ELISA / Multiplex protein analyses
The specific binding between antigen-antibody is the basis for the biochemical technique called Enzyme-Linked ImmunoSorbent Assay (ELISA) which is widely used in immunology to detect the presence of specific proteins in various kinds of samples (serum, tissue homogenates, cell culture supernatants).
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Very simply, antibodies against the protein of interest, which in this case is the antigen, are coated and fixed on the surface of 96-well plates (capture antibody). The sample is added on the 96-well plate and any antigen (protein) present it will bind on the capture antibody. The 96-well plate is washed so excess unbound antigen is removed. Another antibody (detection antibody) against the protein of interest is added in the wells, only this time the antibody is linked to an enzyme. By adding a suitable substrate in the wells the enzyme will produce a visible color signal, which will be measured by a spectrophotometer and it indicates the quantity of antigen (protein) in the sample.
The Multiplex analysis in very simple terms is flow cytometric immunoassay, very much like ELISA, only that the antigen-antibody reactions do not take place on the walls of 96-well plates but on the surface of polystyrene microspheres (xMAP® technology of Luminex® Corp.) and the signals from the detection antibodies are fluorescent and measured by a flow cytometer. The biggest advantages of this “advanced version of ELISA” are that one can simultaneously analyse one sample for up to 100 different proteins.