Immunization protocols for generating activated B-lymphocytes suitable for hybridoma production vary widely. The optimal amount of antigen given, the way of antigen presentation, and the timing between injections must be determined for each antigen. Often, the antigen of interest is not available in a pure form, and the influence of contaminating antigens in the preparation should be considered when the immunization schedule is designed. Generally, a lower immunization dose will give rise to antibodies with higher specificity as compared to the total antibody response obtained after immunization with higher doses. Furthermore, antibodies obtained after one or two injections often show lower affinity than those obtained when a prolonged immunization schedule is used. The number of antibody-producing hybridomas obtained after fusing B-lymphocytes with myeloma cells depends on how well the mouse was immunized, and it is therefore worthwhile to put some effort into optimizing the immunization schedule.