Antibody diversity in amphibians. Noninbred axolotls used the same unique heavy chain and a limited number of light chains for their anti‐2,4‐dinitrophenyl antibody responses

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Abstract
Noninbred axolotls (Ambystoma mexicanum, amphibia, urodela) were immunized with trinitrophenylated sheep red blood cells (TNP-SRBC) and anti-2,4-dinitrophenyl (DNP)/TNP antibodies were individually purified by affinity chromatography. The isolated IgM-like antibodies were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) under reducing conditions. The SDS-PAGE and IEF-separated heavy (H) and light (L) chains were electroblotted onto nitrocellulose, probed with mouse monoclonal antibodies specific for H or L axolotl Ig chains and stained by a rabbit anti-mouse Ig horseradish peroxidase conjugate. The specific detection of axolotl anti-DNP/TNP H chain spectrotypes shows for each of the 14 individually analyzed samples a very similar pattern of 4–5 ordered spaced bands. This suggests that all animals express the same VH chain segment representing the germinal expression of a unique VH gene. When the same analysis was performed starting from a pool of nonimmunized axolotl sera, a low background of natural anti-DNP antibodies was detected. When analyzed by IEF, the H chains of the pooled anti-DNP natural antibodies display the same pattern of restricted heterogeneity when compared to the H chain spectrotypes of the individual immune anti-DNP/TNP antibodies. The specific detection of the axolotl anti-DNP/TNP L chain spectrotypes indicates at the individual level more heterogeneous and polymorphic patterns compared with H chains, although most animals share the majority of their bands. Our experiments indicate that in axolotl, the production of antibodies to DNP results from the germinal expression of a very limited set of V genes, already expressed as naturally occurring anti-DNP antibodies before immunization. This seriously restricts the possible extension of the antibody repertoire and perhaps even the nature of antibody “specificity” in this primitive vertebrate.