Antibody Production FAQ

  • What are the advantages of polyclonal antibodies?
    Polyclonal antibodies will recognize a mixture of different epitopes of an antigen and are more tolerant to small changes in the nature of it, like polymerization or slight denaturation. Polyclonal antibodies are the preferred choice for detection of denaturalized proteins.
  • What is the concentration of the specific, polyclonal antibodies in serum?
    Most commonly, the concentration of specific antibodies in serum varies between 0.05-0.2 mg/ml of serum (based on Agrisera's internal estimation via hundreds of affinity purifications). In special cases, strong polyclonal serum can contain up to 1 mg of specific antibodies per ml of serum.
  • What are the advantages of monoclonal antibodies?
    Monoclonal antibodies recognizes only one chosen epitope of the antigen, which can be a disadvantage in some assays, such as immunoprecipitation, and when making immunoaffinity columns. A monoclonal antibody is as good as a primary antibody in an assay for detection of antigen in a tissue. Theoretically, they should give much lower background than polyclonal antibodies, however that is not always the case. Monoclonal antibodies 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 the C or N-terminal of the protein is used, as those parts of the 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

    The 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?
  • What kind of antigen can be injected?

    Immunization can be done using native proteins, recombinant proteins, peptides, carbohydrates or other compounds of microbial, fungal or viral origin. Minimum molecular weight needed to induce sufficient immune response is 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 (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 denaturing agents).
    • Not all 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 immunogenicity of the antigen. In a standard protocol (for rabbit, goat or hen) you may use around 500 µg of peptide/animal/15 week program, or 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.
  • 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 cases of conserved mammalian antigens, it is beneficial to use a species which is more evolutionarily distant e.g. hens.
  • How should the antigen be prepared for immunization?
    Antigens should be supplied in PBS or carbonate buffer, at physiological pH. If isoelectric point of a protein lays at physiological pH, it will become insoluble. For keeping protein soluble, pH of a buffer should be adjusted below or above. Addatives should be avoided. Insoluble antigens (e.g. inclusion bodies) can also be used for immunization. However, one must keep in mind that other proteins will often also be present in inclusion bodies, not only the target protein. The desired antigen concentration is 1 mg/ml, but lower concentrations are also acceptable.
  • 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.), making double staining possible.
    • A ground rule for immunization is to choose an animal that is genetically distant from the antigen source (e.g. hens are very suitable for production of antibodies against conserved mammalian proteins). For more information about IgY click here.
  • Are antibodies made in rabbit better than those made in chicken?
    Antibody production outcome is a combination of the an antigen, the specific animal immunological response, and testing. For certain proteins, like conserved mammalian targets, antibody production in chickens is recommended. Chickens are more evolutionary distant, and their immune system will respond better to conserved mammalian proteins, which might not be recognized by the immunological system of a goat or a rabbit.
  • If my antibody works in immunolocalization, will it also work in a western blot?
    It depends on which part of the protein the antibody is recognizing, and how this part is exposed after fixation or following the western blot. If a polyclonal antibody is made to a synthetic peptide, it is recognizing a pool of epitopes. If such epitopes are not exposed following fixation, the antibody will not work. In some cases, addition of 6-8 M urea to the loading buffer can help to unfold the protein.
  • How to store antibodies?

    Antibodies present in serum
    Serum 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, the shelf-life can be shorter for anti-peptide antibodies. For very short periods of time, serum may be stored at + 4°C. In some cases, more careful freezing with a first step at -20°C, followed by -70°C is beneficial.

    Total IgG fraction (IgG antibodies purified on Protein G matrix)
    Generally, protein G purified antibodies are stable. They can be stored in -20°C or -70°C for years. For short-term storage, add some azide to a final concentration of 0.02 % (or another preservative).

    Total IgY fraction (IgY antibodies purified by precipitation from egg yolk)
    Purified IgY fractions are 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 µg/ml). Avoid freezing and thawing of IgY, and 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 µg/ml). Egg yolk should NEVER be frozen as this will complicate purification of the antibodies. After 6 months of storage, purifying antibodies present in egg yolk might be somewhat difficult.

    Affinity purified antibodies
    Affinity purified antibodies are the most fragile. Caution 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:

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

    Mammalian, polyclonal antibodies can precipitate following affinity purification. This can occur directly after purification, or overnight during cold storage. Some antigens will stimulate the production of a class of IgG, called cryoglobulins, which precipitate at low temperatures. Heating the cryoglobulins up to room temperature can solve this problem. The antibody solutions can also be centrifuged to remove precipitates.

    Chicken antibodies can also precipitate when stored in the cold, wither directly overnight, or after several weeks. Heating the IgY 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 at the risk of being contaminated by bacterial growth, which is one of the most common reasons for protein inactivation.

    General recommendations:

    • For larger volumes of affinity purified antibodies, filter-sterilize the antibody sample and aliquot the solution to avoid multiple freezing and thawing cycles.
    • Ideal storage occurs at protein concentrations around 0.5-1 mg/ml.
    • In cases of IgM antibody production, 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 dilutions of at least 1:2000.

    Alternative agents for preventing bacterial growth in antibody solution:

    • Thimerosal at 0.01%
    • Gentamicin sulfate at 50 µg/ml