0	Replied by:  New User     Source:  Antagene, Inc

In our company, the injection amount of antigen is 25-50 microgram with smallest volume possible for each immunization per mouse based on the KLH concentration. For rabbit, the injection amount is 0.5 mg/0.5 ml multiple sites / per rabbit. Usually, we used to do 4 times of immunization for each project, test the bleed after the third immunization, and this schedule works well in most projects. We don't care about the allergic reactions in our industry.

We test the immune response of animal to the antigen after the third injection, firstly by ELISA and then by Western Blot or IP. Unlike your protocols, we do the ELISA with different carrier protein conjugated peptide from the immunogen. For example, if the animals are immunized by KLH conjugations, the BSA conjugations should be used for the ELISA. Usually, the ELISA data indicate the immune response of the animal to the peptide, Western Blots and IP will tell us if the antiserum recognizes the native protein. The weakness of the ELISA with same conjugated peptide is that this kind of data only indicates the general response to the whole thing of the antigen, but not the specific peptide and the native protein.

We used to coat 1ug of the conjugated peptide per well for the ELISA test,
and the range is 1-100ug per well in the industry. However, the range will
be 0.1-1ug in most academic institutes.