FREQUENTLY ASKED QUESTIONS
Various purification options offering different levels of purity are available and we would be happy to discuss these on a project-by-project basis with antigen affinity purification being the gold standard for polyclonal antisera.
The concentration of purified antibody available at the end of the project will depend upon many factors, including the method of purification chosen. If a specific amount of purified antibody is required, this should be indicated at the commencement of the project to ensure that this is taken into account at the project design stage. Typically, working concentrations need to be determined empirically.
Custom antibodies typically should be considered to have a shelf life of at least 1 year. However aliquots may be frozen at -20°C or below to increase the shelf life. Preservatives may be added, as required, in order to prevent microbial contamination.
Purification of IgG is typically carried out by Protein A or Protein G affinity purification. However, APS offers a range of purification methods for monoclonal and polyclonal antibodies. The purification methods used are dependent upon the isotype of the antibody for culture.
Other purification methods available include:
– Ammonium Sulphate Precipitation
– Dialysis & Ultrafiltration
– Ion Exchange Chromatography
– Size exclusion chromatography
- SDS-PAGE analysis (H and L chain composition)
- Antibody Fragmentation for generation of fragments including Fab, F(ab’)2 and Fc
- Preparation of affinity matrices using customer-specific immobilised peptide and protein ligands
- Metal chelate affinity chromatography
- Endotoxin testing
- Mycoplasma testing
- Labelling – HRP, Biotinylation, fluorochromes, etc ELISA analysis of serum bleeds
- Immune cell/spleen preparations for mAb production
Antibodies are relatively fragile so repeated freeze/thaw cycles should be avoided. If an antimicrobial agent such as sodium azide is added, product may be stored at +4°C for up to 2 weeks. For long term storage, it is recommended to aliquot the antibody and store at -20°C.
For FITC labelled rabbit clonal antibodies the storage conditions are different. We recommend storing undiluted at +4°C. Do not freeze. Protect from prolonged exposure to light.
Antibodies are supplied as frozen material and will be shipped at -20°C.
For additional materials supplied as part of a custom antibody product service, shipping conditions are
- Test bleeds will be supplied as serum and shipped frozen on dry ice.
- Antisera will be supplied as serum and shipped frozen on dry ice.
- Custom cell lines will be shipped frozen on dry ice..
- Custom peptides are supplied as lyophilised material and shipped at ambient temperature,
usually with a desiccant.
A typical polyclonal project protocol is a 77 day protocol. However, protocols may be adapted to suit the antigen, the antigenic response to the antigen and titre of antibody required.
A project can take from 3 to 9 months depending on the immunisation schedule and overall projectcomplexity. All projects are individually designed to offer the best possible outcome within theconstraints of budget.
Clonal antibodies are monospecific such as monoclonals. The principal difference between these two types of antibodies is that the DB Biotech clonal antibodies recognize solely very specifically selected linear epitope on the antigen molecule after its detailed proteomic analysis. In contrast, the
monoclonal antibodies recognize very often steric epitopes that frequently change their conformation during tissue preparation, protein extraction, etc., making the corresponding monoclonal antibody unspecific, less avid and in extreme cases, non-functional.
Outside of strictly specific linear epitiopes characterisation, clonal antibodies are mature immunoglobulins (IgG), with the complete post translational modification of IgG molecule, mainly glycosylation, which plays an important role in stability of immunoglobulins. There are many reports in the literature describing the incomplete glycosylation patterns of monoclonal antibodies produced from
hybridoma cell lines, which has an important influence on stability, affinity and avidity of antigen/antibody interactions.
When designing the clonal antibody for IHC-P, the severe and irreversible denaturation of the antigen including the conformation of the corresponding epitope against which the clonal antibody is produced is taken into consideration. In most cases, the epitope for this application will be selected on different conformational criteria than for the western blot or ELISA application, where the original structure of the antigen is preserved or where the denaturation is considerably weaker. This is the reason why for most cases, different epitopes are designed for various applications and consequently different clonal antibodies are produced characterizing various epitopal sequences of the target protein molecule.
Clonal antibodies are exceptional because they recognize their target (epitope) with the highest affinity, avidity and specificity that remains unchanged even after the important changes in conformation of the antigen – such as denaturation during the fixation of the tissue, protein transfer in the case of western blot-related procedures, or as a consequence of the protein-protein interactions.
No. Immunoaffinity purified polyclonal antibodies (whether the immunogen is the whole protein or selected peptide) are always represented by numerous fractions of immunoglobulins corresponding to all epitopes in the immunogenic sequence. Rabbit clonal antibody is a homogenous fraction of
immunoglobulin (IgG) corresponding exclusively to one and only specifically defined epitope on the antigen molecule.