Quantitative Relationships in the Phage-Antiphage Reaction: Unity and Homogeneity of the Reactants
Authors: Hershey A. D. , Kalmanson G. , Bronfenbrenner J. 
Published: January 2023

The applications of methods described in the previous paper reveal small but well-defined differences between the reactivity with coliphage of homologous antisera of different rabbits. These differences show themselves as independent variations in the specific rate of neutralization, the neutralizing capacity, and the precipitating activity, each referred to the same measure of antibody-content. It does not seem necessary to ascribe these differences to variations in the specific immune response, since far more noticeable changes of the same kind may occur during storage of individual sera.

By contrast, examination of partially absorbed antisera by the same methods discloses no separation into fractions of differing reactivity. With respect to the properties mentioned, individual antisera behave like homogeneous solutions. This finding is of particular interest with respect to rate of combination of antibody, which might be expected to reflect to a marked degree qualitative differences of any kind.

Crude phage-concentrates contain non-antigenic material rich in nitrogen and phosphorus to which phage is reversibly adsorbed. This material is largely eliminated during purification by ultrafiltration. Since the specific absorbing power per lytic unit of crude and purified preparations is the same, it must be concluded that the lytic unit in dilute solutions is a reproducible entity.

The purified preparations of Kalmanson and Bronfenbrenner contain considerably more protein than is compatible with any reasonable assumption concerning the average particle-weight of the lytic unit. Since the same is true of the specifically precipitable phage-antigen, the tentative conclusion is drawn that the lytic unit is only one of about 30 similar particles of antigenic substance, assumed from diffusion-measurements to have a particle-weight of 1,500,000.

These conclusions are considerably substantiated by the observed combining proportions of phage and antiphage. Thus a single particle of antigen proves on this basis to combine with about 170 molecules of antibody with the latter in excess, to be neutralized by about three, and to be precipitated by about two. Any appreciable departure from the interpretation given the physical data leads to consequences incompatible with the immunologic data. The latter indicate that the isolated material is reasonably pure, and since it contains only traces of phosphorus, the phage does not consist of nucleoprotein.

All the known facts may be reconciled with the assumption that the phage consists of small particles (molecular weight about 25,000) of which some 29 are attached to a single molecule of carrier-protein. The data are not compatible, however, with the larger size indicated by electron-micrographs of this phage. Sedimentation-experiments are described which probably indicate the true nature of the visible material.

Allowing reasonable scope for error, the absolute rate of the phage-antiphage reaction at 0 C may be expressed as 2 to 10 molecules of antibody per second per phage in a “standard” antiserum 10−5 molar with respect to antibody. This corresponds to a bimolecular reaction-constant of 2 to 10 × 105 mol−1 sec−1 liters. Among four antisera, this is the highest specific rate observed, the range of variation being 4-fold.