection of Human pANCA is based on the ELISA (Enzyme-Linked Immunosorbent Assay) technique. The microplate wells are pre-coated with an antigen specific to Human pANCA. The corresponding antibody in the sample or standards binds the immobilized antigen after incubation. After washing the microplate wells to remove all unbound sample material, horseradish peroxidase (HRP) labeled antigen conjugate is added, which binds to the antibody, forming antigen-antibody-enzyme-labeled antigen 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 antibody 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 a cut-off value. |
