5 A’s of Antibody Development
Antibody development is a multi-faceted process and an experiment in itself. To assist in generating an immune response towards your antigen and ensure the antibody works towards your application of interest; we have created the 5 A’s Antibody Development Guide. The Guide is sectioned corresponding to the 5 elements of antibody development: Antigen, Adjuvant, Animal, Antibody, and Application.
Antigens are the target you wish to develop antibodies against. Learn about different antigen types and how different antigen properties affect antibody development success.
In terms of antibody development, an antigen is what the antibody will bind to. Antigens are immunized into host species signaling the start of antibody production within the host’s immune system. Antigens are a crucial step in antibody development because a well defined antigen increases the likelihood of developing the antibody desired for experimental success.
An antigen’s immunogenicity, size, and application of interest are factors to consider when developing an antibody. To start with, generating an immune response is half the battle, so an antigen’s immunogenicity and size matters. The more immunogenic and larger an antigen is the more likely the immune system will generate antibodies against the antigen. In addition to getting an immune response towards the antigen, generating antibodies that work towards your application of interest is also a challenge as not every antibody will work in different applications or assays even if the antigen target is the same because antigen presentation differs depending on the assay.
- Small Molecules
- Proteins
- Carbohydrates
- Peptides
- Antibody Variable Region (idiotype)
To learn more about antigen requirements view the pages for the corresponding service
Simply put, an epitope is the antibody binding site on an antigen. However, to gain a stronger grasp of how all the pieces fit together, it's helpful to start by reviewing the structure and function of an antibody.
Learn more about epitopes.
An immunogen is a specific type of antigen that is able to elicit an immune response. Antibody development is dependent on a humoral immune response mediated by immune cells recognizing a molecule as being foreign.
Learn more about immunogens.
Adjuvants are boosters to elicit and amplify an immune response. Learn how adjuvants affect antibody development towards your experiment.
Learn more about adjuvants.
Freund’s Adjuvant is perhaps one of the most commonly used adjuvants in research today. It is used in animal research to trigger a humoral antibody inflammatory response for the production of high titer antibodies.
Learn more about Freund's Adjuvant.
Antibodies are produced by injecting antigens into an animal host to stimulate the production of antibodies that are directed against the antigens. However, injecting antigen alone may not elicit a desired immune response since the immune system will rapidly remove antigen. Thus, immunizing with only antigen prevents a strong immune response of high antibody titers, and shortening the time required for affinity maturation of high antibody affinity. To prevent this undesired immune response, adjuvant is mixed with antigen before immunization, delaying immediate antigen removal from the immune system and making adjuvants a necessary part of antibody development.
There are many kinds of adjuvants used in antibody development. Below are a few of the most common ajduvants used.
- CFA
- IFA
- GERBU
- Adjuplex
- TiterMax
Animals are hosts whose immune system generates antibodies. Learn how each host’s immune system produces antibodies and which species is right for you.
Choosing the proper animal host is critical to determining which type of antibody you will receive. Each host has specific benefits and drawbacks. For example, rabbits are excellent animal hosts for polyclonal antibody development because of their fast acting immune systems, while goats are larger and have a slower immune response, but provide a greater yield of antibodies. The antibody response to a given antigen will vary between species; each species offers its advantages and limitations. Be sure to weigh the advantage and disadvantage of each species before deciding which one is right for your project or contact us for a consultation.
| Animal | Advantage | Disadvantage | Ideal Antibody | Ideal Application |
|---|---|---|---|---|
| Chicken |
|
|
Polyclonal | Antibody Drug Development |
| Goat |
|
|
Large Volume Polyclonal | Diagnostic Assays |
| Llama |
|
|
Single Domain | Functional Applications |
| Mouse |
|
|
Monoclonal | Anti-Idiotypes |
| Rabbit |
|
Not ideal for highly conserved mammalian proteins | Polyclonal Monoclonal | Small Molecules All Purpose |
Antibodies are the products of antibody development and the heart of what we do. Learn about the nature of antibodies, their size, types, and anything antibody related.
Antibodies are proteins produced by the immune system to bind against immunogens or foreign particles. Due to antibodies’ specificity and ability to bind to unique epitopes, they are an important research tools.
A host is immunized with an antigen and adjuvant mixture causing B-cells generated by the immune system to begin producing antibodies. In the case of polyclonal antibodies, blood serum is extracted to test antibody titers. When the titers are at their highest, more serum is extracted in order to retrieve the largest amount of antibodies possible. The serum is then screened for the desired antibody product. Keep in mind – different hosts reach peak titers at different times, so matching a host to your experiment time frame is an important consideration. For monoclonal antibodies, once antibody titers are at their highest, spleen cells are extracted from the host to undergo hybridoma development. Monoclonal antibodies are then developed either in vitro through cell culture supernatant, or in vivo through ascites using the hybridomas. For recombinant antibodies, llama peripheral blood mononuclear cells (PBMCs) or rabbit plasma cells are isolated and the genetic material of the clones are constructed into a library, screened, and expressed.
- Polyclonal
- Monoclonal
- Recombinant
Recombinant antibodies are a type of monoclonal antibody. The difference is the DNA sequence of the recombinant antibody is known and so they can be reproduced using recombinant protein production methods.
| Antibody Type | Pros | Cons | Species |
| Monoclonal |
|
|
|
| Polyclonal |
|
|
|
| Recombinant |
|
Higher cost of development
|
Llama
Rabbit |
In addition to the difference in antibody type there is also a difference in recombinant antibody technologies:
|
Disadvantage | Technology | |
| Recombinant Rabbit Monoclonal Antibody |
|
New technology not well established | Isolated plasma cell technology |
| Recombinant Llama Single Domain Antibody |
|
Limited success binding small antigens (peptides & small molecules) for llama single domain | Phage Display |
Antibodies are proteins made by B cells of the immune system in response to an immune challenge. They can be defined as immunoglobulins (Ig) capable of interacting specifically with the antigen that caused their formation.
Application is the research assay, diagnostic assay, or biological function that the antibody will be used for. Learn about the different assay protocols, and different antibody properties established through assays.
Different antibodies may work better for one application over another due to how specific the antibody can be when binding to the target. The antigen is presented differently depending on the assay used and may require different binding properties. Be sure to ask antibody experts which antibody best fit your assay needs.
- Western Blot
- Immunohistochemistry
- Immunocytochemistry
- Immunofloursecent
- Flow Cytometry
- ELISA
