SINGLE CELL STUDIES ON 19S ANTIBODY PRODUCTION

Abstract

Rats were immunized with Salmonella adelaide flagella. By zone centrifugation of serum samples in sucrose gradients, it was shown that, as in many other systems of antibody formation, the first response was the formation of 19S, mercaptoethanol (ME)-sensitive antibody. This was quickly replaced by 7S, ME-insensitive antibody. Popliteal lymph node cell suspensions were prepared, and cells with antibody on their surface were identified by the method of bacterial adherence. By micromanipulation such cells were washed, placed into microdroplets, examined under high-power phase contrast and broken to release intracellular antibody. These droplets were then studied in either of two ways. In the first method, each droplet was halved and one half treated with ME. Then both halves were titrated for immobilizing antibody through serial twofold dilution of the half microdroplets. Droplets showing destruction of antibody by ME were classified as 19S; those showing no reduction in titer as 7S; and those showing significant (>1 log2) reduction as double producers; i.e., cells containing both 7S and 19S antibodies. In the second method, droplets were divided into 4 equal quarters, for testing after treatment with either ME, or a specific rabbit anti-rat 7S globulin serum, or both. In these experiments, cells showing some remaining antibody after treatment with either reagent, but not after treatment with both reagents, were classified as double producers. Of 144 cells tested, 123 contained readily detectable amounts of antibody. These comprised 42 19S cells, 64 7S cells, and 17 double producers. The double producers were frequent at times when the switchover from 19S to 7S antibody production was occurring. All except 4 of the cells in the study could clearly be identified as members of the plasma cell series. Though 7S cells became more frequent as the cell population matured, no clear-cut correlation between cell immaturity and 19S production could be obtained. In the primary response many fully mature plasma cells contained only 19S antibody; conversely, in the secondary response many blasts contained 7S antibody. No morphological difference between 19S and 7S cells could be found. The results suggested that many cells or cell clones go through a sequence whereby each forms first 19S and later 7S antibody with identical combining sites.