Equine anti-hapten antibody-VIII. Isoelectric fractions of IgM and 7 S anti-lactose antibody

Abstract

Anti-lactose antibodies induced in a horse with a bacterial vaccine were specifically purified and separated into IgM adn 7 S populations. These were further fractionated by liquid preparative isoelectric focusing. The fractions thus obtained were characterized by their affinity for a monovalent lactose-containing hapten and the deviation from homogeneous binding. In the IgM population there was a major component which exhibited the maximum affinity observed in the IgM fractions and functional homogeneity with a K 0 value of 4 × 10 5 M −1. It was presumably a monoclonal product which persisted as the major IgM antibody during, at least, an 8-week period, from the 6th week to the 14th week after initial immunization. The emergence of this component is regarded as the result of an early maturation process. Another, non-homogeneous IgM fraction appeared to undergo maturation during the 8-week period but did not attain the affinity of the major component. The 7 S population gave rise to several major isoelectric components one of which was isolated in a functionally homogeneous form. All of the 7 S fractions exhibited a striking increase in affinity during the 8-week period and reached a common value of K 0 of 2 × 10 6 M −1. This result provides further evidence for the selective limitation of the affinity of IgM antibody. The binding results with functionally homogeneous IgM component also demonstrate the functional equivalence and independent reactivity of the 10 combining sites of the IgM molecule. Finally, the disparity between the maximum affinities for the IgM and 7 S antibodies raises a question about the notion of an IgM -7 S switch. However, the occurrence of such a transition can be made compatible with our results by introducing special features for this process.