Structure of an antibody molecule.
In the figure below (a crystallographic structure), the heavy chains are blue and gold and the light chains are red. The light chains have two domains: CL and VL, representing constant and variable regions. The heavy chains have a variable domain (VH) and three different constant domains. The stem of the antibody shown at bottom (the portion containing only heavy chain) is called the Fc domain, and the two arms (left and right) are called Fv domains, where the c and v refer to "constant" and "variable". The arms can be cleaved from the Fc domain separately (so that each arm is free) using the protease papain, and that product is called an Fab fragment. A different protease (pepsin) can cleave the antibody so that the Fc domain is removed but the two Fab arms are still connected by a disulfide linkage. Such a bifunctional product is called an F(ab')2 fragment.
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The model |
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The top picture is a labeled ribbon drawing of the first intact antibody (IgG2A) every crystallized. The antibody is assymetric, reflecting its dynamic character. There are two local dyads in the molecule. One relates the heavy chains in the Fc, the other relates the constant domains of the Fabs. |
Polyclonal antisera
As a practical matter, how do we obtain an antibody directed against a purified antigen?
The first thing to consider is the source and purity of the antigen. A recombinant protein can be expressed to very high levels in E. coli, but as we have discussed previously there are legitimate concerns about:
- proper folding
- post-translational modifications
Suppose a researcher is using antibodies to study a eukaryotic protein. The researcher could purify an antigen from its native eukaryotic source, but unless the antigen is expressed at a high level it may be difficult to obtain an acceptable level of purity. How do you get rid of all of the other cellular proteins that you don't want the laboratory animal to see? Is 99.9% purity high enough? This may be a significant problem because the laboratory animal will develop an immune response against any impurities in the preparation, just as it would to the protein of interest. If the antiserum is to be used for identifying the location or amount of an antigen, the presence of antibodies against other native proteins in the same organism would be a problem.
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What a mess!
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There is a considerable advantage to preparing an antigen in a non-native host such as E. coli, because even if the laboratory animal develops an immune response against a contaminant, that contaminant would be an E. coli protein. Antibodies against E. coli proteins are unlikely to interfere with the application of the antiserum against eukaryotic cells, because the proteins from eukaryotes and prokaryotes are dissimilar.
Still, the proteins extracted from E. coli may be improperly folded (particularly if the proteins must be solubilized from inclusion bodies) and may not resemble the native eukaryotic protein with respect to glycosylated or phosphorylated amino acids. That could be a problem, and the researcher may choose to express his protein from a eukaryotic expression vector system (such as in yeast, insect cells, etc.)
Immune response
After the first injection of antigen, the animal will begin to mount an immune response, generally using IgM isotype antibodies. The response peaks and begins to decrease in magnitude about 3-4 weeks after injection. Then, it's time to "boost" the response with another injection. The immune response to the second immunization is much greater in magnitude and arrives after a shorter period of time. In addition, the IgM isotype reaction is lessened and more of the reaction is of the IgG type. A laboratory animal may be "boosted" several more times, with "bleeds" taken a week or so after each injection to check the antibody titer (reactivity). In general, the titer increases with each boost.
Note that serum taken from the laboratory animal is polyclonal at this point. That is, the antibodies directed against the injected antigen are sharing the bloodstream with antibodies unrelated to the experiment. That is, when the rabbit caught the "bunny sniffles" during the experiment, the antibodies against Bunny Sniffle Virus were also generated and are a component in the unfractionated serum.
In the case of antiserum directed against a GST fusion protein, some antibodies will be directed against the Schistosoma japonicum glutathione S transferase enzyme and others will be directed against the C-terminal fusion domain (i.e. the peptide of interest). The anti-GST antibodies are unlikely to interfere with your application of the antiserum (just as the anti-Bunny Sniffle Virus antibodies are unlikely to interfere, unless you are studying a related Human Sniffle Virus).
It is an important experimental control to collect a "pre-immune bleed" from the lab animal before beginning a course of injections, just to be sure that the immune reaction was newly created by the injection.
Antibodies can be purified from crude serum using methods such as ammonium sulfate precipitation, gel filtration chromatography, or affinity chromatography. For example, immunoglobulins tend to have the ability to bind to Protein A (from Staphylococcus A), and protein A-sepharose is a common affinity matrix.
Using the antibody
For example, try out the search engine at AbCam Inc.
Your next concern may be to detect where your antibody is binding, whether on a Western blot, ELISA, or slide of fixed cells. The general approach is to use a secondary antibody that is conjugated with a chemical or enzymatic label, and to detect the secondary antibody binding by a colorimetric (or luminescent) enzyme substrate, fluorescent tag, radioactive tag, gold particle, etc.
Zymed has a list of secondary antibodies that you may wish to peruse. For example, in the list of secondary antibodies to mouse immunoglobulin, you can see that you have a choice of mouse Ig isotype (IgA, IgE, etc.) host of origin (goat, rabbit, etc), and conjugate (biotin, alkaline phosphatase, gold, etc.)
Using fluorescent dyes, it is possible to detect multiple antibody binding events. For example, Molecular Probes Inc. is a major distributor.
Monoclonals
The types of antisera that have been described so far are collections of antibodies. Antibodies raised against a single antigen will tend to have many different specificities, which is to say that some antibodies will recognize one side of the molecule and other antibodies will recognize another side.
You can obtain monoclonal antibodies (a single specificity) by the following method.
1. Immunize rats (or more usually mice) with an antigen.
2. A week or two after the first boost, kill the animal and remove its spleen (containing activated B lymphocytes that are making antibodies). Some of the spleen cells will be responding to the injected antigen.
3. Fuse individual splenic B lymphocytes with a plasmacytoma (a B lymphocytic tumor that secretes large amounts of immunoglobulin). It is common to use a plasmacytoma that does not make an endogenous antibody, but has the capability of expressing antibodies.
4. Grow these hybrid cells (called hybridomas) and sequester groups of them in the wells of microtiter dishes. The hybridomas will secrete antibodies into the culture medium, and these "supernatant" antibodies can be collected and tested.
5. Once a "well" in the plate has been identified as being positive, that is that it has a hybridoma secreting antibody against an antigen of interest, it is important to isolate the hybridoma in pure form. The cell line is diluted to the point where each well contains a single cell, and the culture is "cloned" because it is then single-cell in origin. This means that the antibody being generated by the cells is from a single rearrangement of the immunoglobulin locus and is monoclonal.
6. The hybridoma can be grown in vitro, and the culture supernatant collected and purified. This will yield about 10-100 micrograms of antibody per ml of culture. Alternatively, the antibody can be injected into the peritoneal cavity of a pristane-primed (or mineral oil primed) mouse (or rat), and an ascites tumor will develop. The ascites fluid can be collected and will contain approximately 1 to 10 mg protein per ml. The in vivo methods are frowned upon because they cause pain to animals (not that collecting fetal calf serum for culture is painless), and there is some risk that the ascites will contain endogenous polyclonal antibodies against other specificities, viral pathogens, etc.
7. Hybridoma cells can be cryopreserved
Detection
An antibody binding to an antigen can be detected directly, by conjugation to a marker such as an enzyme or fluorescent dye, or by application of radioactive iodine.
Fluorescent dyes
- Texas Red
- Rhodamine
- Fluorescein Isothiocyanate (e.g. "FITC")
Enzyme-based detections:
- Alkaline phosphatase
- Horse radish peroxidase
- Colorimetric methods
- Chemiluminescent methods
Typically, however, it is easier to detect the first binding event
indirectly, through use of a secondary antibody.
See fig. 9.1 on p. 202 for an example of "structure building"
in detection systems. The secondary detection may be based on things other than
antibodies. For example, protein A from S. aureus can be conjugated to a marker:
A popular connection is biotin-avidin. A secondary antibody may be
biotinylated, for example, and a marker can be avidinylated. See
fig. 9.4 (p. 209) for a related example.
Antibodies may be used as "capture" reagents as well, increasing
the specificity as in this example:
