Equilibrium measurements of interactions of anti-DNP antibodies, prepared using DNP-PLL and several DNP-proteins for immunization, with DNP0.6-PLL240 and with the univalent hapten, ϵ-DNP-L-lysine, were made utilizing the technique of fluorescence quenching. Carrier specificity of anti-DNP-PLL antibodies was demonstrated by a higher average intrinsic association constant (K0) of anti-DNP-PLL antibodies with DNP0.6-PLL240 than with ϵ-DNP-L-lysine. The free energy contribution of the PLL carrier to the interaction of intact anti-DNP-PLL antibodies with DNP0.6-PLL240 was from –0.8 to –2.1 kcal/mole. On the other hand, intact anti-DNP-protein antibodies displayed a lower energy of interaction with DNP0.6-PLL240 than with ϵ-DNP-L-lysine of up to +2.4 kcal/mole.
Fab' fragments of both anti-DNP-PLL and anti-DNP-BGG antibodies have K0's with ϵ-DNP-L-lysine identical to the K0's of the intact anti-DNP antibodies from which they were prepared. However, K0 of interaction of Fab' fragments with DNP0.6-PLL240 (a large proportion of the conjugated PLL molecules in this preparation bear more than one DNP group) is considerably lower than that of the intact antibody. Thus a cooperative effect in the binding of bivalent antibody and bivalent (or greater) antigen exists and is of the order of –1.2 to –2.0 kcal/mole of IgG antibody.
Although the direct contribution of the carrier to the interaction of Fab' fragment of anti-DNP-PLL and DNP0.6-PLL240 is –0.4 kcal/mole, the energy of carrier specificity, based upon consideration of cooperative effects and of repulsion of anti-DNP-protein antibodies for portions of the DNP-PLL determinants, is of the order of –3 kcal/mole (approximately 30% of total binding energy).