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Antibody Development Guide

Home / Antibody Development Guide
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.

Antigen
Antigen

Antigens are the target you wish to develop antibodies against. Learn about different antigen types and how different antigen properties affect antibody development success.

What is an antigen?

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.

What antigen factors affect antibody development?

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.

What antigen types are there? 
  • Small Molecules
  • Proteins
  • Carbohydrates
  • Peptides
  • Antibody Variable Region (idiotype)
What are antigen requirements for developing antibodies?

To learn more about antigen requirements view the pages for the corresponding service

  • Polyclonal Antibodies
  • Monoclonal Antibodies
  • Recombinant Rabbit
  • Recombinant Llama
What is an epitope?

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.

What is an immunogen?

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.

Adjuvant
Adjuvant

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

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.

Why do I need an adjuvant for antibody development?

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.

What kind of adjuvants are used in 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
Animals

Animals are hosts whose immune system generates antibodies. Learn how each host’s immune system produces antibodies and which species is right for you.

Why does host species matter?

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.

What animals are available for antibody development?

There are many animals that can be used for antibody development. For ProSci we offer the following species 

  • Rabbit
  • Mouse 
  • Goat
  • Chicken
  •  Llama 
What are the advantages and disadvantages of each species?
 
Animal Advantage Disadvantage Ideal Antibody  Ideal Application
Chicken
  • Non-mammalian host species
  • Good alternative for highly conserved mammalian proteins
  • Non-invasive (antibodies are purified from egg yolks)
  • Difficult to isolate antibodies from yolks
  • Not ideal for peptide projects
Polyclonal Antibody Drug Development
Goat
  • Large mammalian host species
  • High quantity (250mL) sera for large lots
  • Serum can be used directly in initial assays
  • Not ideal for modified peptides
  • Larger antigen quantities
Large Volume Polyclonal Diagnostic Assays
Llama
  • Large mammalian host species
  • Heavy-chain only antibodies 
  • Serum can be used directly
  • Not ideal for small molecule or peptide antigens
Single Domain Functional Applications
Mouse
  • Large spleen to body ratio makes them ideal for monoclonal antibodies
  • Very common isotype(20)
  • Do not need much antigen
  • Serum can be used directly in initial assays
  • Small serum volumes
  • Not ideal for short peptide antigens
Monoclonal Anti-Idiotypes
Rabbit
  • Most common host species
  • Great host for peptide and most protein antigens
  • Serum can be used directly for initial assays
Not ideal for highly conserved mammalian proteins Polyclonal Monoclonal Small Molecules All Purpose
Antibody
Antibody

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.

What are antibodies?

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.

How are antibodies developed?

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.

What are the different antibody types?
  • Polyclonal
  • Monoclonal
  • Recombinant
What is the difference between monoclonal and recombinant antibodies?

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.

What are the advantages and disadvantages of each antibody type?

 

Antibody Type Pros Cons Species
Monoclonal
  • Unlimited quantity and availability of antibody
  • Lot-to-lot consistency
  • High specificity
  • Quick reproduction of antibody once hybridoma is developed
  • Longer development time
  • Requires a minimum of 4 mgs immunogen
  • Higher cost of development
  • Antibodies less likely to work for multiple applications
  • More difficult if using peptide immunogens
  • Low affinity
  • Mouse
  • Rabbit (Recombinant)
  • Llama (Recombinant)
Polyclonal
  • Quick turnaround
  • Inexpensive
  • High affinity
  • One antibody can work for a variety of applications
  • Requires less than 1mg immunogen
  • Multiple host species, including non-mammalian hosts
  • Quantity available limited by the number of bleeds
  • Lot-to-lot variability in characterization
  • Difficulty to reproduce
  • Chicken
  • Goat
  • Rabbit
  • Llama
  • Mouse
Recombinant
  • Unlimited quantity and availability of antibody
  • Lot-to-lot consistency
  • High specificity
  • Antibody sequence known from the outset
  • Quick reproduction of antibody after development
  • Cost-effective large scale production
Higher cost of development

 

Llama

Rabbit

In addition to the difference in antibody type there is also a difference in recombinant antibody technologies:

 

  • Advantage
Disadvantage Technology
Recombinant Rabbit Monoclonal Antibody
  • Faster screening time 
  • Uses 100% mature plasma cells
New technology not well established Isolated plasma cell technology
Recombinant Llama Single Domain Antibody
  • Small size, ~15kDa, for enhanced tissue penetration
  • Higher neutralization potential
  • Requires less than 1mg antigen
  • Stable under extreme temperature and pH

 

Limited success binding small antigens (peptides & small molecules) for llama single domain Phage Display
Antibody structure and properties

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.

Learn more.

Application
Application

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.

Why does my antibody work in one application but not another?

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.

What are kind of assays are antibodies used in?
  • Western Blot
  • Immunohistochemistry
  • Immunocytochemistry
  • Immunofloursecent
  • Flow Cytometry
  • ELISA

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