Many of the decisions on how to make a successful antibody depends on the materials that a researcher has available. For example, if a researcher has a purified recombinant protein available, it will dictate the options we have to producing antibodies. We can work with researchers to help them fit the best program for their needs. There are four basic methods for producing antibodies. These are 1) Peptide polyclonal antibodies (PPAb), 2) Peptide monoclonal antibodies (PMAb), 3) Recombinant protein polyclonal antibodies (RPAb), and 4) Recombinant protein monoclonal antibodies (RMAb). Genetic immunizations and phage display are not considered in this discussion.
Western Blotting
Western blotting is the most common application used with antibodies. In this assay, protein samples (most commonly cell lysates) are run by electrophoresis on an acrylimide gel in order to separate them out for probing on a nitrocellulose or PVDF membrane. Antibody types to select from:
- PPAb, RPAb if recombinant protein is available
- Comment: Monoclonals do not generally work well for western blotting
Immunohistochemistry
IHC is another very important and common application used with custom antibodies, in which researchers will probe paraffin or frozen tissue sections for endogenous protein. Antibody types to select from:- PPAb, RMAb, PMAb
- Comment: Most commonly used reagents for screening and initial characterization are peptide polyclonals, but monoclonals are many times needed if a more quantitative and expanded analysis are required. Background can be an issue, so specificity is extremely important.
Immunocytochemistry
ICC is very similar to IHC, except that testing is performed on fixed cells. The same cross-linking and antigen presentation conditions apply.
- PPAb, RMAb, PMAb
- Comment: Most commonly used reagents for screening and initial characterization are peptide polyclonals, but monoclonals are many times needed if a more quantitative and expanded analysis are required
ELISA
ELISA is the assay of choice for quantification. Recombinant proteins are the best source for producing reagents for this assay, unless small differences such as post-translational modifications or extreme antigen conservation are issues. Tertiary structure, and therefore, antigen presentation are extremely important. Antibody production methods are:
- RPAb, RMAb, PPAb, PMAb
- Comment: Screening of monoclonals for utility is the key. If conservation or modification detection is necessary, then carefully designed peptides are the only route and should first be validated at the polyclonal stage.
Immunoprecipitation
This same tertiary structural concerns apply, but the larger volumes of antibody are typically not necessary. Antibody production preferred methods:
- RPAb, RMAb, PPAb, PMAb
- Comment: Recombinant polyclonals are almost always the best choice. If no recombinant protein is available, then careful selection of peptide epitopes is essential.
Flow Cytometry
This assay is one of the more difficult for antibody utility. Large numbers of peptides need to be screened or monoclonals may be produced under rigid screening conditions. If researchers need reagents for this application, it is best to have them discuss the details with a technical expert before proceeding.
Polyclonal
Polyclonal antibodies are almost always going to be the best choice for protein characterization. They are far less expensive and they generally have a higher affinity and broader utlity across assays. The major drawbacks are a limited supply and consistency in results. A larger number of species options are also available adding to the value for such applications as double-labeling.
Rabbit: Use 95% of the time. Has the best ability to respond to the broadest classes of antigens. Rabbits also generally provide the greatest yields in the shortest period of time.
Chicken: Good substitute for rabbits when rabbit antibodies have failed to produce the desired reactivity or when researchers need another species antibody to label another protein at the same time or label a different subpopulation of the same protein.
Guinea Pig: Guinea pig antibodies are really popular choices as well for secondary species labeling. They are also well-known for their lower backgrounds in drosophila research.
Rat: Rat is rarely used as a polyclonal antibody source, but they can be useful when only limited amounts of antibody are needed.
Goat/Sheep: The large-scale production option for polyclonal antibodies. Main drawback is that they take longer to respond and maximize titer. They are also quite a bit more expensive to maintain.
Monoclonal
The real value of monoclonal antibodies is their unlimited source and the higher consistency of results they provide. Screening protocols are essential to establishing antibody populations that work in the intended assay use. Their drawbacks include a lower utility and affinity, they take longer to develop and they are more expensive than polyclonal antibodies.
Summary
For certain types of antibodies where researchers want to measure very small differences in proteins such as post-translational modifications, only peptides can be used. This includes, but is not limited to, antibodies that are designed to recognize the following:
