Producing Polyclonal Antibodies

How to Make Polyclonal Antibodies

What is polyclonal antibody production? Polyclonal antibodies (pAbs) are produced by injecting a specific antigen into lab animals, such as rabbits and goats, etc. The animal is immunized repeatedly to obtain higher titers of antibodies specific for the antigen. Within a few weeks, these polyclonal antibodies can be harvested and collected from the antiserum.

Production of polyclonal antibodies is easier and more cost-effective than the production of monoclonal antibodies. Furthermore, polyclonal antisera can be generated in a shorter time (4-8 weeks), whereas it takes about 3 to 6 months to produce mAbs.

Sino Biological provides polyclonal antibody production services which deliver you antisera or purified antibodies in just 45 days. Just send us an antigen sequence, and we'll get you high-affinity polyclonal antibodies for your research or diagnostic purposes.

Rabbit Polyclonal Antibody Production Process

There are several critical steps involved in the production of polyclonal antibodies in rabbits. A four-step procedure of pAb production is outlined below.

Antigen preparation   Animal immunization   Antibody purification   Quality control
Fig 1. How to generate polyclonal antibodies

Step 1: Antigen preparation

The production process starts with preparing the protein or peptide antigens. It's important to ensure the quality of the target antigen, in particular when binding to a conformational epitope is desired. The specificity of polyclonal antibody obtained depends on the purity of the antigen applied. Impurities (<1%) may prove to be immunodominant (e.g., with many bacterial antigens) and may result in antibodies that have more activity against the impurity than against the antigen of interest. Purification of antigen is a time-consuming and laborious work.

Luckily, Sino Biological offers antigen preparation service which is based on our recombinant protein expression platforms. We have rich experience of recombinant protein expressed in CHO, HEK293 and E.coli.

Step 2: Animal immunization

The choice of animals for the production of polyclonal antibodies depends upon the amount of antiserum desired, the evolutionary distance between the species from which the protein of interest has been derived and the species of the animal to be immunized, and prior experience with the immunogens. Rabbits are the usual animal of choice because they are genetically divergent from the human and mouse sources of the proteins most often studied. Rabbits provide as much as 25 ml of serum from each bleed without significant harmful effects.

Adjuvants are used to enhance the immune response for pAb production. Freund's Adjuvant is one of the most commonly used adjuvants in research. In the presence of adjuvant, the protein antigen is injected intramuscularly, intradermally, or subcutaneously into an animal of the chosen species. Booster immunizations are started 4 to 8 weeks after the priming immunization and continued at 2- to 3-week intervals. Prior to the priming immunization, following the primary and each booster immunization, the animal is bled and serum prepared from whole blood. When the antibody titer has reached an acceptable level, the production of polyclonal antibodies should be ended.

Step 3: Antibody purification

Affinity purification is a good choice for purification of polyclonal antibodies.

Protein A/G affinity purification can enrich Immunoglobulin G (IgG) in the raw antiserum and remove the bulk of unwanted proteins. However, there's still a large quantity of non-specific IgGs in these preparations. This will considerably increase the background noise when the antibody is used in assays such as ELISA, immunohistochemistry, and Western blot, etc.

To isolate specific polyclonal antibodies from antiserum, antigen-specific affinity purification is often used. Antigen affinity ​purification results in the elimination of the bulk of the non-specific IgG fraction, and enriches the fraction of immunoglobulin that specifically reacts with the target antigen.

Step 4: Quality control

After purification, a series of quality control tests are performed to ensure the quality of polyclonal antibodies. Antibody concentration is evaluated by absorption at 280 nm (A280). The purity of polyclonal antibody is checked using SDS-PAGE. To estimate the polyclonal antibody titer, an ELISA test was the most suitable method.

Finally, labeling polyclonal antibody with HRP or biotin provides sensitive enzymatic detection in IHC and the flexibility of using a variety of conjugated avidin, respectively.

References

1. Leenaars, P. M., Hendriksen, C. F., de Leeuw, W. A., Carat, F., Delahaut, P., Fischer, R., ... & Lindblad, E. B. (1999). The Production of Polyclonal Antibodies in Laboratory Animals: The report and recommendations of ECVAM workshop 35. Alternatives to laboratory animals, 27(1), 79-102.
2. Leenaars, M., & Hendriksen, C. F. (2005). Critical steps in the production of polyclonal and monoclonal antibodies: evaluation and recommendations. Ilar Journal, 46(3), 269-279.

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