Immunogenicity of biotinylated hapten-avidin complexes.

The efficacy of avidin as a carrier for the generation of anti-hapten antibodies was assessed in mice by immunization with complexes of avidin and synthetic peptides containing biotin and an epsilon-dinitrophenyl (DNP) lysine residue. The synthetic haptens were constructed with 0, 1 or 2 6-aminocaproyl groups as spacers between the biotin and DNP-lysine moieties. Complexes without a spacer did not induce anti-DNP antibody responses, while those with two spacers induced stronger responses than those with only one spacer. However, the anti-DNP responses to avidin-biotinylated hapten complexes were considerably weaker than responses to a conventional hapten-protein conjugate (DNP-ovalbumin), and, like "T-independent" antigens, failed to induce significant immunological memory. The distribution of isotypes in the anti-DNP antibodies produced to avidin-biotin-6-aminocaproyl-epsilon-DNP-lysine-alanine and DNP-ovalbumin was similar, but the former antigen induced significantly lower levels of antibody in (CBA/N X BALB/c) F1 male mice with the xid defect than in phenotypically normal female littermates, and also induced significant responses in nu/nu mice, in contrast to DNP-ovalbumin. These findings suggest that there is at least a "T-independent" or "T-efficient" component in the response to avidin-biotin complexes, perhaps due to the tetrameric structure of the molecule. Estimates of the depth of the receptor site for biotin were obtained by using the complexes to competitively inhibit the binding of anti-DNP antibody to plates coated with DNP-protein. The findings were consonant with the data on immunogenicity (capacity to induce anti-DNP antibody responses) and suggested that the receptor site has a depth of 16-26 A.