Equine anti-hapten antibody—VII. Anti-lactoside antibody induced by a bacterial vaccine

Abstract

Immunization of a horse with a bacterial vaccine of Streptococcus faecalis, strain N led to the sustained and concomitant production of IgM and 7 S antibody with anti-lactoside specificity. Restriction of heterogeneity was apparent in both mol. wt groups with the IgM population exhibiting three major components when analyzed by isoelectric focusing. Equilibrium dialysis studies of the specific binding of a lactoside hapten, p( p-dimenthylaminophenylazo)-phenyl- β-lactoside (Lac dye), were carried out with three preparations of purified IgM antibody. The intrinsic association constants (K 0) ranged between 1 × 10 5 M −1 and 2 × 10 5 M −1 for bleedings taken from 6 weeks to 14 weeks after the initiation of immunization. The availability of 10 binding sites per IgM molecule was clearly evident with no indication of intramolecular heterogeneity although substantial deviation from homogeneous binding was observed. Conversion of the pentameric IgM molecule to the monomeric form resulted in identical binding behavior. The 7 S antibody fraction gave a maximum value of K 0 of 8 × 10 5 M −1. The binding of lactose by IgM yielded a value of K 0 which was 24-fold smaller than that for the Lac dye. Thus, the nonspecific contribution of the aglycoside, arising presumably from its hydrophobic character, to the free energy of complex formation was - 1·8 kcal/mole of hapten. The temperature dependence of the IgM binding of the Lac dye demonstrated that the enthalpy contribution was primarily responsible for the affinity. From this result it was inferred that the specific binding of lactose involves multiple hydrogen bonds, perhaps as many as 10.