Bivalent and multivalent DNP haptens differing in molecular size, degree of substitution, and rigidity were compared for their effectiveness in eliciting Prausnitz-Küstner (P-K) reactions in either newborn colostrum-deprived calves or in those 4 wk of age. The sensitization with reaginic anti-DNP antibody has been accomplished either by feeding colostrum of the immunized dam or by intradermal injection of reaginic serum or colostral whey. It could be demonstrated that equimolar doses of the bivalent ,N-(,N-DNP-aminocaproyl-)-,N-DNP-L-lysine and the multivalent dinitrophenylated bovine serum albumin were equally effective in eliciting reactions in skin sites provided that a high affinity antibody was used for sensitization. By contrast, the comparatively rigid, bivalent hapten, (DNP)₂-Gram consistently failed to induce comparable reactions. Furthermore, it was clearly shown that optimal distances of determinant groups on the haptenic molecule are a prerequisite for positive P-K reactions, since ,,N-bis-DNP-lysine failed to induce comparable reactions.

Concurrent sensitization of skin sites with reaginic anti-DNP and anti-ABA antibodies provides the final proof that cross-linking of two adjacent reaginic molecules on the mast cell surface by a bivalent hapten is required for effective elicitation of immediate-type reactions. This has been accomplished by utilizing the bivalent ,N-DNP-,N-[(4-hydroxy-3-azobenzenearsonic acid)-phenacetyl]-L-lysine (DNP-ABA) carrying noncross-reactive haptenic groups, which was consistently effective in eliciting P-K reactions in doubly but never in singly sensitized skin sites. It is apparent from the results that equimolar doses of monovalent haptens could completely inhibit the response to DNP-ABA. The present studies finally establish that mast cells of newborn colostrum-deprived calves lack IgE molecules on their surface. Thus, mast cells of newborn calves may be unique, to investigate the molecular mechanisms involved in immediate-type reactions more precisely.