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How to generate antibodies by immunizing animals with an antigen (immunogen)

∙ How to generate polyclonal antibodies
∙ How to generate monoclonal antibodies

How to generate antibodies without immunizing animals

∙ Phage display method

Related topics

∙ Difference between polyclonal and monoclonal antibodies
∙ How to purify antibodies

How to generate polyclonal antibodies

Overview of the procedure

How to generate polyclonal antibodies
An antigen (immunogen) injected into animals induces them to produce and secrete high levels of antibodies into the blood.
Several months after repeated immunization, the blood (plasma, serum) is collected, and antibodies are purified (by methods described later). The antibodies generated by this method are called polyclonal antibodies because they are derived from different B cell clones and the resulting antiserum contains numerous different antibodies that react to the injected immunogen.

Animals used for immunization (immunized animals)

Isotypes of immunized animals

In addition to mice and rabbits, various mammals and birds are used for immunization, including rats, hamsters, guinea pigs, chickens, goats, sheep, and donkeys. 

Immunoglobulin isotypes, organization of immunoglobulin genes, mechanism of diversification, and the organ sites of antibody diversification differ between vertebrate animals.

How antibodies are produced

Blood antibody levelsAfter immunization, blood IgM levels increase first.
With repeated immunization, IgG levels increase.

Binding ability of immunoglobulinsThe affinity (binding ability) of IgM for the antigen does not increase even after repeated immunization.

In contrast, the binding ability of IgG increases with repeated immunization.

AZ's point
Dangers of hyperimmunization!
Extending the immunization period, in the hopes of developing a higher affinity antibody to the antigen, is not necessarily beneficial. After an excessively long immunization period, non-specific antibodies, including self-reactive antibodies, increase in amount and the animals’ health may deteriorate. To generate high quality antibodies, it is important to periodically collect a small amount of blood and evaluate the affinity of the antibody for the antigen and non-specific reactions.

How to produce monoclonal antibodies

Overview of the procedure

How to generate monoclonal antibodies
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A single antibody (monoclonal antibody) can be stably produced if a single B cell producing the antibody is isolated and cultured indefinitely.
This is achieved by artificially fusing the antibody-producing B cells with immortalized cancer cells (myeloma) to generate hybridomas that live indefinitely and contain genes encoding specific antibodies, and by selecting the hybridoma clones that produce the desired monoclonal antibodies with high affinity and specificity.
It usually takes 4-6 months from immunization of animals to production of monoclonal antibodies.

Difference between clones in monoclonal antibodies

Difference between clones in monoclonal antibodiesEven though monoclonal antibodies against the same antigen are produced each clone reacts to different epitopes on the same antigen Also, each clone have different suitable applications. It is important to take note of the antigen name and clone name when selecting a monoclonal antibody.

Phage display method

Phage display method
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A phage (bacteriophage) is a virus that infects bacteria. The phage genetically integrated with genes coding a part of antibody is referred to an antibody phage, and displays on their surface the antibody fragment, such as Fab form or scFv form. An antibody phage library is a huge mixed population of antibody phages that can react with various molecules. In the phage display method, antibodies against the target molecule are selected from the antibody phage library by using a molecular affinity of antibodies and the target molecule.

Various sources of the immunoglobulin gene are used in the antibody phage library. For example, naïve antibody phage libraries are prepared using the B cells of healthy individuals. Other examples include B cells from patients whose sera contain antibodies that potently neutralize pathogens or toxins, and B cells from animals immunized with a specific antigen.

Difference between polyclonal and monoclonal antibodies

Difference between polyclonal and monoclonal antibodies
Polyclonal antibodies Monoclonal antibodies
Animal species
Rabbit, guinea pig, goat, sheep, rat, mouse, chicken, etc. Rat, mouse, chicken, rabbit, human, etc.
Antiserum Hybridoma
Class, subclass Mixed classes Single class
Epitope React to multiple epitopes React to a single epitope
Specificity Lower than monoclonal antibodies because multiple types of antibodies are present.
High if good quality antibodies are selected.
Reproducibility Variable among lots. The same antibodies are produced indefinitely.
Stability Binding ability tends to be unaffected by fixation/denaturation of the antigen, because multiple different antibody molecules are present.
Tolerate modifications, such as labeling and removal of the Fc region.
Binding ability may be lost if the epitope is lost by fixation/denaturation of the antigen, because monoclonal antibodies are homogeneous.
Tend to be sensitive to modifications, such as labeling and removal of the Fc region.

Antigen binding

Antigen bindingAntigen bindingMonoclonal antibody reacts to a single epitope. Therefore, only one antibody molecule can bind to an antigen molecule.
In contrast, polyclonal antibody is a collection of immunoglobulin molecules that react against a specific antigen, each recognizes a different epitope. Therefore, multiple antibody molecules bind to an antigen molecule. (if sufficiently large).

Labeled (secondary) antibodiesLabeled (secondary) antibodies
Similarly, only one antibody molecule can bind to a primary antibody molecule if the secondary antibody is a labeled monoclonal antibody. Whereas multiple antibodies can bind to a primary antibody molecule if the secondary antibody is a polyclonal antibody.
Consequently, polyclonal antibodies provide a higher sensitivity of detection (amplification of the signal) and, therefore, are commonly used as a secondary antibody.

Sensitivity to protein-degrading enzymes (proteases)

Sensitivity to protein-degrading enzymes (proteases)
Proteases are often used for antibody modification in order to add a label, or to remove the Fc region to reduce non-specific reaction.

The high sensitivity of monoclonal antibodies to protease digestion can result in digestion outside the intended regions, or loss of antigen-binding ability. Conversely, if the antibody is resistant to the protease, intended modifications would be difficult.

In contrast, sensitivity to proteases is unlikely to cause a problem with polyclonal antibodies, and antigen-binding ability tends to be unaffected because multiple different antibody molecules are present. Thus, polyclonal antibodies are amenable to modification.

How to purify antibodies

Antibodies are usually purified by the following three steps.

1) Partially remove solid materials and proteins other than the antibodies.
Perform centrifugation or filtration.

2) Isolate antibodies by affinity chromatography (purification with Protein A/G / antigen-affinity purification).

3) Remove contaminants remaining after step 2).
Perform gel filtration chromatography to further purify the antibodies and exchange the buffer.

Purification with protein A/G

IgG can be purified using a column packed with immobilized Protein A or Protein G.

Immune evasion by Staphylococcus aureusProtein A is a cell wall protein of Staphylococcus aureus that specifically binds to the Fc region of mammalian IgG. Staphylococcus aureus is a commensal bacterium of the skin and the gastrointestinal tract of humans and other animals, and occasionally causes an infectious disease and food poisoning. Protein A captures IgG, allowing the bacteria to evade elimination by the immune system.

*Protein G: A cell wall protein isolated from group G Streptococci

Antibody purification with Protein A

The ability of Protein G and Protein A to bind to Ig differs between Ig subclasses and between species. The use of Protein G or A should depend on the purpose.

IgM and IgY are purified by different columns.

Antigen-affinity purification

Affinity chromatography is performed using a column packed with immobilized antigen, the same antigen used for immunization. Only antibodies that bind to the antigen are isolated.

This method provides a higher yield of antigen-specific antibodies than Protein A/G affinity chromatography, though the total amount of recovered antibodies is lower.

Protein A/G affinity chromatography and antigen-affinity chromatography cannot be used if the antibodies are inactivated in an acidic solution. In that case, antibodies are purified by ammonium sulfate precipitation (salting out) or ion exchange chromatography.

>> Details of antibody isotypes and subclasses
>> Details of affinity chromatography
>> Details of gel filtration chromatography

Related links

Fractionation and purification of proteins