Clearing the Basics: All You Need to Know About Immunoassay and ELISA

Immunoassays have been extensively used in research, clinics, hospitals, and medical laboratories over the last forty years. Clinical immunoassay testing has led to various discoveries and innovations that have shortened the hospital stays of many patients.

Immunoassay testing helps assess the progression of disease in the human body, which in turn facilitates better therapeutic decisions.

Immunoassays are also largely used to track different antibodies, hormones, and proteins in life science research. The practical, day-to-day applications of immunoassays include quality control during product processing and the detection of contaminants in food and water.

The unique interactions between an antibody and its associated antigens can be used to monitor the concentration of specific molecules, with the help of a well-designed immunoassay.

Enzyme-Linked Immunoassays (ELISA)

An enzyme-linked antibody is used to perform the enzyme-linked immunoassays, also known as ELISA. For this process, the target antibody is incubated with its associated antigen for a specific time.

Into this solution, a substrate is added, using the enzyme to convert it into a colored product. The concentration of the antigen is determined through the extent of color change in the solution. 

ELISA assays are preferred by many researchers since they are quick, accurate, and relatively easy to perform. They can be used in a laboratory or on-site, to detect specific molecules in a given sample.

The inherent ability of antibodies to bind with the structure of specific molecules is used by these immunoassays to produce quick results. Usually found in the blood and tissue fluids, antibodies can bind with antigens (foreign molecules) whenever they are encountered in the body.

Antibodies are highly specific since they are developed to respond to the three-dimensional structure of a particular analyte or antigen. Hence, they can bind only to that particular structure, which makes them the ideal assay reagents for detecting and monitoring specific target molecules. This is because they’re not vulnerable to interference from other substances.

To be used for detection and monitoring, the polyclonal and monoclonal antibodies have to first be extracted and purified from the blood. Four major ELISA formats are used for this purpose. These are known, respectively, as competitive inhibition assays, monoclonal-polyclonal sandwich assays, rapid assays, and antigen-down immunoassays.

Types of Immunoassays

In this section, we will learn some more about the four types of immunoassays mentioned above. These are as follows:

Competitive-Inhibition Assay

These assays only require the binding of a single antibody, so they are often used to measure small analyte samples. A competitive-inhibition assay is not hindered by the steric disturbance that occurs when two antibodies bind simultaneously to one small molecule.

The conjugated analyte and sample are added in steps during sequential competitive inhibition assays, but the reagents are incubated together in a classic competitive inhibition assay. A monoclonal antibody must be coated onto a microtiter plate in this ELISA format.

Later, a specific amount of labeled analyte is added to the plate. The signal generated by the labeled analyte will then facilitate the testing process.

Monoclonal-Polyclonal Sandwich Immunoassay

This immunoassay requires a microtiter plate sandwich, where a monoclonal antibody can be absorbed into the plastic plate. The antibody on the plate binds with the target antigen when the test sample is added to the plate.

In the next step, a polyclonal antibody is added, which also binds with the target antigen. This antigen is already connected with the monoclonal antigen on the plate. Hence, between the two distinct antibodies, an antigen ‘sandwich’ is formed.

Rapid Assay

Immunoassays configured as rapid tests are often used for at-home pregnancy tests and other practical applications. These rapid immunoassays do not make use of microtiter plates. The antigen and antibody reagents in a rapid test are bound to porous membranes which channel excess fluids and react with positive samples.

A flow-through system and lateral flow tests are the two most common configurations of rapid immunoassays. The flow-through system involves adding an analyte conjugate to a sample that has been placed in a well and subsequently washed. The lateral flow test is simpler and involves placing the sample in a well and reading the results immediately after.

Antigen-Down Immunoassay

The antigen is coated onto a microtiter plate, which is subsequently used to bind the antibodies existing within a sample, in antigen-down immunoassays. The antibodies from the sample bind with the antigens on the plate as soon as the sample is added to the microtiter. The higher the signal produced by this process, the larger the number of antibodies present in the sample.

Apart from being used in microtiter tests within laboratory settings, antigen-down immunoassays can also be configured as rapid tests. In this form, they are often used to diagnose allergies. Through an antigen-down immunoassay, a patient’s blood sample can be tested against different allergens. This allows medical professionals to decide if the patient has antibodies to that particular allergen.

In Conclusion

All the above-mentioned ELISA immunoassays can be very helpful in the realm of medical research and diagnostics. However, for the best results, a reliable laboratory, employing experienced researchers, should be hired to perform these complex and delicate immunoassay tests.