6 Important Steps in the Production of Polyclonal & Monoclonal Antibodies


The ability to apply antibodies and peptides to various specific targets has become a powerful tool in medical research, disease diagnostics, therapeutics, and vaccine development. Peptide synthesis has, thus, become a critical technology in this field because peptides effectively yield polyclonal and monoclonal antibodies with very high titers.

When a foreign body stimulates B lymphocytes, they produce antibodies, which are glycoproteins that bind to specific antigens. Naturally, single clone B cells are the production chambers for monoclonal antibodies, while clones of various B cells generate polyclonal antibodies.

Due to their biochemical characteristics, antibodies have become important not only in immunological and immunohistochemistry research but also in the production and control of vaccines. The commercial manufacture of antibodies demands the use of live animals for the procedure, which goes through different invasive procedures to obtain the desired immune reactions. This procedure can take up to six months to obtain the desired antigen-specific antibody, and the process must be elaborately followed to ensure high-quality results.

Steps in Antibody Production

1. Purification of the Immunizing Antigen

The animal’s immune reaction will depend much on the features of the antigen used. It requires confirmation of factors such as the purity of the antigen before application. If there are impurities within the antigen, even as little as<1%, this may result in immunodominance of the impurities, especially if they are bacterial in nature. As a result, the subsequent antibodies may be more active towards the impurities rather than the desired antigen. It requires the antigen to be purified. 

Before application, it is obligatory to examine the antigen for contaminants such as residues of chemicals applied to incapacitate the microorganism, abnormal pH levels, and even endotoxins like lipopolysaccharides. Endotoxins and high/low pH levels may affect not only the immunological outcome but also the wellbeing of the animal. 


2. Selection of the Animal Species

Animals have different genetic characteristics. As a result, the species of the specimen will have an influence on the superiority of the antibody produced. When selecting the animal, one should ensure that the animal species and the antigen have a genetic relationship. The number of antibodies needed, and the use for which the antibodies are produced should also be considered. Rabbits are normally the animal of choice for antibody production. It is because they have a long lifespan, appropriate size, and are easy to handle, which is ideal when requiring high affinity, high-titer antibodies. 

The housing environment, availability of other antigens in the environment, stress levels, and animal diet also affect the quality of the antibodies. Rabbits can be caged in near-natural environments, which reduces their stress levels, resulting in a better immune response. Sex of the animal also plays an important role as it determines behaviour such as aggressiveness, which may affect the results. That’s why female animals are preferred. Next to the rabbit, the guinea pig is also a common specimen animal as well as a rat, and mouse. 


3. Boosting Results Using Adjuvant

An adjuvant helps boost the immune response by providing the immune system with an immune-stimulatory signal, especially if the antigen is poorly immunogenic. It makes it possible to use antigens with low immunogenicity to produce antibodies. It also allows for the use of smaller doses of the antigen. The most common adjuvants include aluminium salts (alum), Freund’s incomplete adjuvant and Freund’s complete adjuvant. Of these, Freund’s complete adjuvant is preferred because it reacts with a variety of antigens to produce high titers of antiserum. However, repeated use can lead to tissue necrosis as a result of the inactivated mycobacterium. It should only be used in the initial immunization. 

The adjuvant should be carefully selected and prepared to avoid contamination. Adulteration of the mixture may present a risk of illness to the animal, and resulting antibodies may be weaker and of lower titer. 


4. Immunization Protocol

Many factors determine the route of administering the antigen. These include the species of the specimen animal, characteristics of the selected adjuvant, and volume and contents of the antigen. Preferentially, the route of injection for most animals is subcutaneous. Subcutaneous injection allows for the monitoring of any possible inflammatory reactions.

Test bleeds are normally done about one to two weeks after the initial injection to measure the level of specific antibodies. 


5. Collection of the Antibodies

Once the desired reaction to the antigens has been noted, exsanguination is performed to collect the antibodies. Production bleeds are performed over some time. The amount of circulating blood volume will be influenced by the animal species being used. 

The collected antiserum is then centrifuged to rid it of cellular debris before conducting quality assurance testing. 


6. Purification of the Antibody

After centrifugation, there will still be some cellular debris, non-specific immunoglobulins, and microorganisms that may lower the potency of the antibodies. Purification helps remove the debris and improve the performance of the antibody. An appropriate purification technique should be considered, which is normally affinity chromatography. 



The production of polyclonal and monoclonal antibodies is a delicate process that requires careful planning to produce antigen-specific antibodies. This procedure requires that researchers use live animals, and thus, additional care must be taken to ensure the welfare of the animals. Monitoring after immunization is essential to observe side effects that may arise at the injection site. Also, blood samples taken for evaluating serum antibodies use invasive procedures and safety should be observed by limiting the blood volume collected to avoid hypovolemic shock to the animal. 


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