FAQ

• recognize only one chosen epitope of the antigen, and that can be a disadvantage in some assays such as immunoprecipitation and in making immunoaffinity columns
• are good as a primary antibody in an assay
• good for detection of antigen in a tissue and theoretically they should give much lower background than polyclonal antibodies, however that is not always a case

• have high homogeneity and give highly reproducible results (if other experimental conditions can be kept
  constant)
  
  

What is important when choosing a peptide for immunization?

Usually C or N-terminal of the protein is used as there are highest chances that those parts of protein are exposed. Also, to mimic protein behavior, the synthesized peptide should have similar structure and charge as the protein it has been "cut out off". Therefore:

• peptides derived from the C terminal should have N terminal modified by acetylation
• peptides derived from the N terminal should have C terminal modified by amidation
• peptides derived from an internal sequence should have both ends modified

Following points should also be considered:

• Are there any other proteins from the family of interest, where cross-reactivity should be avoided?
• Is the crystal structure of the protein (or homologous protein) known? This would be helpful for the peptide chemist in searching for the best peptide for antibody production.
• What is the final application of the produced antibodies? Native or denatured techniques?

More information about chosing an antigen for antibody production can be found here.

What kind(s) of antigen(s) can be injected?

Immunization can be done using native proteins, recombinant proteins, peptides carbohydrates or other compounds of microbial, fungal or virus origin. Minimum molecular weight needed to induce sufficient immune response is from 5 - 10 kDa.
Biohazardous materials for immunizations are not accepted.

Important notes:

• If the antibodies are going to be used on the denatured target protein (example: Western blot, immunohistochemistry on fixed tissues), denatured forms of antigens are preferred (like gel piece, protein in inclusion bodies).
• If the antibodies are going to be used on native target proteins (example: immunoprecipitation), non-denatured forms of antigen are preferred (protein in solution free of denaturalizing agents).
• Not all anti-peptide antibodies will recognize native protein, therefore a careful choice of peptide sequence is of crucial importance.
• Antibodies made against recombinant proteins expressed in bacteria can in some cases fail to recognize native protein. The reason for this might be incorrect folding of the protein antigen when expressed in bacterial cells.

No guarantees can be given in advance for a success of any immunization program.

Recommended references on the subject:

• "Monoclonal antibodies: principles and practice" by James W. Goding, 1996,
  ISBN 0-12-287023-9; Publisher: Academic Press.
• Using Antibodies: A Laboratory Manual, E.Harlow and D.Lane, 1999,
  ISBN: 0879695447; Publisher: Cold Spring Harbor Laboratory Press. 
• Hjelm et al. (2012). Parallel immunizations of rabbits using the same antigen yield antibodies with similar, but not identical, epitopes. PLOS ONE.
  
  

How much antigen is required for immunization?

It depends on the immunogenity of the antigen.
In the standard protocol (for rabbit, goat or hen) we use:

• around 500 µg of peptide/animal/15 weeks program
• around 400 µg of protein/animal/15 weeks program

Lower amounts of antigen (less than 10 µg) are acceptable in cases of low relatedness between the antigen and proteins of the animal chosen for immunization. We can also use your own immunization protocol.  If protein needs to be concentrated, please be aware, that in some cases it can be lost by attaching to the membrane used in a concentrating device.

Why will some antigens only induce response resulting in IgM antibodies?

Responses against highly conserved mammalian proteins are often weak and mainly resulting in IgM antibodies, owning to lack of stimulation of T cells (Goding 1993). However, there are also exceptions. In case of conserved mammalian antigens, use a species which is more distant evolutionary e.g. hens.

How should antigen be prepared for immunization?

Antigen should be supplied in buffered (Tris, MOPS, Hepes) saline solution.
Insoluble antigen (inclusion bodies) is as good as a soluble one.
Desired antigen concentration is 1 mg/ml, however lower concentrations are also acceptable.

You can also send us just a band cut out from SDS-PAGE gel, which contains protein of interest. Destain the gel in water, since acetic acid will contribute to further denaturation of the protein.

• Avoid: Additives toxic to animals, e.g. protease inhibitors such as PMSF, sodium azide etc.
• Acceptable: Low amounts of SDS, imidazole, urea, guanidine, non-ionic detergents, EDTA or EGTA and polyacrylamide. Final amounts depend very much from sample volume/antigen concentration. Please, inquire.
  
  

Which species to choose for immunization?

Advantages of using both IgG (rabbit, goat) and IgY (hen) antibodies developed against the same antigen:

• independent confirmation that the expected target protein is detected.
• antibody pools with distinct properties, complementing each other in different techniques (Western Blot, immunoprecipitation etc.)
• double staining possible
  
  Generally for immunizations choose an animal genetically distant from the antigen source (e.g. hens are very suitable for production of antibodies against conserved mammalian proteins). On this page (IgY) you will find more information about antibodies in hens.
  
  

Can I test pre immune serum or egg yolk from few animals before I start the project?

Agrisera will, on request, send you a few samples of pre immune serum or egg yolk before starting the project. You can test and choose the most suitable animal with the lowest background signal. Our recommendations about pre-immune serum screening can be found here.

How much of serum or eggs can be obtained?

How to store antibodies?

Antibodies present in serum
This is a very stable format for antibody storage. In -20°C or -70°C serum can usually be stored for years. In some specific cases this time can be shorter for anti-peptide antibodies. For very short time periods serum can be stored at + 4°C. In some cases more careful freezing with a first step at -20°C followed by -70°C can be beneficial.

Total IgG fraction (IgG antibodies purified on Protein G matrix)
Generally antibodies in this format are stable. They can be stored in -20°C or -70°C for years. For short-term use some azide to the final concentration of 0.02 % can be added or other preservative.

Total IgY fraction (IgY antibodies purified by precipitation from egg yolk)
Purified IgY fraction is very stable even at room temperature (although we do not recommend it as storage conditions). IgY can be stored at + 4°C with 0.02 % sodium azide (note: azide inhibits activity HRP enzyme) or gentamicin sulfate (50 ug/ml).
Avoid freezing and thawing of IgY or storing it on dry ice. IgY antibodies can be stored at -20°C.

"The IgY preparations were stable over time. No loss of antigen recognition was observed after storage for 3 years at + 4°C. F.De Ceunick et al. Journal of Immunological Methods 252 (2001) 153-161.

Egg yolk
Antibodies in egg yolk should be stored at 4°C with 0.02 % sodium azide (note: azide inhibits activity HRP enzyme) or gentamicin sulfate (50 ug/ml). Egg yolk should NEVER be frozen as this will make difficulties for purification of antibodies. After 6 months of storage it might be more difficult to purify antibodies present in egg yolk.

Affinity purified antibodies
Are the most fragile antibodies. Care should be taken when considering storing conditions, which should be checked experimentally for every single antibody. Affinity purified antibodies against different epitopes can vary in stability. Some will precipitate directly after the purification, while the activity may still remain. It is difficult to predict storage conditions for a given antibody in advance - there are some alternatives to be tested:
Suggested storage conditions - to be tested:

* -20°C or -80°C
* + 4°C with preservatives like azide (0.02%) or merthiolate
* -20°C with glycerol; Final concentration of glycerol 10 or 50%
* -20°C with BSA at final concentration of 0.05-0.5%

Problem: affinity purified antibodies precipitated during storage in 4°C

IgG

Mammalian, polyclonal antibodies can precipitate following affinity purification. This can happen directly afterwards or overnight during cold storage. Some antigens will stimulate a production of a class of IgG, which is called a cryoglobulins, what means that they will precipitate in a cold. However, warming up to room temperature can solve this problem. Antibody solution can be also centrifuged to remove precipitates.

IgY

Chicken antibodies can also precipitate when stored in the cold overnight or after several weeks. Warming up to room temperature often helps to dissolve those precipitates. Otherwise, IgY solution can be centrifuged to remove precipitation prior to use.

Antibody solutions stored without preservatives are in risk to be contaminated by bacterial growth, which is often most common reason for protein inactivation.

General recommendations

* For larger volumes of affinity purified antibodies, filter-sterilize antibody sample and aliquot to avoid multiple freezing and thawing.
* Storage at protein concentration around 0.5-1 mg/ml.
* In case of IgM especially check protein stability in different storage conditions.

Important note: Sodium azide will inhibit horseradish peroxidase as well as interfere with some coupling methods and biological assays. However, the amount present in IgY preparation (0,02 %) can be washed away in ELISA or Western Blot when IgY is used as primary antibody at dilution at least 1:2000.
Alternative agents for preventing bacterial growth in antibody solution:

* Thimerosal at 0,01%
* Gentamicin sulfate at 50 ug/ml