Custom Antibody Production
For over two decades, Aves Labs has been producing custom chicken polyclonal antibodies for customers in academia and industry worldwide.
Our Process Produces Antibodies That Work
Over our long experience in manufacturing custom chicken polyclonal antibodies for diverse customers in academia and the biotech, diagnostic and pharmaceutical industries, we have developed a number of proprietary methods and procedures that optimize the immune response of our host animals and improve the quality of our products. Whereas most competitors work primarily with rabbits, our scientists' deep experience with chickens allows our customers to benefit from unique capabilities, among them improved algorithms for analyzing protein sequences to identify immunogenic peptides.
We Can Support Projects of Any Size
Custom chicken antibodies can require two hens or two hundred hens, which is why we designed our new facilities with flexibility in mind. As a result, Aves can tailor our custom antibodies service to suit your needs, including project sizes up to "bulk" production levels (>100mg).
Comprehensive Support and Service
Using our proprietary Immunogenicity Algorithm®, Aves Labs provides complimentary peptide design help to identify the best sequences for use in applications requiring recognition of native protein structure, such as immunohistochemistry, immunocytochemistry, and immunoprecipitation applications. We then perform all of the steps necessary for production of the final product, including peptide synthesis, conjugation, antibody production and antibody purification.
Responsible Animal Treatment
Aves antibodies are purified from eggs collected from immunized hens. This method allios us to avoid animal restraints and bleeds. In fact, we never subject our hens to restraints, except briefly for the injections themselves.
Our service comes in several packages (prices do not include shipping and handling). Contact us with you project details. We look forward to providing you with a speedy project consultation and quote.
Custom Polyclonal Antibody Production
4 month production protocol
$1,500 (contact us to request a quote)
Antibodies generated against a customer-supplied antigen. Includes:
- Purified immune IgY from two (2) hens (~800 mg IgY / hen)
- Pre-immune IgY from each hen (~150 mg of IgY / hen)
- Four (4) Immunizations per hen
Custom Peptide Antibody Production
4-6 month production protocol
$3,500 (contact us to request a quote)
Affinity-purified antibodies generated against a synthesized peptide. Includes:
- Peptide design
- Peptide synthesis up to twenty (20) residues
- Conjugation to KLH
- Affinity purification pooled from both hens
- ELISA analysis
- Pre-immune IgY from each hen (~150 mg IgY / hen)
- Remaining peptides (~20 mg)
Custom Phosphospecific Antibody Production
4-6 month production protocol
Affinity purified antibodies against a phosphospecific peptide. Includes:
- Peptide synthesis of non-phosphorylated peptide
- Peptide synthesis of phosphorylated peptide
- Conjugation to KLH
- Positive affinity purification
- Negative affinity purification
- ELISA analysis
- Remaining peptides (~20 mg)
Aves Labs is a leading global provider of custom phosphopeptide-specific antibody production services. Chickens are a particularly useful host animal for this production because of the virtually unlimited supply of starting material. Typically, phosphopeptide antibody projects performed in rabbits are limited by the amounts of serum available. With chicken eggs as the starting material, there is considerably more antibody to begin with.
Aves Labs works with customers in phosphopeptide design in order to balance the need for maximal immunogenicity of the phosphopeptide sequences with the need to maximize the proportion of antibody that is specific to the phosphorylated form of the peptide (i.e., doesn’t cross-react with the non-phosphorylated form).
This involves injecting a KLH-conjugated form of the phosphopeptide into laying hens, purifying the IgY fraction from the eggs, then affinity purifying the result using a phosphopeptide-agarose column. We then perform negative affinity purification using a second column containing the non-phosphorylated form of the peptide. This negative affinity purification removes the fraction (typically between 10-50% of the antibodies that cross-react).
We can provide either an "IgY fraction" (i.e., a purified preparation of IgY in PBS, but containing only a small fraction against the antigen of interest) or "Affinity Purified Antibodies" using an affinity matrix (in which the entire antibody recognizes the antigen). For some applications (e.g., Western Blot, immunocytochemistry, etc.), the IgY fraction may be sufficient. For other applications (e.g., immunoprecipitation, etc.), affinity purified antibody is recommended.
Number of Hens
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.
We can perform an ELISA assay, which provides information about antibody titres and antibody avidities for the antigen. This information can help provide guidance about antibody dilutions in your studies.
After delivering antibody preparations to you, we can continue to collect and store approximately twelve (12) immune eggs for a period of two (2) months. These eggs can be used to prepare additional batches of antibodies at a significantly reduced cost. You may also choose to have us continue boosting the hens to collect additional batches of eggs.
Frequent Topics From Our Custom Antibody Service Customers
Where To Start
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.
Boost and Purification Schedule
(approximately 11 weeks from start to delivery*)
Collect preimmune eggs
Perform first injection.
Perform second injection.
Perform third injection.
Perform fourth injection.
Collect immune eggs.
Purify IgY fraction from immune eggs (6).
Ship order (FedEx Overnight).
Collect/store additional 12 eggs during evaluation period.
Affinity Protocol (optional)
(approximately 12 weeks from start to delivery*)
Start ELISA (optional)
Start affinity purification.
End affinity purification.
Ship order (FedEx Overnight).
Additional Boosts (optional)
(approximately 5-6 weeks)
* - Add two (2) weeks for peptide synthesis
Antigen Preparation For Custom Antibody Production
Bear in mind the following general guidance with regard to protein size, protein concentration, vehicles (i.e., buffers), other chemicals in the vehicles, and form of the protein:
In general, the immune system does not recognize small molecules, so there is a minimum size for a protein to become "immunogenic." Although the exact cutoff depends on the protein, 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. This can be done by treating the protein with a bi-functional cross-linking agent, such as glutaraldehyde or paraformaldehyde, or by conjugating the protein to keyhole limpet hemocyanin (KLH).
It is difficult to predict, a priori, what concentration of protein is necessary to inject, but we recommend using at least 2 mg of protein in a total volume of 5000 uL of buffer. This may be generous for highly immunogenic proteins, but with poorly immunogenic proteins, it is likely necessary. There does not appear to be a maximal limit, so the more, the better.
The ideal buffer for injections is phosphate-buffered (10 mM, pH 7.2) isotonic saline (also called "PBS"). It is "ideal" in that Freund used this buffer so many years ago when he was perfecting his adjuvants. Further, it does not contain primary amine groups -- which will interfere with conjugations using aldehydes or NHS-groups -- in the event the protein needs to be coupled to make it bigger. 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 Vehicle
Given the technical nature of this issue, please contact us to discuss your particular vehicle. Generally, if a chemical is toxic to hens, or even if it is an irritant, it should not be present in the vehicle. Needless to say, the hens' health and stress level impact their ability to lay eggs.
Tris and imidazole (anything below 5 mM), chemicals that are commonly found in some preparations, are fine for inclusion.
Detergents are problematic in that they disrupt the emulsification needed to create a good adjuvant. (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.) We strongly recommend dialyzing away detergents, both ionic detergents (e.g., SDS) and non-ionic detergents (e.g., triton X-100, tween-20, etc.).
A common customer concern is that their protein will come out of solution; however, insoluble proteins often make the best preparations for antibody production! The reason is not entirely clear, but it may be that insoluble proteins remain at the site of injection for a longer period, thus 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.
Form of the Protein
We can inject proteins in solution or proteins in polyacrylamide gels. With regard to proteins in solution: the easiest and best way to prepare your protein is simply to put it 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.
With regard to 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. In that case, run a thick gel (at least 1 mm in width) and then use a stain that does not fix the protein inside the gel. We recommend Pierce Zinc Reversible Stain Kit from Thermo. Once the gel is stained, the staining solution should be removed, allowing the protein to diffuse from the gel and stimulate the immune system. Do not use Coomassie stains, as these fix the protein inside the gel. Importantly, minimize the volume of the gel itself. Be sure to cut out the band without extra bits of gel around. It is best to keep the gel volume under 1.0 mL (in a total volume of 5.0 mLs PBS).
What To Do If Proteins Come Out of Solution (Precipitate) When Put in PBS
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.
Sending Gel Slices
It is important not to fix the gels with ethanol-acetic acid. We recommend using Pierce Gel Code Zinc gel staining kit. Alternatively, you can stain the gels with an aqueous solution of Coomassie dye.
Be sure to keep the volume of the gel to a minimum. For each hen, the volume of the gel should be about 1 mL in about 1 mL of buffer, for a total volume of 2 mLs. Divide the 2 mLs into four 500 uL aliquots.
We Can Help You Identify Good Peptide Sequences for Making Anti-Peptide Antibodies
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.
We Offer Non-chicken Host Species through Antibodies Incorporated
Aves Labs works with chickens as its primary hosts. If you require additional primary hosts, such as rabbits, we offer both the monoclonal and polyclonal antibody development services of Antibodies Incorporated, our sister company, to meet your needs.
Agarose-based Matrices for Affinity Purification
We find that such matrices provide the lowest non-specific binding and highest capacities. These matrices are chemically-modified to allow covalent attachment of antigen via either primary amine or sulfhydryl groups.
A Word on Molting
Molting is an annual change in the physiology of birds that normally is triggered by the shortening daylight period in the Fall. It involves changes in dietary habits, loss of feathers, and most importantly, loss of egg production. Although we take steps to avoid molts by carefully monitoring lighting periods and temperatures and by reducing ambient noise levels in the facility, occasional molts do occur. When this happens, we will notify you and provide information about status of the hens. If necessary, we will inject additional hens to replace the molting animal at no extra cost.