Questions & Answers
What are the advantages of polyclonal antibodies?
What is the concentration of the specific, polyclonal antibodies in serum?
What are the advantages of monoclonal antibodies?
What is important when choosing a peptide for immunization?
What kind(s) of antigen(s) can be injected?
How much antigen is required for immunization?
Why will some antigens only induce response resulting in IgM antibodies?
How should the antigen be prepared for immunization?
Which species to choose for immunization?
Can I test pre immune serum or egg yolk from a few animals before I start the project?
Are antibodies made in rabbit better than those made in chicken?
I made a custom antibody production. How do I know that the antibody is recognizing the right protein?
If my antibody works in immunolocalization will it also work in a western blot and the other way around?
How much of serum or eggs can be obtained?
How to store antibodies?
Q: What are the advantages of polyclonal antibodies?
A: 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 dentaturalized proteins.
Q: What is the concentration of the specific, polyclonal antibodies in serum?
A: 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.
Q: What are the advantages of monoclonal antibodies?
A: 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).
Q: What is important when choosing a peptide for immunization?
A: Usually the C or N-terminal of the protein is used, as 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?
Q: What kind(s) of antigen(s) can be injected?
A: 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.
- 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 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.
Q: How much antigen is required for immunization?
A: It depends on the immunogenicity of the antigen. In the standard protocol (for rabbit, goat or hen) we 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. We can also use your own immunization protocol. If protein solutions need to be concentrated, please be aware, that loss can occur from the concentrating device.
Q: Why will some antigens only induce response resulting in IgM antibodies?
A: 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, use a species which is more evolutionarily distant e.g. hens.
Q: How should the antigen be prepared for immunization?
A: Antigens should be supplied in buffered (Tris, MOPS, Hepes) saline solution. Insoluble antigens (e.g. inclusion bodies) can be also used for immunization, however one have to keep in mind that often other proteins will be also present in inclusion bodies, not only a target protein. Desired antigen concentration is 1 mg/ml, however lower concentrations are also acceptable.
You can also send us 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.
Q: Which species to choose for immunization?
A: 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). Find more information about IgY.
Q: Can I test pre immune serum or egg yolk from few animals before I start the project?
A: 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.
Q: Are antibodies made in rabbit better than those made in chicken?
A: Antibody production outcome is a combination of: an antigen, specific animal immunological response and testing. For certain proteins like conserved mammalian targets, antibody production in chickens is to be recommended. They are more far apart evolutionary and their immune system will respond better to conserved mammalian proteins which might not be recognized by immunological system of a goat or a rabbit.
Q: I made a custom antibody production. How do I know that the antibody is recognizing the right protein?
A: First confirmation comes from an indirect ELISA assay. Wells of an ELISA plate are coated with the protein or peptide used for immunization of the animals. The plate is incubated with immune serum followed by a secondary antibody to develop the signal. This shows that immunized animals developed antibodies to injected protein or peptide. However, this result might not correspond directly to detection levels achieved in the native tissue, since the ELISA well is covered with ideally exposed peptide or protein Testing native tissue involves material collection where we know or suspect that our protein of interest is expressed. Controls in the form of extracts from deletion or overexpression mutants needs to be included. Using a recombinant protein as a control in western blotting might provide information but in some cases, produced antibody have recognized only recombinant (not native) protein. Note also that the signal from a recombinant sample will overwhelm and hide the signal from the native tissue, so blots having both types of samples may need to be imaged differently. Some proteins are rather difficult to detect, considering their expression level in specific tissues following certain developmental patterns. All these factors must be taken into account for successful testing.
Q: If my antibody works in immunolocalization will it also work in a western blot and the other way around?
A: Again, 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 and 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.
Q: 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
Q: How to store antibodies?
A: 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, add some azide (or other preservative) to a final concentration of 0.02 %.
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 for storage). IgY can be stored at + 4°C with 0.02 % sodium azide (note: azide inhibits activity of the 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 purification of antibodies difficult. 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:
- -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%
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 are called cryoglobulins, that are likely to precipitate in the cold. However, warming them up to room temperature can solve this problem. Antibody solutions can be also centrifuged to remove precipitates.
Chicken antibodies can also precipitate when stored in the cold overnight or after several weeks. Warming them up to room temperature often helps to dissolve those precipitates. Otherwise, IgY solutions can be centrifuged to remove precipitates prior to use. Antibody solutions stored without preservatives are at risk to becoming contaminated by bacterial growth, which is a common reason for protein inactivation.
- 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