Polyclonal Antibody Development: Production Steps
& Information
Polyclonal antibody development and production methods are faster than monoclonal antibody development and recombinant antibody development and production. In fact, they are often the first step for monoclonal antibody and recombinant antibody development!
By nature, polyclonal antibodies are not monospecific to one epitope like monoclonal and recombinant antibodies; however, the benefit of targeting multiple epitopes is a more robust detection of your target. Major polyclonal antibody development and production factors for optimal antibody performance include:
- The host species,
- Antigen preparation,
- Immunization protocols.
ProSci provides a wide variety of host species, custom configurations, and packaged services optimized to your needs. Outsourcing polyclonal antibody development to ProSci's antibody experts allows you to focus on your research.
Rabbits are the most popular host for antibody development and production. Their immune systems are capable of generating antibodies to a broad range of antigens, from small molecules through large proteins. Their immune response also generates a diversity of antibodies, improving the likelihood to obtain antibodies for multiple applications.
Our 8-week rabbit immunization protocol is optimized to balance turnaround time with immune response. It includes pre-immune serum, four immunizations, test serum collection, production serum collection, and final serum collection, yielding ~75 ml antiserum per rabbit.
Rabbit Polyclonal Antibody Project Antigen Requirements
We request 1 mg of antigen per pair of rabbits. Additional services such as ELISA will need additional quantities of antigen. See below for additional details.
Goats are popular for large-scale polyclonal antibody production. They produce large quantities of antibodies in a short amount of time, are cost effective, and have an abundance of secondary antibodies. Because goats are a larger host species, they take longer to generate an initial immune response compared to smaller host species.
Our 13-week goat immunization protocol includes pre-immune serum, four immunizations, and four production serum collections, yielding ~800 ml antiserum per goat.
Goat Polyclonal Antibody Services Antigen Requirements
We request 1.5 mg of antigen per goat. Additional services such as ELISA will need additional quantities of antigen. See below for additional details.
Chickens are ideal for developing antibodies against highly conserved mammalian proteins. The Fc region of chicken IgY is sufficiently different from mammalian IgG to where background is reduced by not binding to mammalian Fc receptors or protein A or protein G. The antibodies are collected through eggs. However, as titers are similar between serum and the yolks, small test bleeds are collected to monitor production.
Our 8-week chicken immunization protocol includes pre-immune serum, four immunizations, three test serum collection, and egg collection, yielding ~12 yolks on average.
Chicken Polyclonal Antibody Services Antigen Requirements
We request 1 mg of antigen per pair of chickens. Additional services such as ELISA will need additional quantities of antigen. See below for additional details.
Mice are ideal for polyclonal antibodies when there is limited antigen, when not much antiserum/antibody is needed for experiments, or to test antigenicity of a sample. Once an immune response is obtained, mice are also ideal for developing monoclonal antibodies.
Our 8-week mouse immunization protocol includes pre-immune serum, four immunizations, a test serum collection, and production serum collection, yielding ~0.5 ml antiserum per mouse.
Mouse Polyclonal Antibody Services Antigen Requirements
We request 1 mg of antigen per group of five mice. Additional services such as ELISA will need additional quantities of antigen. See below for additional details.
Llamas are another species ideal for large-scale production and require little antigen. In addition to a traditional immune response, llamas generate a heavy chain only antibody. These antibodies lend themselves well to the development of recombinant single domain antibodies.
Our 12-week llama immunization protocol includes pre-immune serum, four immunizations, and three production serum collection, yielding ~800 ml per llama.
Llama Polyclonal Antibody Services Antigen Requirements
We request 0.5 mg of antigen per llama. Additional services such as ELISA will need additional quantities of antigen. See below for additional details.
Polyclonal Antigen Requirements
- Please refer to the specific species above for exact antigen quantities required.
- We can start a custom polyclonal antibody production project with 50% of the total amount of antigen needed. Additional quantities can be supplied during the project’s progress.
- ELISA: 100 µg per bleed per animal tested
- Immuno-affinity Purification: 5 mg for a standard column if the protein is <50 kDa in size, or 8 mg if the protein is >50 kDa in size.
Generally, peptide antigens need to be around 70% purity or greater and protein antigens need to be at least 90% purity or greater. The necessary antigen purity also depends upon project goals. For standard polyclonal antibodies, >70% peptide antigen purity is fine, but for peptide antigens containing modified amino acids >90% purity is recommended.
Antigens should be submitted at a concentration of 1 mg/ml or higher. PBS, Tris, and < 4 M urea are preferred buffers for immunization, but SDS and citrate buffers are also acceptable. Please note for services such as ELISA and immuno-affinity purification the antigen needs to be soluble, and for immuno-affinity purification the protein cannot be in Tris or other buffers containing primary amines.
Lyophilized proteins or lyophilized peptides already conjugated to a carrier protein should be supplied in the quantities listed above. 5 mg of unconjugated lyophilized peptides should be supplied for conjugation to a carrier protein. Include the reconstitution method on the antigen submission form.
Proteins can be submitted as a gel strip, cut from an SDS-PAGE gel. Please note that we cannot perform an affinity purification or ELISA when the protein is supplied in this form. The protein needs to be reasonably concentrated (2-5 mg/ml) and then run into the gel. If it precipitates, use a lower concentration. 100 to 200 µg of your protein. Stain the whole strip with coomassie blue then de-stain it, rinse with water at least twice to remove traces of methanol, and cut the desired strip from the gel. Place the strip in a 15 or 50 ml conical vial with water and ship with an icepack – don’t freeze it.
Please send in an antigen submission form prior to sending your antigens.
Packaged Services & Add-ons
Serum Package
Polyclonal Antibody Production Using Client-Provided Antigen
- Immunization protocol with desired host species
Peptide Package
Polyclonal Antibody Production Using Peptide Synthesized by ProSci
- Synthesis of peptide up to 15 residues (5 mg for customer)
- Conjugation of peptide to carrier protein
- Immunization protocol with desired host species
- ELISA on 1st serum collection to verify titers using unconjugated peptide
Purified Package
Polyclonal Antibody Production Using Peptide Synthesized by ProSci Affinity Purification by ProSci
- Synthesis of peptide up to 15 residues (5 mg for customer)
- Conjugation of peptide to carrier protein
- Immunization protocol with desired host species
- ELISA on 1st serum collection to verify titers using unconjugated peptide
- Immuno-affinity purification of up to 25 ml serum on last scheduled serum collection (includes reusable column)
Add-ons
Peptide Cocktail Antibody Production packages are rabbit-exclusive and include all of the services in the Peptide and Purified packages, respectively. In addition, they include the synthesis, conjugation, and immunization with your choice of two or more peptides. This is an option that increases the likelihood of a peptide-based antibody working for multiple applications, as it doubles or triples the available number of epitopes for the animals’ immune systems to respond against.
Phospho-specific Antibody Production packages are rabbit-exclusive and include all of the services in the Peptide and Purified packages, respectively. In addition, it includes the synthesis, conjugation, and immunization of phospho peptides (or peptides containing other modified amino acids). The Purified Package also offers synthesis of the corresponding non-phospho peptide sequence, used for isolating both the phospho and non-phospho antibodies for your target protein.
Purify Additional Sera includes immuno-affinity purification with preferred serum or pooled serum collected throughout the project.
Additional ELISA includes ELISA testing with preferred serum or additional controls.
Antibody Labeling includes biotin or HRP labeling of purified antibody.
Polyclonal to Monoclonal Upgrades are available: Polyclonal antibody development and production is the first phase for monoclonal and recombinant antibody development. Learn more about converting polyclonal to monoclonal antibodies here. Let’s start your project!
