ELISA is an acronym for Enzyme-Linked Immunosorbent Assay. Assays are tests that are used to determine the presence and potency of various components like peptides, proteins, hormones, etc. Cell-based assays, in particular, are assays that are carried out within cell cultures of a certain kind. ELISA assays stand out because these are primarily plate-based assays.
The chief function of ELISA assays is to find antibodies. But detection is not the only end goal here. ELISA assays also help you to quantify the antibodies. Hence, this assay can be used to determine whether or not you have antibodies related to various infectious diseases. This knowledge is essential for medical professionals to develop an effective treatment plan for you.
How Does an ELISA Assay Work?
The antigen of a particular kind is immobilized on a solid surface (plates) in ELISA assays. Once this is done, this antibody combines with an antibody that is, in turn, connected to a particular enzyme. This entire complex then needs to be detected. This researcher evaluates the activity of the combined enzyme by incubating it with the help of a substratum. This finally produces a measurable product. The interaction between the antigen and the antibody is really specific, and this is the key factor that is required for an ELISA assay.
What Equipment is Needed to Carry Out ELISA assays?
A very specific sort of plate is required to carry out ELISA assays. This plate is also known as a microtiter plate. The plate must either have 96 wells or 384 wells. 96 well plates are most commonly used during ELISA assays. Polystyrene is the material that is most preferred for making these plates. The antibodies and proteins bind with these 96-well polystyrene plates.
The most important step in an ELISA assay is to immobilize the antigen. Since the reagents remain bound, it becomes easier to separate these from the materials that are not bound. ELISA assays are used when the researcher needs to measure particular analytes present in a crude preparation. In ELISA assays, the agents which are not specifically bound are simply washed away.
What are the Various Kinds of ELISA Assays?
There are four different kinds of ELISA assays:
Direct ELISA assays simply follow the rule that has been mentioned already. In direct ELISA, the antigen can be immobilized directly. This enables a specific antibody to detect it automatically. The antibody is paired with a molecule that can be detected. This, in turn, makes the antibody identifiable and quantifiable. HRP is very commonly used as a connector because these molecules are detectable.
Indirect ELISA assays are done when the antibody cannot directly bind with the antigen. Instead, a two-step process is followed here. Primarily, an initial antibody is chosen that is specific to the antigen which is being targeted. Once these two form a complex, a second antibody is introduced. This antibody must be inimical to the original species of the initial antibody. This second antibody is also responsible for carrying the detectable molecule. Once this attaches to the initial complex formed between the first antibody and the antigen, the whole combination becomes detectable. Because of the double binding process that is needed, this is called an indirect ELISA.
This too involves a two-step process. But, this process is named after the sandwich-like formation that is created when the two antibodies sandwich the antigen between themselves. Here too, the two antibodies include one, which is meant for binding, and one, which is meant for making the whole complex detectable.
So, what is the difference between indirect ELISA and sandwich ELISA? In sandwich ELISA, the two antibodies need to be specific to the epitopes of the antigen. Each antibody is specific to one epitope. This is what creates the “matched antibody pairs” that result in the sandwich shape. This is what immobilizes the antigen. Sandwich ELISA assay is the most common kind of ELISA assay that is carried out.
Competitive ELISA is also known as Inhibition ELISA. Here, the process of signal interference is used to measure the concentration of an antibody. Competitive ELISA isn’t a separate kind of ELISA assay. Rather, it must be understood that all the aforementioned ELISA assays can be turned into competitive assays.
What Are Elisa Assays Used For?
These assays are an essential tool that is needed to determine serum antibody concentrations. For example, if a person is HIV positive, ELISA assays can be used to detect the antibodies produced in that person by the specific pathogens that have affected him. When researchers need to identify specific hormones (like HCG) and antigens, they also depend on ELISA assays.
ELISA is also crucial to the field of clinical immunology. Since ELISA can also detect proteins in a sample, they are used to analyze the serum obtained from an infected person. Through the assay, the degree of infection can be ascertained.
Hopefully, by now, you already understand how ELISA assays work in detecting and quantifying the presence of an antibody. Since you are now aware of the various kinds of ELISA assays as well, you will be able to ascertain which one you specifically require.