Antibody Sample Preparation

What makes a molecule immunogenic?

Proteins, peptides, carbohydrates, nucleic acids, lipids, and many other naturally occurring or synthetic compounds can act as successful immunogens. In general, for a compound to elicit a primary antibody response and a strong secondary response it must contain an epitope that can bind the cell-surface antibody of virgin B cell and it must promote cell-to-cell communication between B cell and helper T cell. Binding of the antigen to the surface antibody molecule of virgin B cell is an absolute requirement for an antibody response. This binding determines the specificity of the resulting antibodies, as the antigen binding site on the surface antibody molecule will be identical to the binding site on the secreted antibodies. An immunocompetent animal can produce antibodies with well over 107 different antigen combining sites, and most compounds can bound tightly by at least one of these surface antibodies. The second property of a molecule that allows it to be a good immunogen is that it must promote cell-to-cell communication between helper T cell and B cell. This is achieved by providing a physical link between these two cells. The requirement for both an epitope and a class II- T-cell receptor binding site imposes a minimum size limit on an immunogen size limit on an immunogen, and binding site imposes a minimum size limit on an immunogen, and molecules of less than about 3000-5000 Daltons are generally not good immunogens. Compounds smaller than this can often bind to surface antibodies on the B cell but may not have suitable sites for the simultaneous binding of a class II protein immunogenic molecules overcomes this problem by providing the missing class II - T-cell receptor binding sites and allows the induction of a good antibody response against the small molecules. Small molecules showing this behavior are defined as haptens. The large molecules that render them immunogenic are defined as carriers. In summary, a good immunogen has three chemical features:

  1. It must have an epitope that can be recognized by the cell-surface antibody found on B cells.
  2. It must have at least one site that can be recognized simultaneously by a class II protein and by a T-cell receptor.
  3. Usually, it must be degradable. These three properties are the only intrinsic chemical features needed for a molecule to elicit a strong antibody response.

However, to force an animal to respond to an antigen, other factors need to be considered. These include the dose and form of the immunogen, the use of adjuvants, and potential modifications. Practical advice on these aspects is presented below.

Sources of antigen

The object of preparing the antigen for immunization is to present it to the animal in a form that will induce the strongest and most appropriate response. This section presents protocols for preparing proteins, peptides, and haptens for immunization. In considering the preparation of an antigen, the major decision to be made is how pure does the antigen need to be before starting an immunization schedule. This decision should be based on the intended use of the resulting antibodies. If highly specific antibodies that will only recognize the appropriate antigen are essential, then the antigen must be purified to homogeneity, or the antigen preparation should be used to prepare monoclonal antibodies. If a mixed reaction is acceptable, then further purification may not be warranted. If further purification is needed, standard techniques, including column chromatography, differential extraction, and subcellular fractionation, are the most useful. When using protein antigens, if the polypep tide of interest can be seen as a unique band on a SDS-polyacrylamide gel, then the gel can be used as final purification step. When mixed populations of antigens are used for immunizations, an antibody response to several components of the preparation is expected.

Pure Antigens

Having pure antigens provides the best case for the production of antibodies. However, not all pure antigens will elicit a good response, and there are no tests to determine whether an antigen will be immunogenic other than the obvious empirical test of injecting the preparation and testing the sera, as long as the antigen is larger than the minimal size range.

Purifying Antigens from Polyacrylamide Gels

If the antigen is not available in pure form, one simple method for purifying a protein antigen further is to separate the samples on SDS-polyacrylamide gels. Antigens purified this way often induce good antibody responses. Since the antigen is denatured by this route of preparation, the resulting antibodies are usually particularly good for techniques that need or benefit from denaturation-specific antibodies. However, some of the resulting antibodies will not bind to the native antigen. Run your protein sample on any of the standard gels. Locate the antigen using any of the methods below and process for injection.

Location the Antigen after Electrophoresis

After electrophoresis, the protein band of interest must be located in the gel, and the gel slice excised for injection. A variety of identification methods can be used, all of which are designed to avoid excessive fixation of the protein in the gel matrix. The choice of method depends partly on the abundance of the polypeptide. The methods are commonly used:

  1. Staining of side strips cut from the edge of the gel.
  2. Light staining of the gel itself.


Many small chemicals can be used to raise antibodies, if they are coupled to larger protein molecules. The small compounds are known as haptens, while the proteins which they are coupled to are called carriers. The haptens themselves serve as epitopes for binding to the antibodies on the B-cell surface, and the carriers provide the class II - T-cell receptor binding sites. In general, haptens should be coupled to soluble carriers such as bovine serum albumin (BSA) or keyhole limpet hemacyanin (KLH). The coupling mechanism will vary with each hapten. The use of synthetic peptides as immunogens has been an important technique in the elucidation of the prope rties of an antibody response.

Bacterial Expression

As DNA sequences and their corresponding protein sequences have become known, recombinant protein have been used to prepare antibodies specific for previously uncharacterized proteins. Recombinant protein fuse to carrier protein can be expressed in Bactria cells, and even crude extract can be used for immunization, if you are not going to test the antisera on bacterial proteins.

Synthetic peptides

Also synthetic peptides can be use as an antigen. The synthetic peptides are purified and coupled to carrier proteins, and these conjugates are then used to immunize animals. In these cases, the peptides serve as haptens with the carrier proteins, providing good sites for class II- T-cell receptor binding. Peptide -carrier conjugates seldom fail to elicit a response because of tolerance. Consequently, the peptides can usually be seen as epitopes and high-titered antisera commonly are prepared. Characteristically, these antibodies will bind well to denature proteins, but may or may not recognize the native protein. The two most important advantages of anti-peptide antibodies are that they can be prepared immediately after determining the amino acid sequence of a protein (either from protein sequencing or from DNA sequencing) and that particular regions of a protein can be targeted specifically for antibody production. Rapid conversion from DNA sequence information to antibodies has enormous potential for application in molecular biology. Similarly, the production of site-specific antibodies has immediate implications for functional and clinical studies. Polyclonal Antibody Preparation