Producing polyclonal antibodies is easier, faster, and more cost-effective than producing monoclonal antibodies. Polyclonal antibodies consist of heterogeneous collections of antibodies each generated by cloning different B cells raised in response to an antigen, and these antibodies target different epitopes of the same antigen.
The polyclonal antibody production process consists of the following steps:
- Preparing the Protein or Peptide Antigen of Interest
The first step in the polyclonal production process is to prepare the antigen of interest. You can use a full-length protein to generate antibodies against or a specific peptide sequence that has been derived from this protein. The purity of an antigen directly impacts the specificity of the polyclonal antibody. Even minute impurities can cause generated antibodies to produce more activity against the impurities in a sample than the antigen, so validating antigen quality is crucial. A too small or too large an amount of the antigen can result in sensitization, suppression, tolerance, or other unwanted responses.
- Immunizing the Host Animal and Measuring Immune Response
After an antigen is prepared, it is injected into a host animal of the chosen species. The animal is also injected with an adjuvant to enhance the immune response to the antigen and increase the intensity and duration of antibody generation. Four to eight weeks after the initial priming immunization, the animal’s immune response will begin to wane so additional booster immunizations are administered to increase antibody titer. Researchers will regularly monitor animals after each booster immunization for side effects and evaluate the immune response by obtaining blood samples and measuring antibody titer. Immunization continues until the target concentration of antibodies in the bloodstream is reached, usually within two to four months.
- Isolating and Purifying Antibody Population
When an animal produces the required level of antibodies, researchers will harvest the blood from the immunized animal, centrifuge it to isolate the serum from the red blood cells, and further process the serum to isolate the desired antibodies. Because the serum will contain multiple immunoglobulins against other targets or molecules in the serum, it must be purified to increase sensitivity and specificity for the assay.
Antibodies can be isolated through protein A/G purification or antibody affinity purification. In protein A/G purification, the serum is exposed to protein A or protein B, which binds to the immunoglobulin G (IgG) antibodies and enriches them while removing unwanted non-specific proteins from the serum. This method is simple and cost-effective, but often leaves behind a large quantity of non-specific proteins in the preparation and can cause background noise when using the antibody in an assay.
A second method, antibody affinity purification, is often used to isolate polyclonal antibodies in the serum that are specific to the protein of interest. Researchers expose the serum to a column containing the target protein, and antibodies that recognize this protein remain bound to the column while the non-specific, unbound antibodies are removed via dissolution. The antibodies are then dissociated from the column and the target protein. This method eliminates most of the unwanted immunoglobulins and enriches the antibodies that only react with the target antigen.
- Pre-adsorbing Antibodies to Reduce Cross-Reactivity
In experiments involving antibodies raised against several different species, researchers can pre-adsorb polyclonal antibodies to remove antibodies that may potentially cross-react with antibodies of another species. Pre-adsorption of the antibodies requires passing them through a column that contains immobilized serum proteins from other species. The antibodies that do not cross-react with these proteins will pass through the column while the cross-reacting antibodies will be left behind, resulting in an antibody population specific for the target protein. If the experiment uses several polyclonal antibodies that detect different antigens, a common technique for reducing cross-reactivity is to use pre-adsorbed secondary antibodies.
- Performing an ELISA Test for Quality Control
After antibody purification and re-adsorption, researchers can perform ELISA testing to ensure they have recovered antibodies with the appropriate binding capabilities (specificity and sensitivity) in the target application. ELISA is the ideal method for measuring polyclonal antibody titer to ensure antibodies will consistently and reproducibly bond to the antigen.