Phase I: Immunization
- A minimum of four mice (6-8 weeks old) is 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 42 with an additional final boost prior to fusion.
- Selection of the best responding mouse 10 days after the last injection using the client's ELISA test.
If the antibody titer is too low after three booster injections, you may request additional injections. If the ELISA report remains negative, the immunization should be terminated because the probability of success is too low.
If the antigen has a low immunogenicity, it can be coupled to an appropriate protein carrier.
Duration of this standard program: 11 weeks.
The above description is the Eurogentec standard protocol. However, a personalized procedure may be designed and adapted to your needs.
Phase II: Fusion
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. If any fusion cocktail remains, it will be stored in liquid nitrogen.
Duration: 2-3 weeks
Phase III: Screening and amplification of hybridomas
- IgG producing hybridomas will be screened against the target antigen and two vials of every positive hybridoma stored in liquid nitrogen
- The positive colonies are expanded and tested again for antibody production with the antigen used for immunization
- At this point, positive colonies can also be screened against additional antigen for cross-reactivity or against a region of the antigen of interest
- Other screening tests can be used such as Western blots or immunofluorescence. Please contact us for a price offer
- In addition, 5-10 ml supernatant of the 20 most positive hybridomas will be sent to you for screening confirmation and additional tests. This will ensure that the positive ELISA results identify antibodies useful for your purpose
Duration: 5-7 weeks
Phase IV: Cloning and isotyping
- The specific hybridomas you have selected at the end of Phase III will be cloned by limit dilution. The assay can be carried out as in Phase III (screening for the antigen used for immunization and additional screening of positive clones with additional antigens). After cloning, three vials of every cloned hybridoma are stored in liquid nitrogen. We send you 15-20 ml of every hybridoma supernatant together with the frozen hybridomas.
- Subsequently, the subclass of every monoclonal antibody will be determined by ELISA testing.
Duration: 4 weeks