If you’re reading this blog there’s a high chance you’re using antibodies in your work, in some form of immunoassay. However, many of us rarely stop and think about where these antibodies come from and how their development has been fundamental for the evolution of life sciences.
A brief history of the antibody
Antibodies as an idea were first hypothesized in the 1890s when Shibasaburō described what we would now call antibody activity against diphtheria and tetanus toxins, suggesting that some molecule in the blood was driving this effect. This led to Paul Ehrlich to propose the theory for antibody-antigen interaction and indeed coin the term antibody, although it was several years before this usage was accepted.
The first molecular evidence of these interactions occurred in the 1920s when Heidelberger and Avery demonstrated that antibodies were indeed proteins produced by cells and that they could be used to isolate their antigen out of a solution. This led to a rapid expansion of the field with the seminal work by Linus Pauling in the 1940s confirming the lock-and-key theory and showing the antibody-antigen interactions are more dependent on their shape rather than their specific chemistry. At the same time Astrid Fagreaus discovered that it was circulating B cells which were the major source of antibodies, and together these two discoveries kick-started the antibody industry as we know it today.
Building on from this significant work has been performed in identifying and refining our understanding of antibody structure with the discovery of the heavy and light chain structure and the immunoglobulin groups A, D, E, G and M. Which are driving the development of multiplex assays further improving our technical capabilities.
So now we have an understanding of the history of antibodies how do they relate to immunoassays including perhaps the most currently widely used enzyme linked immunoassay, or ELISA. While there are many variations of immunoassays such as ELISAs, radio-immunoassays, blood typing assays etc… they all rely on two specific antibody functions, capture and detection.
Capture or Detection
Immunoassays rely on the sensitive and specific detection of antigens by antibodies, however such assays also need to be repeatable and relatively quick to perform. These two contrasting aims and the varying affinities of antibodies for their antigens allows us to utilise antibodies with different capabilities for different immunoassays, or steps within an an immunoassay.
Antibodies with a very high affinity and selectivity for their antigen are ideal for “pulling” and “holding” their antigen out of a mixed solution, allowing non-specific proteins or other molecules to be washed away. Antibodies like this make ideal capture antibodies and are usually fixed onto a surface allowing sample to be added and washed away, as in an ELISA. Such antibodies are usually monoclonal providing a high degree of specificity with the potential to under detect some samples.
Conversely antibodies with a quicker binding rate, but with perhaps reduced affinity make ideal detection antibodies working on samples which have already been immobilized. As they don’t require high affinity these antibodies are often polyclonal which allows for a greater detection range often at a cheaper price. However, this is often dependent on the way in which the sample has been immobilized.
Direct, Indirect and Sandwich
These two families of antibodies can then be applied in a variety of ways depending on assay requirements.
Direct detection immunoassays are the most rapid but are less stringent and so are ideal for tests which require a rapid readout, but where ultimate accuracy is not required. In these assays the substrate is immobilized onto a surface and then primary detection antibodies are added. These antibodies are directly conjugated with a measurable signal such as a fluorescent or radioactive probe, or an enzyme such as HRP which can drive a colorimetric change.
Indirect detection immunoassays improve upon the specificity of direct detection immunoassays by introducing a conjugated secondary antibody. This step also has the added benefit of allowing for signal amplification, which is especially important for low abundance samples. Secondary antibodies are usually designed to target the constant region of the primary antibody, but does so over a variety of sites meaning that a single primary detection antibody can be bound by several conjugated secondary antibodies.
Sandwich immunossays provide the greatest level of sensitivity and detection, the capture antibody is immobilised onto a specific surface and exposed to the sample. This can then be washed off to remove all unbound and any weakly bound product. The sample is then incubated with a detection antibody which can display much reduced specificity but increased binding efficiency to amplify signal. This detection antibody can itself be conjugated or as a further step a secondary antibody can be used. The downside with all these steps is the increase in assay length and the difficult steps of finding antibodies that bind to the same target efficiently and without interference of each other.
The vast majority of immunoassays on the market today use these basic techniques, and understanding how they interact is key for the development of future assays, including the exciting development of in-cell Western blotting, sometimes called cell based ELISA or in-cell Western, which I hope to cover in a later post.