Antibodies are one of the most important tools in molecular biology. Basic researchers use antibodies to identify, locate, isolate and quantify specific proteins. Clinical researchers use antibodies to target a specific drug (such as a chemotherapeutic agent) to a particular cell. Because of the cell specificity provided by the antibody, a higher amount of antibody drug conjugate (ADC) can be delivered to the cell without systemic toxicity. However, ADC characterization must be rigorous for this same reason.
Regardless of what species, cell type, or organ system you are studying, it is almost inevitable that you will use an antibody-based assay at some point.
Here is a quick review of the key concepts for antibody use in your molecular biology lab:
What Is an Antibody?
Antibodies are globular proteins produced by cells as part of their defenses against pathogens. When pathogens invade the body, antibodies specifically recognize pathogen proteins and attract other immune cells to destroy them. When an antibody recognizes a pathogen, its production is up-regulated.
Each antibody binds to one specific protein sequence, called an epitope. Epitopes are both sequence and structure specific. Some antibodies recognize a protein in all its forms by recognizing a common epitope – other antibodies only recognize very specific post-translational modifications, such as phosphorylation of a particular amino acid. So you need to be very careful to pick an antibody that fits your assay perfectly.
Antibodies consist of a pair of “heavy chains” and two “light chains.” When the heavy and light chains assemble to form the antibody molecule, they form two regions: the constant region, which is highly conserved, and a highly variable region. The variable region is also called the antigen recognition domain. It recognizes and binds specific epitopes.
What Types of Antibodies Are There?
In molecular biology labs, you use a variety of antibodies, depending on the assay type and the particular molecule you are studying. You also use antibodies that are generated in different species. A good rule of thumb is to never use an antibody that was raised in the same species your assay is studying, i.e. if you are looking at a rabbit brain, you should not use rabbit antibodies.
Commercial antibodies generally come in two types, Monoclonal or Polyclonal.
Polyclonal antibodies are simple to create. You inoculate an animal with your epitope-of-interest, collect the animal’s blood over the course of a few weeks, and fractionate this blood to separate out the antibodies from the other blood components. The resulting antibody fraction will contain all the animal’s circulating antibodies, including some antibodies that are specific for your epitope-of-interest.
Monoclonal antibodies are created in a similar, but more labor-intensive manner. After inoculating the animal with your epitope-of-interest, you remove the blood and separate out the white-blood cells (which produce antibodies). You then fuse the white blood cells with myeloma cell lines, forming cells called hybridomas. This steps allows you to culture the antibody-producing cells indefinitely. Each individual hybridoma cell produces a specific antibody. To find your perfect antibody, grow individual colonies of hybridomas and screen for their ability to bind to your epitope of interest. Harvest the best clones and simply culture them to produce more antibody, which is released into the culture medium.
So What Is Better? Polyclonal or Monoclonal?
It is important to appreciate the difference between polyclonal and monoclonal antibodies. Owing to the differences in their production, polyclonal antibodies are much less expensive than monoclonal antibodies. They also tend to give more robust results in certain assays as they can bind multiple areas of the same protein. However, monoclonal antibodies are usually more specific, and have less off-target binding than polyclonal antibodies. The choice of a polyclonal vs monoclonal antibody is therefore a balance between the required specificity for a given assay, the sensitivity of the antibody and the cost.
What Are Antibodies Used For?
In nature, antibodies are important for targeting proteins produced by pathogens. In the lab, we use antibodies to identify, purify and target proteins as well as for drug delivery.
Protein identification is the most common use of antibodies in molecular biology research labs. For example, Western blots and ELISA assays rely on antibodies to identify specific target proteins. Medical pathologists use antibodies to identify proteins associated with disease states.
Protein purification is another common antibody-based assay. Antibodies are used to capture single proteins from the complex molecular soup that is a cell lysate.
Well there you go, my brief practical primer on antibodies and their uses is over. Hope that helps!
Read on to learn more about ADC analysis and early drug screening assays.