6 simple secrets for polyclonal antibody production

Careful planning and preparation is essential to produce exceptional polyclonal antibodies (pABs). Polyclonal antibodies are produced from a mixture of B lymphocytes with different epitope affinities toward the same antigen. From start to finish, pAb development takes two to six months, though custom schedules may take more time.
The first step is to select the immunizing antigen. pAbs are not suitable for some applications and monoclonal antibodies (mAbs) may be more appropriate, as discussed in the next blog article.

The first step is to select the immunizing antigen. pAbs are not suitable for some applications and monoclonal antibodies (mAbs) may be more appropriate, as discussed in the next blog article.

Selecting the animal

Following antigen selection and preparation, it's time to select the appropriate animal species. Rabbits are often the first choice for pAb development due to their size, long lifespan, ease of handling and ability to produce high-titer, high affinity antibodies.

Animal diet, housing environment, stress levels and the presence of other antigens in the animal’s environment also affect polyclonal antibody production. Rabbits, for example, are easily housed in controlled laboratories and researchers can obtain superior pAb yields by housing the rabbits in group housing with strict barrier environments. The former reduces stress levels associated with individual cage housing and allows for more natural behavior patterns, which in turn results in better immune responses and optimal polyclonal antibody production. Strict barrier environments, in turn, can minimize the level of undesired antibodies in the rabbits and may lead to lower background immunoglobulin levels.

Use of adjuvants to boost results

An adjuvant provides an immuno-stimulatory signal to the immune system that boosts the immune response. Antigens that have low immunogenicity can still be used to generate pAbs if they are administered with an adjuvant. In addition, adjuvants enable smaller doses of antigen to be administered.

Some examples of common adjuvant formulations used in polyclonal antibody production are: Freund’s complete adjuvant, Freund’s incomplete adjuvant and aluminum salts (Alum). Freund’s complete adjuvant is extensively used in polyclonal antibody production due to its ability to induce high titers of antiserum against a wide variety of antigens (Hancock et al., 2005b). Its use is generally reserved for the initial immunization since the inactivated mycobacterium present in this adjuvant can cause tissue necrosis following repeat administration.

Careful aseptic preparation of the antigen-adjuvant mixture is essential in order to avoid the introduction of contaminating microorganisms and to maintain the stability of the antigen. If the antigen-adjuvant mixture is poor quality, the animal is at risk of illness and the antibodies may be of lower titer and functionality.

Protocol for immunization

The route used to deliver the antigen depends on a number of factors including the animal species, the specific characteristics of the adjuvant and the composition and volume of the antigen. In rabbits, the most common immunization protocol is to make multiple subcutaneous injections (SC) on the back. This method allows for potential inflammatory reactions to be visually monitored. Following immunization, animals should be monitored daily, which often involves palpation of the injection site to monitor the development of potential pathological lesions, as well as clinical observation for general health. The actual antibody response should also be monitored.

Seven to fourteen days after the primary and boost injections, test bleeds are usually performed. The level of specific antibodies is measured at this time using assays similar to those created for the intended use of the antibody.

Antibody collection

After an appropriate response to the immunization has been observed, serum can be collected. Different species of animals have unique allowable circulating blood volume percentages. Production bleeds, which are multiple larger volume bleeds, are carried out over time. Prior to quality assurance testing, collected antiserum is pooled and centrifuged to remove cellular debris.

Polyclonal antibody purification

Remaining cellular debris, microorganisms and non-specific immunoglobulins can impact antibody performance. As a result, it is critical that researchers purify the pAb. Investigators should carefully consider which purification technique is most appropriate. Most pAbs are purified using affinity chromatography, which relies on Protein A or Protein G which is immobilized in a chromatographic matrix. (Grodzki et al., 2010; Roque et al., 2007).

Although Protein A/G can increase the purity of pAbs, some pAbs may still suffer from high levels of background when utilized in certain assays (Grodzki et al., 2010). In these cases, antigen affinity purification should be conducted, either alone or in conjunction with Protein A/G.

Resins have recently been developed that will purify several species of IgM and IgA antibody molecules. This also enhances antibody stability by removing contaminants such as proteases. (Grodzki et al., 2010; Roque et al., 2007).

The production of pAbs for use as reagents in research, diagnostics and clinical development consists of many distinct and important steps. By carefully considering variables such as animal species, adjuvants, injection protocols, and methods of antibody collection and purification, researchers can develop high quality pAbs.

Considerations in polyclonal antibody production

Overall, careful planning and preparation is required to produce exceptional polyclonal antibodies for use as reagents in research, diagnostics and clinical development. pAbs are often selected for various applications where their ability to recognize multiple epitopes on a given antigen is advantageous. Read more in our white paper: Considerations in the production of antibodies as reagents: Polyclonal antibodies.