Immunizations are normally tailored to the specific immunogen to maximise formation of high affinity IgG. In general, we require 1-5 mg of antigen for immunizations (depending on the immunogen) and 1-3 mg for screening. Synthetic peptides will be conjugated to carrier proteins for both immunizations and screening.
Project strategies can be adjusted if the antigen is difficult to isolate or not readily available. We use standard protocols to immunize three Balb/c mice per project. Each animal typically receives 3-6 injections over 2-3 months.Usually, the animals are immunised every three weeks and the antibody titre tested after the third injection. The route of immunization used is usually a combination of sub-cutaneous and intra-peritoneal using an appropriate adjuvant. The final challenge, before fusion, is intravenous.
Sometimes, the protein supplied requires some processing before immunizations. For examples, immunoblots or SDS-PAGE samples will need to be homogenised before injection.
Co-injection of two peptides or more from the same or two different target proteins into one animal will improve the chances of success.
The sera from the immunised animals will be tested by ELISA using the same antigen used for immunization (synthetic peptides will be coupled to different carrier proteins). Sera can also be sent to the client for testing against native protein using methodologies normally employed in their laboratory. This is very useful if antibody are being produced against synthetic peptides since antibodies that react against the synthetic peptide, might not recognise the same sequence within the native structure of the protein. Also, antibodies might need to be screened for their usefulness in Western Blot tests or in immunostaining, as well as ELISA since they might react differently to the various type of tests. Once an acceptable titre is obtained, and after consultation with the client, hybridoma fusion will be performed using the spleen cells from the mouse with the best titre.
The remaining two animals will continue to be boosted so that can be used for a second and even a third fusion, if required.