ELISA or enzyme-linked immunosorbent assay is a plate-based technique designed for the detection and quantification of immunological substances such as proteins, peptides, hormones, and antibodies. Here, you have an enzyme-conjugated with an antibody reacting with a colorless substrate to yield a colored product. These colorless substrates are known as chromogenic substrates. Horseradish peroxidases, alkaline phosphatases, and beta galactosidases are key enzymes involved in the ELISA assay technique.
ELISA principle –
ELISA technique principally involves a 96 well-polystyrene plate. The first step is to incubate your serum samples into the wells – one serum sample per well. Likewise, both a positive and a negative serum sample would be incubated amongst all the 96 well polystyrene plates. Antigens or antibodies within the biological serum will find a corresponding antigen or antibody immobilized on to the solid surface and form conjugation with them. After some point of time, the 96 well plates are washed off to remove all the serum particles and any unbounded antigens or antibodies present within the wash buffer. The bounded antigens or antibodies are detected with the help of a secondary antibody, which is primarily attached to an enzyme. Once the incubation period is over, unbound antibodies, if any, are washed out of the good contents. With the addition of a suitable substrate, there occurs a reaction between a conjugated enzyme and the substrate. After that, the color produced is measured as a number of antibodies or antigens present within the given sample.
ELISA types –
There are three key types of ELISA which helps in drug development process –
Indirect ELISA –
Antibodies can be quantitatively detected by an indirect ELISA method. Here, the antigen gets coated on to the surface of the microtiter well. After that, serum or any other sample containing your primary antibody of interest is added to the microtiter well. This added serum is after that allowed to react with the coated antigen. Any free antibody, if present, is washed off. This is further followed by the detection of the enzyme-conjugated secondary antibody binding with the primary antibody. All your unbound secondary antibody is subsequently washed off from the surface of the microtiter well. Your added conjugated enzyme hydrolyzes your substrate, thereby forming colored products. The amount of the end colored products is further read by measurements derived from the spectrophotometric plate readers.
Sandwich ELISA –
In the sandwiched ELISA technique, your antibodies are coated on to the surface of the microtiter well. A serum sample containing your antigen is added to your polystyrene well. Further, it is allowed to react with the immobilized antibodies, facilitating the formation of the antigen-antibody complex. Once the well has been washed off, a secondary enzyme-linked antibody-containing binding sites specific for different antigenic epitopes are added to the well. After that, all the unbound antibodies are washed away. Finally, you add on your substrate, which is further hydrolyzed by the conjugated enzymes.
Competitive ELISA –
In the competitive ELISA technique, your antibody is first incubated into a solution containing a sample with antigens. A mixture containing antigen-antibody complex is later added to your microtiter well, which has been previously coated with your antigen. The more antigen concentration is within your sample, the lesser the free antibodies are available for binding to your immobilized antibodies. Once the well is washed off, the enzyme-conjugated antibodies, which are specific for primary antibody isotypes, help determine the presence of antibodies bound to your microtiter well.