If you are supplying ready-to-inject antigen (e.g., recombinant protein), please provide us with a description of the antigen (type, size, buffer, etc.) and simply ship us the antigen along with a copy of our Polyclonal Antibody Production Order Form.

• For research scale production (i.e., two hens), we suggest 4.0 mg of protein in a total volume of 4.0 mLs of PBS or TBS (i.e., 1.0 mg/mL). It is best if the protein is placed in four 1.0 mL aliquots and frozen on dry ice. If you only have a precious amount of protein, we likely can use a concentration of 0.5 mg/mL.
• If your ready-to-inject antigen is in a buffer solution other than PBS or TBS, please be sure that the buffer does not contain high concentrations of urea (greater than 0.5 M), SDS (greater than 1% (w/v), imidazole (greater than 10 mM), EDTA/EGTA (greater than 100 mM), or sodium azide (greater than 0.01%). All of these agents are irritants and may cause the hens to cease egg production.
• If you would like us to affinity purify the antibodies, send an additional 5 mg of antigen in PBS. Since we use primary amine chemistry to conjugate most proteins to matrix, these preparations must not contain any Tris buffer, which interferes with this chemistry.
• If you supply us with peptide, we will need to conjugate the peptide to a carrier protein such as keyhole limpet hemocyanin (KLH). Please include sequence information to help us choose the best conjugation options and peptide solubilization strategy. We require 5-10 mg of peptide for conjugation to carrier protein, and another 5-10 mg of peptide for conjugation to affinity matrix.

Please contact us with questions about these or other chemicals in your antigen preparations.

Since chickens are outbred species (like rabbits), we highly recommended injecting at least two hens. From that starting point, the number of hens to be injected depends on the amount of antibody you require as well as the hens' inherent immunogenicity.

Yes! Just send us the sequence or the accession number. We will perform the analysis and present you with options. We provide this service to new clients for a $200 analysis fee, which we credit to your account should the project move forward.

Here are some general guidelines on protein size, protein concentration, buffers, other chemicals in the buffers, and form of the protein:

Protein Size

In general, the immune system does not recognize small molecules, so there is a minimum size for a protein to become "immunogenic."

• We recommend that the proteins under 20 kDa in size (about 200 amino acids) be coupled to something bigger in order to make them maximally immunogenic.
• Treat the protein with a bi-functional cross-linking agent, such as glutaraldehyde or paraformaldehyde, or conjugate the protein to keyhole limpet hemocyanin (KLH).

Protein Concentration

It is difficult to predict what concentration of protein is necessary to inject due to differences in the immunogenicity of the proteins. In general, the more, the better.

• We recommend using at least 2 mg of protein in a total volume of 5000 uL of buffer.
• This is high for highly immunogenic proteins, but with poorly immunogenic proteins, it is likely necessary.

Buffers

The ideal buffer for injections is phosphate-buffered (10 mM, pH 7.2) isotonic saline (also called "PBS").

• Freund used this buffer so many years ago when he was perfecting his adjuvants.
• It does not contain primary amine groups -- which will interfere with conjugations to smaller molecules that use aldehydes or NHS-groups.
• Tris buffered saline ("TBS") is fine for injections as well, but it contains a primary amine group, such that it cannot be used when conjugations are required.

Other Chemicals in the Buffer

Given the technical nature of this issue, please contact us to discuss your particular buffer.

• If a chemical is toxic or an irritant to hens, it should not be present.
• Tris and imidazole (anything below 5 mM), chemicals that are commonly found in some preparations, are fine for inclusion.
• Detergents disrupt the emulsification needed to create a good adjuvant, and should be dialyzed away. This includes both ionic detergents (e.g., SDS) and non-ionic detergents (e.g., triton X-100, tween-20, etc.).
• Don't worry if your protein comes out of solution, insoluble proteins often make the best preparations for antibody production!  

Form of the Protein

We can inject proteins either in solution or in polyacrylamide gels.

• For proteins in solution: put your protein into PBS at a concentration of 2 mg in 5000 uL (400 ug/mL). Do not put in anti-microbials, such as sodium azide, or other agents that will harm or irritate the hens.
• For proteins in polyacrylamide gels: if your protein is contaminated with other proteins, you might want to run an SDS-polyacrylamide gel to separate the protein of interest from the contaminants. Please see our FAQ for details on sending gel slices.

Insoluble proteins generally make great antigens, so don’t worry if your protein comes out of solution in PBS. Simply make an even suspension before aliquoting your antigen.

The reason is not entirely clear, but it may be that insoluble proteins remain at the site of injection for a longer period, allowing them to interact with the antigen-presenting cells. Therefore, it is good news if your protein comes out of solution while making your injection mixture!

Emulsification is the process by which miscelles of aqueous buffer that contain the protein become surrounded by oil. This process allows the protein to be released slowly into the hen's connective tissue, which is the preferred time for delivery for antibody production. This process is important to make a good adjuvant.