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.


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