Knowledge
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Frequently asked questions about antibody production

What are the advantages of polyclonal antibodies?


Polyclonal antibodies:
  • will recognize a mixture of different epitopes of the antigens
  • are more tolerant to small changes in the nature of antigen, like polymerization or slight denaturation
  • are a preffered choice for detection of denaturated proteins


What is the concentration of the specific, polyclonal antibodies in the serum?


Most commonly the concentration of specific antibodies in the serum is between 0,05-0,2 mg/ml of serum. In special cases, strong polyclonal serum can contain from 1-3 mg of specific antibodies per ml of serum (or even more).

What are the advantages of monoclonal antibodies?


Monoclonal antibodies:
  • recognize only one chosen epitope of the antigen, and that can be of the disadvantage in some assays as immunoprecipitation and 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 homogenity
  • 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, synthesized peptide should have similar structure and charge as a protein it has been "cut out". Therefore:

  • peptides derived from C terminal shoudl have N terminal modified by aceetylation
  • peptides derived from N terminal should have C terminal modified by amidation
  • peptides derived from internal sequence should have both ends modified
Following points should also be considered:
  • Are there any other proteins from a family of interest, where cross-reactivity should be avoided?
  • Is crystal structure of a protein (or homologus protein) known? This would be helpful for peptide chemist in searching for a best peptide for antibody production.
  • What is a final appliction of produced antibodies? Native or denatured techniques?

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 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 antibodies are going to be used on native target proteins (example: immunoprecipitation), non denatured forms of antigen are preferred (protein in solution free of denaturating 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 it might be uncorrect folding of the protein antigen when expressed in bacterial cells.
In other words: 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.



How much antigen is required for immunization?


Depends upon the immunogenity of the antigen.
In the standard protocol (for rabbit, goat or hen) we use:
  • 200 µg of protein or peptide for the first immunization
  • 100-200 µg of protein or peptide for the following one
In total:
  • around 800 µg of peptide/animal/14 weeks program
  • around 500 µg of protein/animal/whole 14 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 some antigens will only induce response resulting in IgM antibodies?

Responses against highly conserved mammalian proteins are often weak and mailny 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 immunisation?

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 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.

How to send the antigen?

In Sweden: we recommend Post Office service for over night delivery as: Express (letter) or Express 0700 (package).

In Europe: please, use a reliable carrier.

Which species to chose for immunisation?

Advantages of using both IgG (rabbit, goat) and IgY (hen) antibodies developed against the same antigen:
  • independent confirmation that expected target protein is detected
  • antibody pools with distinct properties, complementing each other in different techniques (Western Blot, immunoprecipitation etc.)
  • double staining possible

Generaly for immunisations 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?

Yes, AgriSera on request will send you few samples of pre immune serum or egg yolk before starting the project. You can test and choose most suitable animal with the lowest background signal.

How much of serum or eggs can be obtained?

Goats: up to 200 ml serum/month
Rabbits: up to 50 ml serum/month
Hens: up to 25 eggs/month