Detection of Immune Response Against Synthetic Polymers of Amino Acids Employing the Plaque-Forming Cell System

Abstract

Incorporation of homologous polypeptide (10^-5 M) into plaque assay plates containing anti-GLA³⁰ (LLL) immune lymph node cells specifically inhibited these PFC. However, at lower levels of antigen incorporation an enhancement of PFC was noted. The enhancement was complement-dependent, did not occur with non-immune lymph node cells and was inhibited by specific anti-allotype serum. Cross-reaction studies suggested that the polypeptide did not enhance by merely optimally coating the erythrocytes. Serum titers were measured on modified localized hemolysis-in-gel plates. Titers were increased when certain concentrations of antigen were incorporated into these plates, thereby implicating the increased hemolytic efficiency of complement or antibody in the presence of excess antigen. Puromycin (5 to 25 µg/ml) did not inhibit the production of unenhanced plaques, but prevented the optimal enhancement of plaques by antigen. These same concentrations of puromycin inhibited (40% to 96%, respectively) the incorporation of ¹⁴ C-L-leucine into trichloroacetic acid precipitable protein. The inclusion of polypeptide in the incubation medium increased the above incorporation by 20% to 170%; puromycin in addition to polypeptide prevented this increase, although the radioactivity was above that obtained when puromycin alone was present. A similar effect was noted with chloramphenicol. The results suggest that enhancement of anti-polypeptide plaques by incorporation of polymer in the medium is a complex phenomenon and is probably due to at least two factors: the increased hemolytic efficiency of immunoglobulin or complement in the presence of excess antigen, and the stimulation of protein synthesis in immune lymph node cells in the presence of excess antigen.