What is an Enzyme-Linked Immunosorbent Assay and How Does it Work?
ELISA or Enzyme-Linked Immunosorbent Assay is a plate-based assay technique employed mainly for detection and quantification of proteins, hormones, peptides, and antibodies. It is a technique wherein an antigen is immobilized onto a solid surface followed by complexing it with an antibody which is previously linked to the desired enzyme. The conjugated enzyme activity is further detected through incubating it along with a substrate when it produces a measurable product. One of the major crucial elements employed in the detection strategy here is the interaction that takes place between an antibody and an antigen. This is a highly specific reaction.
These assays are typically accomplished within a 96 well polystyrene plates which facilitates passive binding of both the antibodies and proteins. It is due to this binding and immobilization reaction that renders the ELISA method as one of the easiest ways to design and perform.
General procedure for ELISA assay development –
The first and the foremost step in ELISA is the coating step where the first coating layer consists of a target antibody or an antigen. This first layer is adsorbed onto a polystyrene plate exhibiting 96 wells. The next step is known as the blocking step. Here, all unbound antigens (if any) are coated with a blocking agent to prevent them from interfering with the detection. This is immediately followed by a series of washes which helps in removing all the unbound antibodies. Later, a suitable substrate is added, which emits a colourimetric signal and finally, the plate is read.
Types of ELISA assays –
There are four different types of ELISA methods –
Direct ELISA –
In the direct ELISA procedure, an antigenic layer is first coated to a multi-well plate which is then detected by addition of the suitable antibody. This antibody should be previously conjugated with the desired enzyme. This can be a good selection when the commercial kits are unavailable for your target kit.
Indirect ELISA principle –
In the indirect ELISA detection method, there are two stages, or layers followed to detect the antigen-coated to the 96 well polystyrene plate. Here, you need to first apply an unlabelled primary antibody specific to your target antigen. The addition of an enzyme follows this labelled secondary antibody, which detects and binds the primary one. The secondary antibody used here is usually an anti-species antibody and is often labelled to be polyclonal.
Sandwich ELISA principle –
These ELISA’s typically involves the use of matched pairs of antibodies. Here, each antibody is designed to be specific to one of the non-overlapping part present on an antigenic molecule. Sandwich ELISA begins with the addition of the first antibody to the well; followed by the addition of a sample solution. This step is later followed by the addition of a second antibody which is usually known as the detection antibody.
Competitive ELISA principle –
Also known as the Inhibition ELISA, the competitive ELISA is a surface-based technique where you have plate pre-coated with a captured antibody. These captured antibodies quickly react to the molecules of interest. This technique is so named because here both the labelled and unlabelled ligands compete with each other to search for the limited number of antibody binding sites.