CST BLOG: Lab Expectations

The official blog of Cell Signaling Technology (CST), where we discuss what to expect from your time at the bench, share tips, tricks, and information.

What makes an antibody monoclonal, polyclonal, or recombinant?

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When you’re shopping for antibodies, there are so many factors to consider. For example, will it work in my cell or tissue model? Has it been tested in the application I want to use? Sometimes, it’s a struggle to find what you need because your options are limited, but in other instances, there may be several reagents that seem like they could work in your experiment.

Monoclonal, Polyclonal, or Recombinant antibodies

What if you’re looking at a monoclonal, polyclonal, or recombinant antibody against the same target? What’s the difference? Is one better than the other or are they all designed for different purposes? It’s a complicated question, but one definitely worth asking.

What is a polyclonal antibody?

A polyclonal antibody is a heterogeneous mixture of antibodies derived from distinct B-lymphocyte populations that detect different epitopes within the same immunogen. Polyclonal antibodies are typically produced in rabbits, but can also be made in ungulates (sheep, goat, horse, etc.), rodents, and chickens, depending on the researchers’ need. Production of polyclonal antibodies typically involves the collection of blood from the immunized animal, isolation of the immunoglobulin fraction, and affinity purification to remove non-specific antibody populations. Rabbits offer significant advantages over other species due to their larger antibody diversity and ability to generate immune responses to a wide range of antigens including small molecules and peptides with or without post-translational modifications.

What is a monoclonal antibody?

A monoclonal antibody is a homogeneous antibody derived from a single B-cell clone that detects a single epitope within the immunogen. All monoclonal antibodies begin as a pool of polyclonal antibodies but are isolated through a selection or cloning process to identify and expand the desired monovalent clone. Monoclonal antibodies are typically produced from rodent hosts, rabbits, and camelids, and are produced via a variety of methods depending on the species and type of antibody desired (as detailed below). Traditionally, monoclonal antibodies are generated via stable clones of immortalized B-cells, either by injecting and collecting ascites from a mouse or culturing the antibody expressing B-cells and collecting the supernatant. More recently, newer techniques have enabled recombinant cloning of the immunoglobulin heavy and light chain genes from immunoreactive B-cells for expression and production of recombinant monoclonal antibodies in mammalian cell lines.

Antibody Production

As mentioned above, there are many ways to produce an antibody. Generally, polyclonal antibodies are purified from the serum of the immunized animal. Monoclonal antibodies can be manufactured using a number of different strategies. Most monoclonal antibodies are produced by culturing the antibody-producing B-cell hybridoma and collecting the supernatant off the cells. Antibodies generated this way can be used with or without additional purification depending on the titer and affinity of the antibody.

Alternate methods include injection of the B-cell hybridoma in to the peritoneal cavity of a suitable host and collecting the ascites fluid after a period of time. This method has the advantage of the rapid production of large quantities of antibody, but has the disadvantage of requiring an animal host and may require an additional purification step to remove lipids and other debris.

Alternatively, the heavy and light immunoglobulin chains of a monoclonal antibody can be cloned into an expression vector, transfected into a mammalian cell line suited for antibody production, and expressed ectopically for collection and purification. Recombinant production of antibodies is a growing trend among antibody manufacturers due to its inherent consistency and the ability to genetically engineer the antibody.

Regardless of the clonality or production method, all antibodies must be properly validated in the intended application prior to use in an experiment. Even the best antibody, regardless of clonality, host species, or isotype, can yield spurious or misleading results when used incorrectly.

   Polyclonal Antibodies  Monoclonal Antibodies
Cost to develop and produce Inexpensive to develop and produce Expensive to develop, inexpensive to produce
 Technical expertise to develop and produce Low High
Development time Short (immunization to purified polyclonal antibody within four months) Long (requires multiple rounds of cloning/selection to isolate to homogeneity)
Time to validate After initial validation, each new bleed or lot must be re-validated to ensure consistency Production method ensures greater consistency but re-testing between production runs is highly recommended
Host species  Rabbit, sheep, goat, horse, chicken  Mouse, rat, rabbit, guinea pig, alpaca, llama
Production method Collection and purification from serum

- Collection and purification from ascites or cultured cell supernatant

- Recombinant expression of heavy and light chains in mammalian cell lines

Sensitivity Increased sensitivity due to recognition of multiple epitopes; ideal for low-abundance proteins Sensitivity is dependent on the affinity of the antibody for the target epitope – most clones are selected based on affinity and functionality
Specificity Potential for off-target reactivity due to multivalency Higher degree of specificity due to monovalent interaction
Consistency Risk of lot-to-lot variability due to immunogenic drift and other factors High degree of lot-to-lot consistency due to selection and production methods
Affinity    Can only be estimated due to the heterogeneous nature of the antibody Measurable due to the monovalent nature of the epitope and antibody
Ability to conjugate to dyes and other functional groups? Yes   Yes
Stability High  

Rabbit mAbs: High
Mouse mAbs: Moderate

Use as companion or clinical diagnostics?  Rarely  Yes
Use as therapeutic agent?  Rarely  Yes
Ability to engineer antibody at genetic level (humanization, etc.)?  No  Yes

 

Chris Sumner
Chris Sumner
Chris Sumner was the Editor-in-Chief of Lab Expectations. When he's not reading/writing about curing disease, he's hiking in the woods, playing guitar, or searching for the world's best lobster roll.

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