After giving examples for practical examples of Biosensors, today’s article classifies the differences between heterogeneous and homogeneous assays. To understand the differences between the two, a few definitions are needed. Assay: testing procedure for estimating the concentration of a pharmaceutically active substance in a formulated product or bulk material (1).
Antibody: specialized Y-shaped proteins produced by our immune system. They bind like a lock and key to the body’s foreign invaders: viruses, bacteria, fungi or parasites (3). When antibodies bind their specific targets, our immune cells recognize the invader covered in antibodies more efficiently, and can then eliminate it.
Antigen: any substance, living or not, that triggers an immune response.
Both assay classes are mostly immunoassays, “bioanalytical methods that measure the presence or concentration of analytes in a solution through the use of an antibody or an antigen as a biorecognition agent.”(1) Homogeneous assays give accurate readouts without separating the analyte from the biomolecules used to detect it, such as labeled antibodies. On the other hand, heterogeneous assays require one or more steps of separation, where unbound antibodies and/or unbound analyte must be washed away. This makes heterogeneous assays longer and more complex to run. They are often more precise than homogeneous assays, and they help with more complex analytes. Homogeneous assays are usually reserved for the detection of small, simple molecules.
Enzyme Multiplied Immunoassay Technique, otherwise known as EMIT, is an assay used to detect the presence or the amount of analyte in a solution. As its name implies, it uses enzymes to yield this result. The main component of the assay is a complex made of an enzyme (“reporter enzyme” in figure below), attached to the analyte of interest. The enzyme’s active site (the part that binds) is specific for a substrate that becomes fluorescent when it binds the enzyme. In short, when this substrate and the enzyme-analyte complex bind, the substrate changes color (yielding a detectable product). These complexes are added to a sample solution, usually from a patient. When the substrate changes color, the whole sample changes color. This however still does not help us explain how we can determine the presence of the analyte of interest in the sample. To this end, we add an antibody specific for the analyte of interest to the sample (anti-analyte antibody). Here, there are two scenarios: either the sample contains the analyte of interest, or it does not.
If the sample does not contain the analyte, the antibody can only bind the analyte attached to the enzyme, in the enzyme-analyte complex. There is no free analyte in the sample. When this happens, the antibody blocks the active site of the enzyme. This means that no other molecule can bind this site, because there is no access possible. The color substrate that would normally bind there can no longer do so. It never becomes fluorescent, and the sample does not change color. If the solution does contain the analyte, the added antibodies have many more molecules to bind. They bind mainly the free analyte in solution, which is easy to reach, and some of the enzyme-analyte complexes. Some complexes remain unbound by the antibody. There the color substrate can bind and become fluorescent, and the sample changes color.
Mechanism of EMIT
For a more animated explanation, go watch the video at : EMIT immunoassay
ELISA stands for <strong>Enzyme Linked Immunosorbent Assay. Like its homogeneous counterpart EMIT, it determines the presence or the quantity of an analyte, but as a heterogeneous assay.
There are four types of ELISA: direct, indirect, sandwich, and competitive (see figure below). All four involve an analyte, at least one antibody, and an enzyme that changes color when a desired reaction occurs. What makes every ELISA heterogeneous is the washing steps (also depicted). They remove all unbound molecules, whether analyte or antibody.
A widely used ELISA is the indirect assay. Typically, it is used to detect the presence of an antigen, for example a virus. The test begins by attaching the antigen to the bottom of a plate. After washing the plate to remove any unbound antigen, we add an antibody specific for this antigen (primary antibody). After another wash step to remove any unbound antibodies, we add another antibody (secondary antibody), this one specific for the first antibody. The secondary antibody is usually issued from an animal (e.g. rabbit), so that it recognizes the human antibody administered first. It is also conjugated to an enzyme that will react with a substrate that we administer in the final step of the assay. Before we add this substrate, we wash the full sample one more time to remove any unbound secondary antibody. Finally, we add the substrate: when reacting with the enzyme, a color change is induced, and the solution changes color. The color change indicates the presence of the analyte. It is proportional to the amount of primary antibody, and therefore to the amount of analyte.
As the figure on the left shows, there are other types of ELISA. Their advantages and disadvantages are listed in the table on the right.
One of the biggest disadvantages of ELISA in the past was its low throughput readout. Roche has simultaneously solved this problem and created a fast detection method of SARS-Cov-2 based on their general Elecsys assay technique. It is “an immunoassay for the in vitro qualitative detection of antibodies (including IgG) to SARS-CoV-2 in human serum and plasma”. It utilizes a principle very similar to ELISA called ECLIA, pictured below.