Detection and Localization of Antibody-Forming Cells to a Bacterial Endotoxin by an Immunobioautographic Procedure

Specific antibody-forming cells to E. coli endotoxin were detected as discrete foci of bacteriolysis with intact spleen sections from mice immunized with varying concentrations of the antigen. Normal nonimmunized mice had an average of 1–2 antibody foci/spleen. Following immunization with 0.1–50 μg of endotoxin there was a rapid increase in the number of bacteriolytic foci, with a peak by the fourth to fifth day after antigen injection. Spleens of mice injected with 10–50 μg of antigen usually had confluent areas of bacteriolysis on the peak day. The animals receiving lower doses had fewer and more discrete antibody foci. Specificity was readily demonstrated by inhibition tests with specific antigen. In addition, the number of foci per spleen correlated with the increased number of single antibody plaqueforming cells, as detected by similar bacteriolytic antibody assays with dispersed cell suspensions. However, the increase in the number of single PFCs occurred later than did antibody foci. Similarly, the rise in serum antibacterial antibody, as detected by agglutination tests, occurred at a later time, with the peak usually between the fifth and eighth day after immunization. There was little or no increase in the level of serum antibody during the first 2–3 days after immunization.