L-Tyrosine-p-azobenzene-p-arsonate (RAT) is immunogenic and serves as a carrier for anti-hapten antibody responses in guinea pigs, rats, and mice. However, the murine anti-N-2,4-dinitrophenyl (DNP) plaque-forming cell (PFC) response to the bifunctional antigen 2,4-dinitrophenyl-6-amino-caproyl-L- tyrosine-p-azobenzene-p-arsonate (DNP-SAC-RAT; or BI-1) is extremely weak (2,000-4,000 PFC/spleen) and exclusively IgM in both primary and secondary responses. The 6-amino-caproyl group serves as a spacer in this antigen between the DNP haptenic and RAT carrier epitopes.

In view of recent evidence indicating that different T helper cells synergize for optimal antibody responses, a trifunctional antigen, N-2,4- dinitrophenyl-6-amino-caproyl-L-tyrosine-p-azobenze-p-arsonate-(proline)9-L- tyrosine-p-azobenzene-p-arsonate (DNP-SAC-RAT-PRO(9)-RAT; or TRI), was prepared to investigate the effect of adding a second RAT epitope to BI-1. The nonaproline spacer between the two RAT epitopes in TRI is assumed to be a rigid rod of approximately 28 A. TRI induced about twice as many PFC as BI-1 in primary responses of A/J mice, and induced both IgM and IgG PFC in secondary responses. Furthermore, TRI induced IgG PFC responses in mice primed with p-azobenzene-p-arsonate-keyhole limpet hemocyanin, BI-1, or RAT, whereas boosting with BI-1 failed to induce IgG PFC, even in mice primed with TRI. These findings indicate that the minimum antigen structural requirements for inducing IgG PFC in mice are two carrier epitopes and one haptenic epitope. In addition, priming with a mono-epitope carrier (RAT) is sufficient preparation for IgG responses to a trifunctional immunogen.

Because TRI differs from BI-1 by the (proline)(9) spacer as well as the additional RAT epitope, two other compounds, N-2,4-dinitrophenyl-6-amino- caproyl-(proline)(9)-L-tyrosine-p-azobenzene-p-arsonate (DNP-SAC-PRO(9)-RAT; or BI-2) and N-2,4-dinitrophenyl-6-amino-caproyl-(proline)(9)-L-tyrosine-p- azobenzene-arsonate (DNP-SAC-RAT-PRO(10); or BI-3), were prepared to evaluate the possible role of the spacer in the observed responses. BI-2, but not BI-3, induced IgG as well as IgM PFC in TRI-primed mice. However, BI-2 failed to induce IgG responses in RAT-primed mice, indicating that TRI and BI-2 were not equivalent immunogens. Because anti-prolyl antibodies had been found in guinea pigs immunized with N-2,4-dinitrophenyl-(proline)10-L-tyrosine-p- azobenzene-p-arsonate (DNP-PRO(10)-RAT), it seemed possible that priming with TRI might induce anti-prolyl antibodies, which, in turn, could cross-link BI-2 molecules into aggregates containing at least two carrier epitopes. To help resolve this question, mice were immunized with acetyl-(proline)10-L- tyrosine-p-azobenzene-p-arsonate and boosted with BI-2. IgG PFC responses were detected, suggesting that anti-prolyl antibodies were indeed responsible, because priming with RAT and boosting with BI-2 did not induce IgG formation.

Accordingly, the observations that IgG responses in RAT-primed mice were induced only by TRI and not by any of the bifunctional antigens indicate that two carrier epitopes per antigen molecule are indeed required for IgG induction. They also provide indirect evidence for synergistic help in the switching of immunoglobulin isotypes.
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