Protocols for antibody handling that really work. Profit from our long experience in antibody development, production and handling. The hand picked protocols and information about antibodies was made by our scientists. Feel free to print the protocols and share or collect them with your colleagues.
|ELISA Titer Determination||Protocol|
|Production of Chemically Competent Cells and Transformation of E. coli||Protocol|
|Monoclonal Antibody Development||Information|
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|
The amount of antibodies, that are generated with the antigen specific affinity purification varies. Depending on the antigen, the titer and the antiserum amount, one rabbit (40-90 ml Antiserum) can produce approximately 0.5 - 10 mg specific antibodies. To receive the same amount of antibodies in chicken, approximately 10 eggs are needed. For each rabbit affinity purification you receive a small aliquot of the raw antiserum as BackUp. When you do not need a BackUp please let us know when you order the antibodies. In this case we purify the whole antiserum.
Sie erhalten ein Preimmunserum mit den finalen Antikörpern. Bei vielen Immunisierungsprotokollen ist die Entnahme eines sogenannten Testserums (in der Regel am Tag 35) enthalten. Mit diesem Testserum überprüfen wir die Antikörperentwicklung und passen das Immunisierungsprotokoll gegebenenfalls an. Sie können ein Aliquot des Testserums (0,5 - 1 ml) erhalten. Sie zahlen nur die Versandkosten.
Die finale Antiserummenge beträgt bei Kaninchen zwischen 40 und 90 ml. Nach der finalen Blutung können keine weiteren Antikörper gewonnen werden. Wenn Sie mehr Antikörper benötigen können Sie große Testseren erhalten. Der Vorteil von Antikörpern aus Hühnereigelb besteht darin, dass Sie auch nach der ersten Lieferung weitere Antikörper erhalten können. Hühner produzieren dann ca. alle 14 Tage die Antikörpermenge, die Sie von einem Kaninchen erhalten. Über mehrere Wochen oder Monate erhalten Sie so eine große Anzahl an Antikörpern.
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.
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.
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. More information to our peptide antibodies are here.