Difference between ELISA and Western Blot

An immunoassay is a biochemical or bioanalytical technique that measures the concentration or presence of a macromolecule in a solution using antibody-antigen reaction. ELISA and western blot are such techniques that are used to detect the presence and concentration of protein ligands. Read this article to find out important differences between ELISA and Western blot.

ELISA

ELISA, short for enzyme linked immunosorbent assay, is a gold standard of immunoassays that was first illustrated by Eva Engvall and Peter Perlmann in 1971. It is a highly sensitive test that is used to detect and quantify hormones, glycoproteins, proteins, antibodies and antigens. It uses the interaction between an antibody and an antigen to identify the specific molecules.

In most simple words, ELISA can be performed by attaching antigen from the sample to a surface (usually a polystyrene plate). Next, a matching antibody attached with an enzyme is applied so that antigen can bind to it. All the unbound antibodies are washed off from the plate. Lastly, a substance that acts as a substrate for the enzyme is added which gives a detectable signal such as colour change when binding happens.

ELISA can be performed in many different ways depending on the molecule and the binding of antibody and antigen. There are four major types of ELISA:

• Direct ELISA
• Sandwich ELISA
• Reverse ELISA
• Competitive ELISA

Refer: What is ELISA? – Types, Procedure, Principle and Applications

Western Blot

Western blot or western blotting is a widely used analytical technique for separation and detection of proteins from a mixture. Majorly, it is a three step process that includes: separation of proteins on gel, transfer of proteins on a solid membrane, and visualisation of protein by marking it with primary and secondary antibodies.

The technique was first described by Harry Towbin in 1979 and named by W. Neal Burnette in 1981. The procedure of western blotting can be understood by the following points:

• Gel Electrophoresis: The protein samples are separated using gel electrophoresis. SDS-PAGE is commonly used for this step.
• Transfer: The separated proteins are transferred on a solid support as nitrocellulose or polyvinylidene difluoride membrane.
• Total Protein Staining: The proteins transferred on the membrane are visualised by staining the membrane using dyes such as Ponceau S, Coomasie R-350 or amido black.
• Blocking: This step is done to block the interaction between the membrane and the antibody that will be applied in the next step. Since the target molecule and antibodies are both proteins, there are chances that the membrane might interact with the antibody.
• Incubation: An antibody with a reporter enzyme is probed on the membrane. A substrate that is specific to the enzyme is applied on the surface. If binding happens, a colourimetric reaction is produced.
• Detection and Visualisation: The unbound probes are washed away and the gel is visualised for the bound proteins.

ELISA vs Western Blot

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Also see:

• Difference between Direct and Indirect Elisa
• Difference between Antigen and Antibody
• Difference between Active and Passive Immunity