Polyclonal and Monoclonal Antibodies: Production and Purification

Polyclonal Antibodies

Polyclonal antibodies recognize multiple epitopes on an antigen because they are derived from multiple antibody-producing B-cells. As a result, polyclonal antibodies have both advantages and disadvantages for the end user over monoclonal antibodies, listed in Table 2 below.

Table 2:Key features of polyclonal antibodies

The features in Table 2 can make polyclonal antibodies the preferred choice for some applications. For example, polyclonals are often preferred for immunoprecipitation because they tend to have a higher chance of capturing the target protein. Their availability from multiple host animals also makes them suitable for multiplexed IHC and ICC.

Monoclonal Antibodies

Monoclonal antibodies correspond to one epitope for a given antigen because they are derived from a clonal population of B-cells. Advantages and disadvantages of monoclonal antibodies are outlined in Table 3.

Table 3:Key features of monoclonal antibodies

Monoclonal antibodies are often the preferred choice in laboratories requiring large amounts of identical antibody or performing the same procedure repeatedly over time due to the consistency in monoclonal production. They are also ideal for designing matched pairs of antibodies in sandwich ELISAs, because known binding sites makes it easier to find two complementary antibodies.

Clone number

Monoclonal antibodies are always given a clone number, which represents the specific cell line that the antibodies were produced from. All antibodies with the same clone number are derived from the same clonal population. This means that all antibodies with the same clone number will have the same structure, affinity and specificity as each other. Because multiple monoclonal antibodies may exist for a single target, clone numbers are used to identify antibodies to ensure that exactly the same antibody is being used as in previous experiments.

Mouse vs Rabbit Monoclonal Antibodies

Monoclonal antibodies tend to be made in either mouse or rabbit host cells. The availability of only two hosts can limit multiplexing in application such as immunohistochemistry, forcing researchers to consider a sequential labelling and stripping strategy, or directly conjugated primary antibodies.

For many researchers, the choice between mouse and rabbit will mostly come down to cost and availability of antibodies against their target of interest. However, there are some differences to be aware of.

A significant implication of monoclonal antibodies being produced in mouse is that using mouse antibodies with mouse tissue (mouse-on-mouse) can produce significant non-specific interactions. This is an important issue given how prevalent mice are as a model organism in some areas of research and as disease models.

Rabbit can also be preferred because rabbits are able to mount an immune response against a wider range of antigens, including small peptides or other molecules. This means that an antibody against a small molecule is more likely to be available from rabbit. Rabbit antibodies can also exhibit higher affinities for their targets, producing a stronger signal for end users.

Rabbit monoclonals are more expensive to produce, however. Mouse monoclonal technology has been used for much longer than rabbit, meaning that there is more likely to be a mouse monoclonal antibody against a given target than rabbit.

The ability to engineer class switching is therefore more established in mice as well. Class switching refers to when the cells producing the antibodies switch the constant region of the antibody, and therefore its isotype, while retaining the variable region and its specificity against the epitope. Using multiple isotypes can be one solution to the issue with multiplexing mentioned earlier, and can be easier in mice because they have multiple IgG subclasses (IgG1, IgG2a/2c, IgG2b, and IgG3) whereas rabbits only have one.

Finally, the technology for producing recombinant antibodies is also more established in mice.

See our comprehensive guide on Rabbit Monoclonal Antibodies for more information on their advantages and uses.

Recombinant Antibodies

Recombinant antibodies are monoclonal antibodies that are generated using recombinant DNA technology, meaning the exact DNA sequence needed to produce an antibody with a given target specificity is known and can be expressed in cell lines. This is in contrast to the hybridoma technology usually used to produced monoclonal antibodies (see Antibody Production).

In general, recombinant antibodies retain all of the benefits of monoclonal antibodies, but provide a greater deal of control over their production. For example, while traditional monoclonal antibodies exhibit little lot-to-lot variability when compared to polyclonal antibodies, some genetic drift can occur leading to slight variation due to acquired mutations. Knowing the DNA sequence avoids this drift. Recombinant antibodies can also be easily modified, such as to different isotypes or formats.

More information can be found on our Recombinant Antibodies page.

Multiclonal Antibodies

Multiclonal antibodies address the issue of monoclonal antibodies targeting only one epitope. While this is often seen as an advantage, targeting a single epitope does make monoclonals less robust to conformational changes in the antigen, and can limit the strength of signal produced on binding.

Monoclonal antibodies are produced as normal, and then combined together into a tube to produce multiclonal antibodies. This gives the beneficial property of polyclonals of being able to bind to multiple epitopes to produce a stronger signal, while retaining the known binding sites and high specificity of monoclonal antibodies.

Polyclonal Antibody Production

Polyclonal antibody production (Figure 2) tends to be easier than for monoclonal antibodies. The general steps involved in their production are as follows:

1. The antigen against which antibodies will be made is prepared and purified.
2. A host animal is chosen, which will be based on cost, animal availability, typical immune responses of different animals, and the species from which the antigen was obtained.
3. An animal is immunized with a purified target antigen, triggering the animal’s B-cells to produce antibodies against the antigen. The antigen can be combined with adjuvant to boost the immune response if the antigen is only weakly immunogenic.
4. This is repeated at set intervals over several weeks to increase the number and affinity of the antibodies.
5. The animal is then bled, meaning some of its serum is collected. The fraction that contains immunoglobulins can be isolated, but it will contain other antibodies in addition to the target antibody.
6. While the serum can be used in this form, it is generally then purified by affinity purification to isolate the target antibody. The antibodies can also be cross-adsorbed against similar antigens to further enhance their specificity by removing cross-reactive antibodies.

Figure 2: Polyclonal antibody production.

How Monoclonal Antibodies are Made

Monoclonal antibody production (Figure 3) usually begins with similar first steps as polyclonal production. Antigens are purified and immunized into a selected host (usually mouse or rabbit). The titer of antibodies in the serum is determined by collecting a small amount of blood from the immunized animal and determining antibody concentration by ELISA or other method. The production procedure then follows these steps:

1. Once the titer is sufficient, cells from the spleen are harvested. These cells will include the antibody-producing B-cells, which mature in the spleen and other lymphoid tissues.
2. B-cells can proliferate, but not indefinitely, meaning isolated B-cells will eventually dye. To overcome this, B-cells from the spleen are fused with an immortal tumor of lymphocytes (myeloma) to produce hybridomas. Myeloma cells, and therefore hybridomas, can grow and divide indefinitely, giving a limitless supply of antibody. Fusion is achieved either by chemical fusion (polyethylene glycol) or electrical fusion (electroporation), both of which cause the cell membranes of the cells to fuse with each other.
3. Using special culture conditions, called HAT medium, to grow the new hybridomas means that only fused cells survive. Unfused myelomas die because they cannot tolerate the culture conditions, while unfused B-cells die because they cannot proliferate forever. This step is important because unfused myeloma cells have the potential to outgrow and outcompete new fusions.
4. The hybridomas are cloned by ‘limiting dilution’, meaning they are diluted into multi-well plates to such an extent that each well contains at most one cell. This ensures that antibodies produced in each well are from a single clonal population.
5. Hybridomas are then screened to find fusions that are producing antibodies of appropriate specificity. This screening is usually done by ELISA, whereby the antigen is immobilized to the ELISA plate, or by flow cytometry, where a fluorophore-conjugated antigen will bind to hybridomas expressing the antibody on their cell surface.
6. The antibodies are characterized to determine their binding properties, specificity, and target epitope.
7. Based on this characterization, certain hybridoma clones are chosen for commercial or scaled-up production of the antibody.

Figure 3: Monoclonal antibody production.

Recombinant Antibody Production

Recombinant antibodies are not made by hybridomas, and do not require a live animal at any point. Instead, they are made using display technology, typically phage display for antibody fragments or display systems in higher organisms (yeast, insect, mammalian cells) for more complex antibodies.

Briefly, phage display involves creating a library of millions of bacteriophages where each phage expresses and presents a single antibody on its surface. These phages are then screened against an antigen that has been immobilized on a plate. Phages expressing antibodies that are specific for that antigen will remain bound, while others are washed away. The selected phages are eluted, amplified in E. coli, and can be subjected to screening again to find the highest affinity antibodies. Once the target-specific phages have been validated, the DNA sequence of highly specific antibodies, derived from selected phages, can be used for recombinant antibody production in an expression system of choice.

More details of recombinant antibody production can be found on our Recombinant Antibodies page.

Antiserum

Antiserum is blood serum containing polyclonal antibodies of interest, as well as other serum proteins and immunoglobulins. This can be further enriched by isolating just the immunoglobulin fraction of the serum. Antiserum is more likely to show off-target activity due to contaminants.

Ascites

This is a historical method of producing monoclonal antibodies that has mostly been superseded by recombinant technology. Once hybridoma cells had been produced, they were injected into and grown within the peritoneal cavity of a mouse or rat. After proliferation, they produce a fluid called ascites, which contains a high antibody concentration and can be harvested. The ascites method can also produce cell lines that show enhanced antibody production at a later time.

Tissue Culture Supernatant

Supernatant from hybridoma cultures can be directly used in antibody applications, assuming the concentration of the antibody is high enough.

Purified

Most researchers use purified antibodies, particularly if purchasing a commercial antibody. Nonetheless, there are different degrees of purification.

Protein A and Protein G bind to the Fc region of antibodies with high affinity, and can therefore be used to isolate all immunoglobulins from antiserum. They will not show any selectivity for a target antibody, however, so there may still be off-target activity.

Affinity purification purifies proteins by making use of strong and specific interactions between the protein of interest and a resin coupled to a ligand. In the case of antibodies, the target antigen can be coupled to the resin on a column. The crude sample containing the antibody is then washed over the resin; antibodies will bind to the antigen and stay on the resin, while everything else will be washed through. The antigen-antibody interaction can then be disrupted to elute the pure antibody of interest.

Pre-adsorption is a final step to distinguish closely related antibodies, and is usually applied to secondary antibodies (e.g. antibodies that are specific for another type of antibody). A sample of secondary antibodies is washed across a column containing immobilized serum proteins from non-target species that are likely to be cross-reactive. Non-specific antibodies will bind to the proteins, leaving only the highly specific antibodies to flow through.