Humoral immune response to 2,4-dinitrophenyl -- keyhole limpet hemocyanin in antigen-free, germ-free and conventional BALB/c mice

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Abstract
The B cell immune response to 2,4‐dinitrophenyl (DNP) keyhole limpet hemocyanin was compared in antigen‐free, germ‐free and conventional BALB/c mice. The numbers of total and of DNP‐specific IgM‐, IgG‐ and IgA‐secreting cells in the spleen were determined by enzyme‐linked immunosorbent plaque assays after primary, secondary and hyperimmunization. Three days after primary immunization a peak of DNP‐specific IgM‐secreting cells was seen in conventional mice only. However, this specific response was accompanied by a rise in the total number of IgM‐secreting cells. At day 6 after primary immunization the total number and the frequency of DNP‐specific IgG‐secreting cells were higher in antigen‐free mice, compared to germ‐free and conventional mice. After secondary immunization in conventional mice only, a considerable bystander IgG response was seen together with the DNP‐specific IgG response. At the end of the secondary response 90% of all IgG‐secreting cells were DNP specific in antigen‐free mice, while the corresponding figure in germ‐free and conventional mice was 63% and 14%, respectively. After hyperimmunization, the absolute number of DNP‐specific IgG‐secreting cells in the spleen was 5‐fold and 11‐fold higher in antigen‐free mice then in germ‐free and conventional mice, respectively. Approximately 48% of all IgG‐secreting cells were DNP specific in antigen‐free mice, while the corresponding figure in germ‐free and conventional mice was 17% and 12%, respectively. The results show that the exogenous antigenic load of animals influences the immune response to newly introduced antigens. The higher absolute and relative numbers of antigen‐specific IgG‐secreting cells after hyperimmunization in antigen‐free mice compared to germ‐free and conventional mice may provide a better source for antigen‐specific B cells that eventually can be used for hybridoma production.