Antibodies, special proteins that are produced by the immune system to bind to foreign macromolecules, are an important reagent for researchers in the molecular biology and biochemistry fields. Antibodies are extremely useful because they can recognize and bind only one protein, even if the protein is in a mixture with other proteins or in a cell. This allows the researcher to tag and monitor their protein of interest. Antibodies are created by injecting an animal with either the whole protein or a representative peptide fragment. This macromolecule activates the immune response in the animal, which causes B lymphocyte cells to produce antibodies that will recognize and bind to the protein of interest.
Scientists use two types of antibodies called "monoclonal" or "polyclonal". Monoclonal antibodies are derived from one B lymphocyte cell and are able to bind to one specific region, called an epitope, on a protein. The antibody is produced from a special fused cell called a hybridoma. This hybridoma can produce the antibody almost indefinitely, meaning the same monoclonal antibody can be obtained over a long period of time. Polyclonal antibodies are a mixture of antibodies produced by a mixture of B lymphocyte cells. All the antibodies recognize the protein of interest, but they recognize different epitopes. Polyclonal antibodies are obtained by isolating antiserum (serum from an animal that is producing antibodies) from the animal's blood. This source gives a much more limited supply of antibodies than hybridomas. Both polyclonal and monoclonal antibodies are useful for various laboratory techniques that analyze proteins, including Western blots, immunoprecipitation, and immunofluorescence. While monoclonal antibodies tend to be much more sensitive for these techniques, polyclonal antibodies are cheaper and easier to develop.
While monoclonal antibodies are mainly created in mice, polyclonal antibodies can be created in a wide variety of animals. The most common animals include rabbits, chickens, rats, sheep, and mice, as well as odder choices such as llamas and raccoons. The animal used to make the antibody depends on several factors. For one, the larger the animal the larger the amount of antibody obtained. Mice, rats, and hamsters produce 1 to 5 milliliters of antiserum per bleed and rabbits produce about 25 milliliters. If a larger amount of antiserum is needed scientists often use goats, sheep, or even horses. Another important factor is ensuring that the protein or fragment will produce an immune response in the animal. This is done by using an animal that is evolutionarily divergent from the animal that has the protein of interest. For example, if you are making an antibody that will recognize a mouse protein, injecting it into a mouse would not stimulate the immune response since the mouse would recognize the protein as self. Likewise, injecting the mouse protein into rats may also not give a response because the rats probably have a protein (ortholog) similar or identical to the mouse protein and therefore will also recognize the mouse protein as self. A species that is more distant from the mouse, such as a goat, would have a better chance of detecting the mouse protein as foreign and producing an immune response. Along these lines, rabbits are commonly used when developing antibodies that recognize human proteins. Finally, different animals produce antibodies with varying specificity. For example, chicken antibodies are notorious for their nonspecific binding which often give dirty results when used in experiments.
How to make a polyclonal antibody:
1. Either the full-length protein or a fragment of its peptide sequence, roughly 15 amino acids in length, will be injected into the chosen animal. These fragments are often too small to activate the immune system. To overcome this, scientists add a larger protein, called a carrier protein, to the peptide fragment. Carrier proteins include KLH (keyhole Limpet hemocyanin) and BSA (bovine serum albumin). This mixture is often combined with a solution called Freud's complete adjuvant, which contains mycobacteria to additionally stimulate the immune system.
2. The protein or fragment is then injected into the animal. The area of injection varies, but is generally intramuscular, intradermal, or subcutaneous. This injection will weakly stimulate the immune system. Four to eight weeks after this initial injection the animal is given more injections, called boosting, that additionally activate the immune system. These boosts occur about every two to three weeks. Blood is collected from the animal roughly two weeks after boosting and the antiserum is isolated by centrifugation and analyzed. If the antiserum shows a low level of the desired antibody then the animal is given more injections. If the antiserum does not show any antibody production after boosting the procedure is repeated in another animal.
Once the antiserum has acceptable levels of the antibody large amounts of antiserum are collected by bleeding the animal several times over the course of several weeks. The antiserum is then tested to ensure it recognizes the protein in various laboratory techniques. If the results are promising the antibody is normally purified from the antiserum by affinity chromatography. The antibody and the antiserum can be stored for long periods of time in a refrigerator or freezer until they are needed.
Current Protocols in Molecular Biology, volume 2, 1998.