The discovery of antibodies by Paul Ehrlich in 1900 immediately led to the use of antibodies for the detection and analysis of specific compounds in complex samples. From the earliest ABO blood typing and precipitin tests, through the more sensitive detection by radioimmunoassays (RIA) and enzyme-linked immunosorbent assay (ELISA), antibodies have been at the heart of immunoassays. The purpose of this blog post is to focus on the basic role of antibodies and antibody specificity in immunoassay development and detection. In future blogs I will focus on and discuss specific assay formats and applications, so stay tuned if this is a subject that interests you.
In the simplest terms, an immunoassay is any technique that utilizes antibodies to capture or detect a specific target for qualitative or quantitative analysis, typically in a complex mixture. One of the greatest aspects of antibodies, for both immune defense and bioanalytical applications, is the high degree of specificity that antibodies have for their targets. As a result, they are extremely useful for the identification of an individual target or epitope even when this makes up a very small proportion of the total mixture. This property was recognized immediately upon the discovery of antibodies, in the earliest part of the 20th century, and immunoassays have been used since that time for diagnostic, research, and analytical purposes.
II. Immunoassay Properties
While specific immunoassay platforms vary in their methods, there are really only two functions performed by antibodies in any antibody based technique (see Figure 1): capturing a target or detecting a target. The fundamental structure of antibodies generates specific recognition of a region, or epitope, of a target molecule which makes them outstanding tools for developing quantitative assays. While immunoassays are most commonly used for protein evaluation, all biological molecules and many small inorganic and organic compounds are able to be recognized by an antibodies in immunoassays.
The specific characteristics of the antibody binding to its target is the determining factor on how well it will perform in a given immunoassay and also whether it will be capable of capturing the target or if it will only work in the detection of the target. A capture antibody binds to a target in solution, in effect “grabbing” the target out of a complex mixture and holding onto it such that all the other molecules in the mixture can be washed away. Therefore the binding properties of a capture antibody requires high affinity binding with a fast “on rate” and a slow “off rate” in order to grab and hold onto the target. A detector antibody binds to a captured target – either on a solid surface (i.e. membrane or plastic surface) or is captured by another antibody. The detector antibody may be labeled with an enzyme or other molecule that can generate a signal capable of being measured. The properties of the detector require specificity for the target, however it doesn’t necessarily need to be the highest affinity as the target is already immobilized and the antibody can come on and off and still detect the target. The detector antibody is usually in excess so that the avidity of the target-antibody interaction allows for an antibody to always be bound to the target at all times, allowing for detection and measurement.
III. Immunoassay Design
The entire purpose of an immunoassay is to be able to measure a specific target. All immunoassays have the ability to: 1) immobilize or capture the target and 2) detect and measure the target. The target can be trapped on a membrane, plastic plate, or some other solid surface or free in solution. There are three general formats for an immunoassay (see Figure 2): direct detection, indirect detection, or a sandwich assay. The direct detection uses a detection antibody that has the ability to be directly measured. An enzyme or other molecule allows for the generation of a signal that will produce a color, fluorescence, or luminescence that allow for the signal to be visualized or measured (radioisotopes can also be used, although it is not commonly used today). An indirect assay will have a primary antibody to detect the target on the plate or membrane, which is then followed by a secondary antibody (sometimes referred to as a tracer) that recognizes the primary antibody (this would also be classified as a detector antibody). The secondary almost always has specificity for the constant region so it could recognize all mouse IgGs for example. The secondary (or tracer) antibody generates the measurable signal. This can serve two purposes, first it can be set up in a standardized format: all antibodies do not need to be labeled but rather can be detected by a secondary antibody that can be used for many different assays. Second, the secondary or tracer antibody can usually bind to multiple sites on the primary detector antibody, which will enhance the signal and increase the sensitivity of the assay. It may also increase background, so this must be assessed and controlled for either with assay controls or through washes and blocking that will reduce or eliminate the background signal.
The third assay format is the sandwich assay. This format uses a capture and detector antibody. The capture antibody is used to trap or capture the target in solution which allows the target to be isolated from all other components in the solution. The target is now in effect bound to the antibody and a detector antibody can be used to generate a signal (either directly or indirectly as described above). The sandwich format requires two antibodies each with a distinct epitope on the target molecule. In addition, they must not interfere with one another as both antibodies must be bound to the target at the same time.
While there are many immunoassay platforms (we will discuss these in future blog articles), nearly all of them use one of these three immunoassay formats. The format and the platform is often determined by the source of the target, the desired sensitivity of the assay, and the complexity of the assay (are you measuring a single target or multiple targets). As the structural availability of an epitope will vary in solution vs. on a solid surface, generally different antibodies would be required for each of these formats. In fact the target structure may vary from a nitrocellulose membrane to a plastic plate to the surface of a cell, which means the epitope availability and structure may vary considerably among different assay formats.
When developing an immunoassay, all of these factors need to be considered. In developing antibodies (monoclonal or polyclonal) for use in an immunoassay all of these components need to be taken into account, as well. In fact, it is best to incorporate the role of the antibody in the hybridoma development process to be able to generate the best antibody for that specific purpose. It may not be practical or necessary to use the actual assay platform during the isolation of hybridoma clones, however if it is known that the antibody is going to be used as a capture or detector in a specific platform then this role should be mimicked during the hybridoma development process. Specific consideration must be given to the antigen as well as the assay during the hybridoma development planning process in order for both the successful generation of a monoclonal antibody, or antibodies, as well as the creation of an effective immunoassay.