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Association Constant and Heterogeneity Index of Antibodies Specific for a Natural Determinant of a Bacterial Protein

Time-Course Studies during the in Vitro Immune Response to a Distinct Site of Escherichia Coli -D-Galactosidase¹

From the International Agency for Research on Cancer, Biological Carcinogenesis Unit, 150, cours Albert Thomas, 69008 Lyon, France

Abstract

The evolution of the immune response to a bacterial antigen was studied in a closed in vitro system, whereby the physiologic architecture of the responding tissue is preserved and communications with central organs (thymus, bone marrow), circulating cell and antibody pools, and antigen depots are cut off. The time-course changes in binding properties of antibodies directed to a distinct determinant of native, wild type, Escherichia coli -d-galactosidase were determined by measuring their association constant for a naturally occurring ligand, a defective point mutant enzyme (AMEF), at various intervals during the response of rabbit lymph node fragments, which had been challenged in vitro, washed, and placed in culture, individually or in groups of five. Early secondary antibodies were distributed over a wide range of affinities, although antibodies in the serum of the donor rabbit were restricted at the moment of sacrifice and initiation of cultures. Later on, a gradual increase in affinity with progressive restriction occurred more often in multifragment cultures and in cultures challenged with 50 µg/ml than in those challenged with 5 µg/ml of -d-galactosidase. The magnitude of the affinity rise was inversely correlated with the association constant of early antibodies and the peak affinity reached in each culture did not go over the highest value recorded in the serum of the donor, with only few exceptions. This indicates that although there is a strong tendency to maturation, there is a certain ceiling which is rarely surpassed. In some cultures a late fall in K₀ was observed together with increased antibody heterogeneity. The overall data indicate that during microevolution of the immune response in vitro against a natural determinant in its native molecular configuration, a progressive contraction of high-rate antibody-forming clones occurs toward high affinity. Expansion of the memory potential over a wide range of affinities takes place in parallel. The limits of these processes, top affinity reached, and width of memory spectrum seem to be mainly determined by a prefixed array of precursors or memory cells. However, generation of new affinities during the immune response also seems to be possible.

Footnotes

¹ Part of this work was done at the Laboratory of Cell Biology, Rome, and supported by the Consiglio Nazionale delle Ricerche, Italy.