Antibody Protocols and Information
Antibody handling protocols that work effectively. Take advantage of our extensive knowledge in antibody creation, manufacturing, and handling. Our scientists created the hand-picked methods and antibody information. Please feel free to print the procedures and distribute or collect them among your colleagues.
Learn more how to store antibodies properly.
Take advantage of Davids protocols and methods.
General information on antibodies and Davids.
Antibodies by Davids
Information on buffers and preservatives Davids uses for their antibody fractions.
|Antibody Storage||Storage Protocol|
Application of Antibodies and Protocols
|ELISA Titer Determination||ELISA Protocol|
|ELISA with HRP||HRP ELISA Protocol|
|IHC with frozen tissue||IHC Frozen Protocol|
|Production of a single cell suspension from spleen or lymph nodes||Cell Suspension Protocol|
|Protein Determination with Bradford||Bradford Protocol|
|Production of Chemically Competent Cells and Transformation of E. coli||Competent Cells Protocol|
|Protein Expression in E. coli||Protein Expression Protocol|
|PCR (Polymerase Chain Reaction)||PCR Protocol|
|Davids Cell Membrane Conservation KIT for Immunization||K105 Manual|
|Monoclonal Antibody Development||Monoclonal Information|
|Tagged Antigens||Tagged Antigen Information|
Antibodies by Davids: Buffer
Please find attached the standard buffers and supplements for our antibody fractions. If you have special requirements please ask before the immunization start.
|Affinity Purified Antisera||0.1 M Phosphate Buffer|
10 mM Na-Acetate
|ProteinA Purified Antisera||0.1 M Na-Acetate||0.02% Na-Azide|
|PrepI Chicken Egg Yolk||1 x PBS||0.02% Na-Azide|
|PrepII Chicken Egg Yolk||1 x PBS||0.02% Na-Azide|
Antibodies by Davids
The quantity of antibodies produced by antigen specific affinity purification varies. One rabbit yields approximately 40-90 ml Antiserum with around 0.5 - 10 mg specific antibodies depending on the antigen, titer, and antiserum volume. It takes around 10 eggs to get the same amount of antibodies when chicken is used for antibody production. You will receive a small backup from the raw antiserum before purification. When ordering antibodies, please let us know if you do not require a backup. In this situation, the entire antiserum is purified.
You will receive a preimmune serum, that is taken before the first immunization together with the final antibody fraction. Most of our immunization schedules include a so called test bleed (usually at day 35 after the third immunization). With this bleed we determine the antibody development and may adapt the immunization protocol if necessary. You can receive an aliquot of this test bleed (0.5 - 1 ml for rabbits). We only charge the shipping costs in this case.
The final antiserum volume is between 40 and 90 ml from one rabbits. After the final bleed you cannot receive additional antibodies. Please let us know at the start of the project when you need more antibodies and we can adapt the immunization protocol. The main advantage of antibodes from chicken egg yolk is, that you can receive more antibodies after the first batch. Chicken will produce the same amount of antibodies compared to the immunization of one rabbit every 14 days. Within several weeks or month you are able to receive a very large quantity of antibodies from the same animal.
B-Cells and Antibody Production in living bodies
B-Lymphocytes can produce antibodies. The name B-Lymphocyte comes from their origin: Bursa fabricii in birds, respectively bone marrow in mammals.
To induce the immune response an antigen is needed. The antigen (i. e. bacteria or virus particles) is recognized as a foreign body from macrophages. These macrophages digest the foreign bodies and dissolve them. Pieces of the dissolved antigen (epitopes) are presented on the surface of the macrophage. T-Helper Cells recognize the Epitopes and are activated. Simultaneously B-Cells comes in contact with the antigen and present it on their surface. The activated T-Helper-Cells produce interleukines, when they recognize teh antigen, that is represented from the B-cells. This leads to the "clonal expansion" of the B-Cells (Reproduction) and to the specialization of the B-cells: An antibody producing plasma cell.
Antigens (ANTIbody GENerating) are molecules, that are recognized from lymphocytes and antibodies. It is the molecul, the antibodies are produced against. The piece of the antigen, where the antibodies bind is called epitope. One antigen can have multiple epitopes. Lymphocytes can recognize nearly any antigen. This is secured due to the somatical recombinantion of the lymphocytes. The antibody coding genes are recombinated and a huge amount of different antibodies are generated to detect nearly any antigen.
Antigens can for example be peptides, proteins, virus particles, cells, bacteria, DNA or small molecules.
The titer determination is an indirect method to determine the concentration of the antigen-specific antibodies in blood. The antigen is covalently bound to wells of a microtiterplate. The antiserum is pipettet in the microtiterplate and diluted in multiple steps (i. e. 1:5, 1:25, 1:125, ... 1:300000). The highest dilution, where you can see a specific antibody reaction is called the titer. Good titers begin at 1:16000.
Similar to a vaccination the titer established against an antigen, when the animals are immunized multiple times with the antigen. Antibodies against this foreign molecule are generated from the animal.
Polyclonal and Monoclonal
Every healthy animal has antibodies against multiple antigens. For this reason the antiserum contains antigen-specific and unspecific antibodies. The antiserum can be purified by antigen-specific affinity purification to receive only the antigen specififc antibodies. As an antigen has multiple epitopes, there are multiple antibodies that recognize the antigen. For this reason you call them polyclonal antibodies as they are derived from multiple cells.
In contrast to polyclonal antibodies, the monoclonal antibodies come from one B-cell. For this reason one fraction of monoclonal antibodies contains only one antibody against one epitope.
Application of Antibodies
Antibodies are used in science to detect molecules or proteins. Applications like WesternBlot, Fluorescent Activated Cell Sorting (FACS), ELISA (enzyme-linked immunoabsorbent assay), ChIP or other assays need antibodies. With Immuno-Histo-Chemistry (IHC), it is possible to detect localize in cells or tissues. With this method cancer cells can be identified or localized.
In diagnostics antibodies are used to detect substances, cells or nutritions in blood. With this method lipids, drugs, virus particles, hormones or other antibodies can be detected. This is usually done with ELISA, Agglutination or Immunoprecipitation.
Antibodies can be used in therapy or as a vaccine as well. Antibodies can be used to fight cancer, Auto-Immune Diseases and AIDS.
When you want to produce antibodies against a protein where you only know the sequence from, we can synthesize peptides that represent your protein. Our scientists help you to find peptides with our prepdiction program. It calculates the antigenicity, solubility and epiope propability of the peptides.
As the 3D-structure and protein modifications like glycosilation or lipids on the surface plays an important role in antibody recognition, we recommend to use 2 or even 3 peptides from one antigen and immunize with a mixture of the peptides. With this method the chances to receive the best antibody for your application raise. Please visit our peptide antibodies for further information.