The immunoblotting (western blotting) has been useful in identifying specific antigens recognized by polyclonal or monoclonal antibodies and is highly sensitive.
The immunoblot technique is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. The technique consists of much the same steps as an ELISA but the antigen is here attached to a membrane on which the patient sample is applied.
If there are specific antibodies in the sample, they will bind to the antigen. In the next step an enzyme labeled antibody is added and this antibody is directed against human IgG.
The enzyme labeled antibody can only bind, if there is an antibody against the antigen in the patient sample. Then a substrate is added to the enzyme. The enzyme converts the substrate chemically and the substrate changes color.
Staining in the form of dark bands indicates that the sample contains antibodies with certain antigen specificity.
The western blot is extensively used in biochemistry for the qualitative detection of single proteins and protein-modifications. It is used as a general method to identify the presence of a specific single protein within a complex mixture of proteins. A semi-quantitative estimation of a protein can be derived from the size and color intensity of a protein band on the blot membrane.
The technique uses three elements to accomplish this task:
The technique has also found important clinical applications in autoimmune disease, allergy, infectious diseases and others.