Inhibition of plaque-forming cells with anti-idiotope or hapten: variation due to hapten density on indicator red cells.
Phosphorylcholine (PC)-specific antibody plaque-forming cells (PFC) were enumerated in the spleen of BALB/c mice immunized with S. pneumoniae R36a bacterial vaccine. Two indicator red blood cells were compared: RBC coupled with the hapten, PC-RBC, and cells coupled with PC-bearing polysaccharide from the pneumococcus, PnC-RBC. Equal numbers of direct (IgM) PFC were detected with both types of indicator cells. However, a significant difference appeared in the attempt to inhibit the respective PFC either with hapten (monovalent PC chloride, PCCl) or with monoclonal antibodies against T15 idiotopes (anti-Id). At optimal coupling concentrations, inhibition of anti-PnC-RBC plaques required a 10-fold higher concentration of the hapten when compared to anti-PC-SRBC plaques. Also, the inhibition of anti-PnC-SRBC plaques with anti-Id required much higher concentration of the antibody. The phenomenon may be explained by a higher epitope density on PnC-RBC than on PC-RBC. Heavily coupled PC-RBC were more difficult to inhibit (much like the PnC-RBC) by either the hapten or the anti-Id than lightly coupled PC-RBC. A mathematical analysis of the experimental curves supports the notion that under the assay conditions used, inhibition by either the hapten or the anti-Id is influenced primarily by antibody secretion rate and epitope density on indicator cells. If the 2 variables are too high, a significant inhibition of PFC with a low affinity anti-Id may not be possible. The theory also predicted that addition of a small amount of hapten into the assay would be roughly equivalent to secretion rate reduction and would facilitate the plaque inhibition by anti-Id. Indeed, we show a synergism between a minute (non-inhibitory) amount of PCCl and anti-Id in the inhibition of PC-specific PFC. These results point out that the detection of an idiotype-bearing PFC by plaque-inhibition assay is greatly influenced by technical variation in the assay, in particular, the preparation of the indicator red cells.
D000818:Animals; D000921:Antibody-Producing Cells / Q000276:immunology*; D003979:Diazonium Compounds; D000939:Epitopes / Q000276:immunology; D004912:Erythrocytes; D006241:Haptens / Q000276:immunology*; D006462:Hemolytic Plaque Technique; D007130:Immunoglobulin Idiotypes / Q000276:immunology*; D007202:Indicators and Reagents; D051379:Mice; D008807:Mice, Inbred BALB C; D010758:Phosphorus; D017553:Phosphorus Compounds; D010767:Phosphorylcholine / Q000276:immunology; D011135:Polysaccharides, Bacterial / Q000494:pharmacology; D012756:Sheep
D003979:Diazonium Compounds; D000939:Epitopes; D006241:Haptens; D007130:Immunoglobulin Idiotypes; D007202:Indicators and Reagents; D017553:Phosphorus Compounds; D011135:Polysaccharides, Bacterial; C027158:polysaccharide C-substance (Streptococcus); D010767:Phosphorylcholine; D010758:Phosphorus; C013196:phosphoryl chloride