ELISA is a technique that allows the identification and quantification of known proteins to be ascertained by immunological means. The key to ELISA is linking an antibody to an enzyme that is capable of catalyzing a colorimetric or chemiluminescent reaction. ELISA was first developed in the early seventies and replaced radioimmunoassays which used radioactivity as the reporter mechanism. For biological products, ELISA is a common method for determining the presence and concentration of products. It is also used in cell based assays, immunogenicity studies and as a screening method. There are several common types of ELISA, direct ELISA, Indirect ELISA, Sandwhich ELISA, and Competitive ELISA.
In direct ELISA, an antigen is bound to the bottom of a 96 well plate and a rest of the well is blocked with a blocking agent (usually BSA or Milk). After washing, a primary antibody that is conjugated to an enzyme (such as horseradish peroxidase) binds to the antigen at the bottom of the well. The unbound primary antibody is washed away and when the chemical substrate is added which the enzyme acts upon to produce a colorimetric or chemiluminescent reaction which can be measure by a plate reader. Direct ELISA is used to detect and quantify the amount of antigen present in a sample.
Indirect ELISA is similar to direct ELISA but requires a secondary antibody that binds the primary antibody. Secondary antibody typically binds the Fc region of the primary antibody and will be conjugated to an enzyme that is able to catalyze a colormetric or chemiluminescent reaction wen exposed to the appropriate substrate. Secondary antibodies are easy to find commercially and therefore most studies are performed using indirect ELISA over direct ELISA which would often require the user to conjugate the enzyme to the primary antibody themselves.
When performing an indirect ELISA, the antigen is attached to the bottom of the plate and the plate is blocked just like direct ELISA. The primary antibody is then added and binds to the antigen. Excess primary antibody is washed away and the secondary antibody is added which binds to the primary antibody. Substrate is then added and the enzyme linked to the secondary antibody catalyzes a colormetric or chemiluminescent reaction which is detected using a plate reader.
In sandwich ELISA, the primary antibody, which is bound to the bottom of the plate, binds the antigen and then a secondary antibody also binds the antigen forming an antibody-antigen-antibody sandwich. Because the antibody that binds the antigen (primary antibody) is often not commercially available with an enzyme conjugated to it, a secondary antibody is added which is conjugated to an enzyme that can catalyze the colormetric or chemiluminescent reaction when exposed to substrate.
The name “Competitive ELISA” derives from the competitive binding between the sample antigen and antigen that has been added in. The procedure for competitive ELISA differs from the other types of ELISA. For competitive ELISA, the primary antibody is added to the sample which contains the antigen. The primary antibody will bind the antigen forming an antibody-antigen complex. The sample is then added to 96 well plates which has antigen bound to each well. Primary antibodies have already been bound to the antigen in the sample can not bind to the antigen on the plates and are therefore washed away. The more antigen in the sample the more primary antibody gets washed away. A secondary antibody is then added to the wells which binds the primary antibody. The secondary antibody is again bound to an enzyme which can catalyze a colormetric or chemiluminescent reaction. For competitive ELISA, a low signal from the enzyme means that there is high amount of antigen in the sample.