In vitro immunization (IVI) possesses a number of advantages over conventional immunization. However, the number of positive clones derived from IVI is limited, and the affinity of the antibodies from derived clones is relatively low. Moreover, the majority of immunoglobulins produced in culture are IgMs instead of IgGs, which limits the application. Here, we report an improved protocol for IVI using mouse spleen cells. This protocol consists of multiple cycles of repeated antigen stimulation followed by cell expansion, which increases the frequency of plasma cells that produce antigen-specific IgG antibodies. The culture conditions, including the cell density, the type of stimulants, and the initial cell preparation, were found to be important for inducing the IgG response. In addition, an analysis of the genes and cytokines expressed during the IVI showed that the antigen-specific B cells were specifically activated via CD4-positive helper T cells. As evidence for this concept, our IVI protocol enabled us to establish an IgG antibody against keyhole limpet hemocyanin with a dissociation constant in the order of 10(-7)M.
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