What is an ELISA test?

ELISA tests are antibody-based experiments that measure macromolecular interactions. Just a warning: we use a lot of antibody terminology here, so it might be good to read our previous blog, What is an Antibody first to refresh your memory! So, what is an ELISA? To start, let’s look into the history of the technique.

A typical ELISA plate

The History of ELISA

The only way to determine antigen-antibody binding prior to the ELISA was the radioimmunoassay (RIA) developed in 1960 by Rosalyn Sussman Yalow and Solomon Berson (1). Diagnostic researchers had to label antigens with radioactive carbon or sulfur in vivo (in the cell) or wash the antigen with isotopes of iodine in vitro (in a test tube). Both labeling methods would expose them to radioactivity; a well-known carcinogen. An alternate method was necessary to avoid the health risks inherent in RIA. The idea of molecule labeling was one such alternative, but it had been met with skepticism because it was believed molecule labeling would interfere with the binding event. However, between ’66 and ’69, Dr. Stratis Avrameas and his team at the University of Paris in Villejuif found successful results after coupling antigens or antibodies with enzymes(2). By 1971, three independent research groups published papers outlining techniques using enzyme labeling(3,4,5,6). The team under Peter Perlmann and Eva Engvall at Stockholm University coined the term ELISA in their paper Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. For their contribution, Perlmann and Engvall shared the 1976 Nobel Prize in Biochemistry with Doctors Anton Schuurs and Bauke van Weemen.

Types of ELISA Tests

The word ELISA actually encompasses several different types of test, but all of these have a few factors in common. First, they all use an Enzyme-Linked antibody — the “EL” of ELISA — which can induce a color change in a solution. Because the enzyme is linked to an antibody (immunoglobulin) which binds (adsorbs) to some target, these tests are collectively referred to as ImmunoSorbent Assays. The general principle is that an antibody solution is added to a container which has a surface — called the “plate” — coated with some kind of immobilized antigen. Antibodies that fail to bind to the antigen on that surface are washed away. Any remaining antibodies are exposed to a chemical marker, which is chemically altered to induce a color change by the linked enzymes. The intensity of the color change will correspond to how many enzyme-bound antibodies are in the solution, which in turn tells you how much of the target is present!

An indirect ELISA (left) and direct ELISA (right). The antigen (green) is bound to the plate. The antibody (yellow/red) binds to the antigen directly. An enzyme attached to the primary antibody (direct) or secondary antibody (indirect) induces a color change when its substrate is added.

The first and simplest type of ELISA is the direct ELISA. In a direct ELISA, the antigen is bound to the surface, and then enzyme-linked antibody binds it directly.

The indirect ELISA is functionally similar to the direct ELISA, but with one extra step — instead of the first antibody being linked to the enzyme, a second enzyme-linked antibody is added to bind a spot on the end of the first antibody. Because the tail-end of the “primary” antibody can be the same for many antibodies with different antigen binding properties, one enzyme-linked “secondary” antibody can be used in the detection of many different antigens — without affecting the binding abilities of the primary antibodies.

The sandwich ELISA is less common than other ELISAs. This test starts with antibodies bound to the plate. The plate is exposed to an antigen, some of which will bind to the antibodies. After the excess is washed away, the signaling antibody is added, ‘sandwiching’ the antigen between itself and the plate antibody. This method is common for home-use ELISA kits, as it doesn’t need the antigen to be purified to provide high sensitivity.

A sandwich ELISA. Antibody A (yellow/red) is bound to the plate. Antibody B (cyan/red) is in complex with the antigen (green) and an enzyme (tan) which will signal when its substrate is added.

The competitive ELISA is a variation of the indirect ELISA, where the binding of a new add-in antigen (or a sample that may contain this antigen) is measured in comparison to the original antigen. Briefly, the primary antibody (unlabeled) is incubated with an original antigen, and then this mixture is added to the wells which are pre-coated with the same antigen. Then the samples are processed the same way as in the case of the indirect ELISA. The add-in antigen binds to the primary antibody leading to lower signal than in the absence of the add-in antigen. This assay allows the assessment of the antigen presence in crude or impure samples.

Another variation of the competitive ELISA is when an antibody is immobilized on the surface of the well, and a fluorescently tagged antigen is added to detect the presence of this antibody. When the antigen is mixed with an unknown sample which may contain the antibodies that bind to this antigen, the signal produced by the original antibody-antigen interaction will be reduced, thus providing evidence for the presence of the antigen-interacting antibodies in the sample. This latter variation is often used to detect the HIV antibodies in patients serum.

Everyday Uses of ELISA

A pregnancy test is a streamlined, home-use ELISA

ELISA have been used in labs since their invention to measure protein concentration and binding affinity as well as detect target proteins in mixtures. However, since ELISA kits are fairly inexpensive to make, they have been adapted for use outside the lab. There are kits to detect food allergens that are useful for producers, restaurants, and individuals who suffer from extreme allergic reactions. Pregnancy tests are sandwich ELISAs that use capillary action to automate the detection of a pregnancy hormone. The home HIV test uses direct or indirect ELISA in a similar process to detect HIV antigens or antibodies, respectively. Many other diseases can be detected with ELISA kits, too. These include lyme, chagas, and heartworms to name a few. In fact, all you need is a target associated with the disease, an antibody that will bind to it, and a way to transfer the target to a test strip. With the ever decreasing price of technology, we may not even need to visit our doctors’ offices for a diagnosis in the future.

Links and Citations:
1. Yalow RS, Berson SA. Immunoassay of endogenous plasma insulin in man. Clin Invest 1960;39:1157–1175
2. Avrameas S. Coupling of enzymes to proteins with glutaraldehyde. Immunochemistry 1969;6:43–52
3. Engvall E, Perlmann P. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 1971;8:871–874
4. van Weemen BK, Schuurs AHWM. Immunoassay using antigen-enzyme conjugates. FEBS Letts. 1971;15:232–236
5. Avrameas S, Guilbert B. [Enzymo-immunological determination of proteins with the aid of immunoadsorbants and enzyme-labeled antigens]. C R Acad Sci Hebd Seances Acad Sci D 1971;273:2705–2707
6. Avrameas S, Guilbert B. A method for quantitative determination of cellular immunoglobulins by enzyme-labeled antibodies. Eur J Immunol 1971;1:394–396.

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