monoclonal antibody

A key technology in modern biology.

A monoclonal antibody is a preparation of antibody that all have the same exact specificity, and in fact, is all the identical protein.

When vertebrates are vaccinated with an antigen, one of the responses that the immune system comes up with is to make antibodies to the vaccine. Because many B-cells are responding at the same time, the blood of the vaccinated animal contains many different antibodies. Even the antibodies that actually bind the antigen are all different, and potentially bind different parts of the antigen.

For an immune response to a pathogen, or for having a good response to a vaccine, this is not important. But researchers have known the potential use of antibodies as a tool in research. Since vertebrates can make antibodies that bind to pretty much anything, making a specific probe to bind a molecule of interest is potentially as simple as shooting whatever you want the antibody to bind into a rat.

Monoclonal antibodies are made in such a way as to obtain antibodies that are all the same and see the same antigen in the same way. Basically, one immunizes an animal, removes their spleen and purifies the B-cells that make antibodies. These cells are then separated from one another, and each are fused to another cell that confers the ability to replicate and survive outside of the animal. The culture supernatants of these hybrid cells are tested for the antibody that you want. Once you find a hybrid cell making the antibody you want, you can grow as much of the now monoclonal antibody as you need.

Niels K. Jerne, Georges J.F. Köhler, and Cesar Milstein all won the 1984 Nobel Prize in Medicine for coming up with this technology.

---

The writeup below talks about the biomedical uses of monoclonal antibodies, many of which didn't pan out. Monoclonal antibodies have proven fantastically useful, however, in the pursuit of molecular biology. As the method let's one make monoclonal antibodies to any particular molecule, the monoclonal antibody itself is used to determine if the antigen exists, whether in a particular sample (Western blot) or on the surface of a cell (FACS). The antibody can even be used to purify the antigen it was raised against (via immunoprecipitation, or chromatography techniques).

Monoclonal antibody production not only gave us great insights into the biology of B-cells and antibodies, but provided one of the key techniques that have helped the progress in biological sciences for the past 20 years.

In the writeup above, fhayashi gives a great description of what monoclonal antibodies are, but never mentions what they are good for. Monoclonal antibodies are useful in medicine because they enable the immune system to fight a specific pathogen without expending the time and energy required to make its own antibodies.

Some modern monoclonals are even better, because they can fight a specific pathogen without any help from white blood cells. They do this by carrying a toxin or radioactive isotope, rather than the white blood cell binder that's part of a normal antibody. These elements damage the pathogen when the antibody attaches to it, not involving the immune system at all. Normal antibodies (and monoclonal antibodies acting in the normal way) serve as attractor sites for white blood cells which eventually come and destroy the pathogen.

Since monoclonals were invented in 1975 (resulting in the 1984 Nobel Prize mentioned above), and seem to have all the advantages over vaccination, you may wonder why they aren't in drastically wider use. As it turns out, monoclonals simply haven't proven viable in human trials until eight or nine years ago, for a variety of reasons. First, the monoclonals developed in the 80s were themselves interpreted as pathogens by the immune system, and dealt with accordingly. Thus they were worse than useless, since the immune system inevitably killed some of its own antibodies while purging the monoclonals. Also, many monoclonals had been developed using mouse antibodies, which are more often than not rejected by the body in the human anti-mouse antibody (HAMA) reaction.

B lymphocytes, a variety of white blood cell, are isolated from mice that have been exposed to an antigen/pathogen. These lymphocytes are then fused with a human lymphocyte isolated from myeloma, a cancer of the bone marrow. These "hybridomas" have the antibody properties of the mouse cells along with the ability to reproduce infinitely from the cancer cells, so they are perfect to grow a large culture of. The culture is exposed to the original antigen, and those hybridomas which bind to it (i.e. those which contain the antibody against it) are isolated. They can then be cultured in a petri dish or in living mice, with monoclonal antibodies eventually harvested from either.

Unfortunately, these harvested antibodies will still suffer from the problems above, since they are made completely of mouse protein. One way to get around this is to fuse the antigen binding regions from the mouse antibody with the basic structure of a human antibody, resulting in a so-called chimeric antibody which is around 66% human. Another, related, method is taking the antigen binding sites themselves off of the mouse's binding regions, and attaching them to a fully formed human antibody. This is known as a humanized antibody, and is 90% human protein.

Making human monoclonals seems an even better approach than rebuilding mouse ones, but has turned out to be very difficult in practice. Researchers have (finally, after 25 years of work) fused human B lymphocytes with immortalized cells, but haven't tested antibodies produced by these new cultures. Taking the opposite approach, two companies have engineered mice to produce human antibodies rather than murine ones. Some antibodies made this way are currently undergoing human drug trials.

Antibodies are proteins that are created by the immune system to recognize and bind to certain foreign macromolecules, such as other proteins. These antibodies are extremely specific and will generally bind only to that macromolecule. Because of this specificity, researchers often use antibodies as a sensitive way to monitor an individual protein in a cell. Antibodies play a major role in several different laboratory techniques that analyze proteins. For example, antibodies can be used to tag a protein in order to see where it is located in the cell by a technique called immunofluorescence. Antibodies can also analyze the presence and quantity of the protein by performing a Western blot. They can also be used to isolate a protein from cells with a technique called immunoprecipitation and can help purify a protein from a mixture by using affinity chromatography.

Researchers use two different types of antibodies, polyclonal or monoclonal, to analyze proteins. Both types of antibodies are produced from cells called B lymphocytes. They are created by injecting mice with a foreign protein or a peptide. The B lymphocyte cells of the mouse then produce antibodies that recognize and bind to the protein or peptide. These cells are isolated and the antibodies are collected from the cell medium. Polyclonal antibodies are created from a mixture (poly) of different B lymphocytes that all create antibodies that recognize the protein. The different antibodies can recognize and bind to several different regions, called epitopes, on the protein of interest. Monoclonal antibodies are cloned from a single (mono) B lymphocyte cell that produces the antibody. Therefore, the monoclonal antibody will recognize only one specific epitope on the protein of interest. Because of this monoclonal antibodies are often more sensitive and specific than their corresponding polyclonal antibodies and generally give cleaner experimental results. However, monoclonal antibodies are much more difficult to create and can take several months to develop. They are also very costly to produce.

Scientists spent many years developing a way to make monoclonal antibodies. A major problem was that the B lymphocyte cells that produced antibodies are somatic cells and therefore died after a few generations of growth in cell culture. This was a severe drawback as only a small amount of antibody could be created at a time. The solution was discovered in 1975 by the scientists Georges J.F. Köhler and Cesar Milstein. They found that they could fuse together a B lymphocyte cell and a myeloma cell from a tumor to form a cell called a hybridoma. This hybridoma produced antibodies and was immortal, meaning it could be grown indefinitely. Now scientists could make an unlimited supply of monoclonal antibodies. Köhler and Milstein's discovery proved to be so valuable to research that the two scientists were awarded the Nobel Prize in Medicine in 1984.

How to make a monoclonal antibody:

Monoclonal antibodies can be designed to recognize not only proteins but also carbohydrates, and nucleic acids. This is done by injecting the macromolecule of interest into mice. When an antibody is going to be made that can recognize a protein the scientist has the option of either injecting the full-length protein or a small peptide sequence from the protein. Injecting the peptide is often the better choice because it is smaller and will have an easier time being absorbed than the bigger, complete protein. It is therefore more common to inject a peptide. However, when using a peptide there is a slight chance that the resulting antibody will not be able to recognize the full-length protein.

1. For this situation, let's say we're going to make an antibody that will recognize a peptide sequence from a full-length protein called "protein A". Peptides used to make antibodies are generally about fifteen amino acids in length. The three dimensional structure of the folded protein A is often examined before choosing the sequence. Sequences that are hidden in the folds of a protein are generally bad choices, since antibodies created from these sequences will probably not be able to find the sequence on the folded protein, and therefore will not recognize protein A.

2. Next, the peptide is injected into mice. The peptide is often injected along with a compound called Freud's complete adjuvant, which contains mycobacteria to additionally stimulate the immune system. The mice are injected with this solution every one to two weeks. The presence of both the bacteria and foreign peptide activates the mouse's immune system, and the B lymphocyte cells present in the spleen create antibodies that recognize and bind to the peptide. After several weeks, samples from the mouse are taken and the immune response is analyzed. If the response is weak more injections are given.

3. When the immune response is strong the spleen is removed from the mouse and the B lymphocyte cells are isolated from the organ. These cells are then fused with myeloma cells in order to make hybridomas. The fusion is aided by a solution called polyethylene glycol, or PEG. The hybridomas are isolated from the other cells that did not fuse by placing the cells in a medium called HAT medium. This medium only supports hybridomas and leftover B lymphocytes and myeloma cells will slowly die over the course of several weeks. The hybridoma cells can now be passaged in cell culture indefinitely.

4. Next, the hybridoma cells are diluted so that only one hybridoma cell is present in a dish. This cell then grows and divides to make a colony of its clones that produce the monoclonal antibody. Once the colonies are grown they are screened using a technique called ELISA to determine which colonies are producing the desired antibody. Positive colonies are often again diluted down to one hybridoma cell and cloned to further purify and strengthen the antibody. This process is called subcloning.

5. Finally, the monoclonal antibody needs to be tested on the full-length protein A to make sure it recognizes the protein. This is done by using the antibody to detect protein A either in the cell by immunofluorescence or in cell lysates by analyzing a Western blot. It is certainly possible (but somewhat uncommon) that the antibody will not be able to recognize protein A and the entire process must be started over with a different peptide sequence. If the monoclonal antibody passes this stage, the hybridoma cells can be grown to create more antibody or frozen away for storage.

Sources:

• Antibodies - A Laboratory Manual, 1988.
• Current Protocols in Molecular Biology, Volume 2, 1994.