The E2 transcriptional activator of the human papillomavirus regulates the expression of most viral transcripts. Its binding to specific target DNA sequences involves large conformational changes in the interacting macromolecules. The high stability of the E2:DNA complex prompted us to analyze the role of macromolecular interactions and adjuvant emulsions in the appearance of conformation‐specific antibodies. We demonstrate that immunization with free or DNA‐complexed E2 emulsified in an oil‐in‐water adjuvant elicits a humoral response shifted to the recognition of discontinuous epitopes. Epitope mapping and functional analysis of the generated anti‐E2 mAbs reveals that two separate antibodies populations can be obtained: those able to form a stable ternary complex with protein and DNA, and those which recognize the DNA‐binding surface of the transcription factor, interfering with E2 binding to DNA.