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Polyclonal Antibody Information
B-cells and Antibody production
B-lymphocytes are able to produce antibodies. The name of the B-lymphocytes is derived from their destination:
Bursa fabricii in birds, respectively bone marrow in most mammals.
First an antigen is needed for an immune response. The antigen (i. e. bacterials or virus particles) are recognized as foreign bodies from macrophages. They engulf and lyse them and present the pieces of the antigen (epitopes) on their surface. T-helper cells recognize this epitope and are activated. Simultaneously a B-cell gets in contact with the antigen and present it on the surface. The activated T-helper cell produces interleukins when recognizing the antigen, which is represented from the B-cell. That leads to a clonal expansion of the B-cell (reproduction) and to a specialization into an antibody producing B-cell: plasma cell.
The titer is an indirect measurement method to determine the concentration of antibodies in the blood. First the antigen is coated on a microtiterplate. Then the serum is diluted in steps like ( 1:5, 1:25, 1:125, ..., 1:325000) and pipetted in the coated microtiterplate. The highest dilution where the antibodies are detectable is the titer. So a very good titer is 1:50000 to 1:300000.
Like in human vaccination a titer is gained after the animals are immunized with the antigens and antibodies are produced against that molecule.
As there are not only the antibodies against the molecule, but all antibodies the animal produced, in the serum we call them polyclonal antibodies. That means that there is a pool of antibodies that recognize multiple epitopes of different antigens. With purification methods the antibodies can get more specific.
What animals are used to generate antibodies
Usually the custom antibody production is done with rabbits, chicken, mice, rats or guinea pigs. But even larger animals like horses or goats can be used for antibody production.
Antibodies can be used to detect molecules or proteins. You can use a second antibody, which detects the first one to improve the signal and/or to dye the antigen-antibody-complex. With this method a lot of proteins and molecules can be detected. Applications: WesternBlots, fluorescent activated cell sorting, ELISA (enzyme-linked immunoabsorbent assay) or other assays.
The antibodies bind proteins in a tissue or cell. You can use fluorescent antibodies or use secondary antibodies to dye the antigen-antibody complex. With this method you can locate proteins in cells or tissues. You might even locate and classify cancer cells, which express certain antigens. With antibodies that recognize the expressed antigens you can identify the tumor cells.
Antibodies can be used to detect substances and cells in blood or food like lipids, drugs, virus particles, hormones or other antibodies. The diagnostic is usually done with ELISA, agglutination or immunoprecipitation
There are several methods to use antibodies in science like ChIP.
Therapy & Vaccination
Antibodies can be produced for vaccination like hepatitis or flue. They can be used to produce and purify hormones like insulin.
In addition antibodies can be used for therapy against cancer, auto-immune deseases or AIDS.
For the polyclonal antibody production animals are immunized with the antigen so that they produce antibodies against the antigen. The antibodies are in the serum of rabbits, mice or guineapigs or in the eggs of chicken.
Antigens (antibody generating) are molecules where lymphocytes and antibodies can bind. It is the molecule you want to generate the antibodies against. The part of the antigen, where the antibody binds is called epitope. Lymphocytes can detect nearly any kind of antigens. This ensures the somatic recombination of the lymphocytes. The antibody-encoding genes are recombinated to generate a vast amount of different antibodies, recognizing any kind of molecules.
Please have a look at our Antigen Section for more information.
Antigens used for antibody production
Usually one of the following antigens is used in a custom antibody production:
- Protein or another antigen provided by the customer
- Recombinant protein provided by customer or produced by us
- Synthetic peptide, synthesized and conjugated to a carrier by us
- Small molecules like DNA, virus particles
- Whole cells
- Other molecules: just ask
Animals are immunized with the antigen. Usually after 3 - 5 immunizations a good antibody titer is detected. ELISA titer tests give a hint if more immunizations are necessary or if it is recommended to collect the antibodies. You can have a look at our standard immunization protocols here, including SuperFast protocols, where a good titer is usually established after 28 days. Of course we can use your own protocols.
Improve the activity of your antibody fraction
Sometimes it is necessary to purify the antibody fraction for your special applications. You have the choice to test your antisera and decide afterwards if you want to purify them. Just contact us for further information and a schedule.
With the affinity purification the antigen specific antibodies are purified from a pool of polyclonal antibodies. This is highly recommended when you want to use the antibodies in immunohistochemistry or immunofluorescence applications or in any other assay where you need a low background.
First the antigen (protein, peptide, ...) is bound covalently to an affinity matrix. After this the antigen-specific antibodies bind the affinity matrix. The unbound antibodies are removed and the specific antibodies are eluted afterwards. Usually a small backup from the raw serum is not purified and sent to the customer with the purified fraction. Move your mouse over the picture to enlarge.
ProteinA and ProteinG are proteins from the cell wall of S. aureus. ProteinA binds the Fc part of the antibodies, especially from IgG. With the ProteinA or G purification all IgG's are purified from the antiserum. Most of the lipids, polysaccharides, IgE or IgM are removed from the fraction.
With a depletion you can remove antibodies from a fraction you do not want. For example antibodies that recognize a HIS/MBP/Strep-tag or antibodies that recognize E. coli proteins. The tag is bound to an affinity matrix and the anti-tag-antibodies bind to the affinity matrix. All unbound antibodies are used for further applications.
We keep the order process as simple as it can be. Use one of the following ways to state your order:
- Send us your antigen with an order form: Custom Antibody Order Form
- Contact us via email or phone to get your individual offer, suggestions and help
- Send us your protein sequence. We analyze the sequence and suggest you some peptide sequences, that are useful for antibody production. After a confirmation from you we start the synthesis and the immunizations.
Information that helps us to speed up the antibody production process are the following:
- Antigen concentration [mg/ml]
- Antigen amount [mg]
- Antigen name/sequence
- Did you (try to) produce the antibody in the past? Were there any problems?
- Have a look at our Antigen Section for more information