FAQ POLYCLONAL ANTIBODIES

For anti-peptide projects a total amount of 10-15 mg is sufficient for immunisation and screening. Anti-protein projects depend on the animal involved in the protocol: little animals (rabbit, mouse, rat, guinea pig and chicken) require at least 400 µg of protein while bigger animals (goat and llama) require at least 1.2 mg of protein. These values include only the minimal material required for immunisation and screening. Larger quantities may be asked if additional injections are to be performed during immunisation, and if you wish to immunoprecipitate the final bleed as well. Please contact us if only smaller antigen quantities are available.

The protein can be supplied lyophilised, in solution, included in gel slices or attached to sepharose beads. For the immunisation of one rabbit we require 200 µg of antigen (4 injections). If the antigen is supplied in solution, the minimum protein concentration required is 100 ug/ml.

Our standard protocol using rabbit as host animal is 88 days long. Shorter protocols (53 or 67 days) are also available.

Our standard protocol includes four injections, three test bleeds including the pre-immune bleed and the final bleed.

We can assist you in designing your peptide. We use a combination of prediction software and in-house expertise to select the most relevant immunogenic peptide sequences. We select peptide sequences for immunisation based on a number of criteria:

• antigenicity, hydrophobicity and accessibility,
• sequence alignment (BLAST) to confirm peptide specificity,
• secondary structure analysis (signal peptide, transmembrane domains, DNA binding domain…),
• predictive study to determine potential difficulties with the synthesis.

Phosphorylation is one of the major post-translational modifications. Visit the section of our website dedicated to post-translational modification as immunogens for more specific information. Briefly, the phosphorylated peptide is first conjugated to a carrier protein and used to immunise the host animals. Following the final bleed phospho-specific antibodies will be purified by a 3-step affinity purification procedure:

• the immune serum is loaded onto a column with the control peptide coupled to agarose beads, thus retaining unmodified peptide-specific antibodies,
• the flow-through is then loaded onto a column with the modified peptide coupled to agarose beads, thus retaining the modified peptide-specific antibodies,
• after elution, the eluate is assayed by ELISA against both peptides to control its immunoreactivity and its specificity against the modification.

We offer a wide range of antibody labelling, such as biotin, fluorophores and enzymes. See our dedicated webpage about antibody labelling for more details.

Sodium azide is a preservative which prevents bacterial growth. We recommend adding sodium azide to the serum for long-term storage. Please note that sodium azide interferes with some biological assays. However, it can be easily removed by dialysis or buffer exchange.

For rabbit projects you will receive 4 to 5 ml for the first test bleed, 10 to 15 ml for intermediate test bleeds and 50 to 70 ml for the final bleed per animal.

During collection and processing of the sera, haemolysis of red blood cells may occur which leads to the release of haemoglobin. The immunoreactivity of the serum will not be affected.

For short-term storage (up to one month) the serum can be kept at 4°C. For long-term storage we recommend aliquoting the serum in small vials and storing the aliquots at -20 °C.

The results of the ELISA tests are reported on a graph. The antibody titre is defined as the reciprocal dilution giving an optical density (OD) equal or greater than 1 at 450 nm. When the titre is greater than 8000, the immunoreactivity of the sample is considered good. The pre-immune serum of the animal represents the negative control.

The titre of an antibody sample is a measure of the antibody concentration determined under a defined set of conditions. Serial dilutions of the antibody sample (test bleed or purified antibody) are allowed to react with a known amount of antigen. The antibody titre is defined as the lowest dilution to bind significantly to the antigen and is expressed as the reciprocal dilution giving an optical density (OD) equal or greater than 1 at 450 nm.

ELISA titration is a simple method to assess whether an immune response to a specific antigen has occurred in the immunised animals. We can perform ELISA on all test bleeds (including the pre-immune bleed) for you.

Due to the unpredictable nature of the immune response in host animals, we cannot guarantee the efficiency of your antibody in your specific applications. We follow the project and help customers throughout the protocol and do all that is possible to induce a significant immune response.

If the titre is below 500 at the end of the standard protocol a project extension and further boosts are unlikely to increase the antibody titre.

If the immune response against your antigen is starting to increase at the end of the protocol, we recommend extending the project for another boost. If you require a greater volume of immune serum, maintaining the project is also a good option when the titre is high.

A project can be extended to up to one year. This is of interest if the titre is satisfactory and large quantities of serum are required.