To start immunisation programme please send us:
- your purified protein of interest
- OR your peptide sequence
- OR your protein's accession number
Phase I: Immunization ~ 6 weeks
- A minimum of four mice (6-8 weeks old) are used for immunization.
- Preimmune serum is taken on day 0.
- A total of four injections are administered (50 µg antigen per animal per injection).
The first injection on day 0 with equal parts (v/v) of complete Freund's adjuvant and antigen.
The following three boosts are carried out with equal parts (v/v) incomplete Freund's adjuvant and antigen on days 21 and 36 with an additional final boost prior to fusion.
- Selection of the best responding on the basis of Customer's test results.
If the antigen has a low immunogenicity, it can be coupled to an appropriate protein carrier.
Phase II: Fusion/Hybridoma production~ 2-3 weeks
The splenic lymphocytes of the best responding animal will be fused with the cell line Sp2/O-Ag-14 using PEG (polyethylene glycol), and the resulting hybridomas will be seeded in eight 96-well plates in HAT medium.
Duration: 2-3 weeks
Phase III: Screening for positive hybridomas ~ 4-5 weeks
- Screening for positive antibody producing hybridomas (ELISA against the target antigen)
- Positive colonies are expanded and tested for antibody production
On request, positive colonies can also be screened against other antigens for cross-reactivity or against a specific region of the antigen of interest
Different screening techniques are available and can be used on request. Please contact us for more information
- > 1 ml of positive supernatants (maximum 30) will be sent to customer for screening confirmation and additional tests.
Phase IV: Cloning and isotyping of positive hybridomas ~ 4-5 weeks
- The specific hybridomas selected by the customer at the end of the Phase III are cloned by serial dilution.
- Positive hybridomas are collected in vials
- Monoclonal antibody isotype is determined
- > 10 ml of supernatant from 1-2 positive clones and frozen hybridomas are delivered