Comparison of three hemolytic plaque assays using sheep and mouse erythrocytes as antigens in rats.
Several hemolytic plaque assays using spleen cells from rats immunized with sheep or mouse erythrocytes were compared. Modifications of the assays were made to define optimal conditions. Mrbc in assays with guinea pig complement yield a much reduced number of plaques than is observed when human complement is used. In contrast, no difference in numbers is noted with Srbc regardless of complement source. The use of Srbc in an assay technique that does not employ agarose is more sensitive than techniques that require agarose; the result is a 50-100% increase in the number of plaques. This increase in plaque number was observed with either guinea pig or human complement.  In contrast, there was considerable clumping of Mrbc in the absence of agarose which tended to obscure distinct plaque formation. Increased sensitivity of plaque development for Srbc in the absence of agarose was due to increased numbers of direct (IgM) plaque detection and not to concomitant detection of cells producing IgG (indirect plaques). Maximal detection of plaques with Mrbc was observed when human complement was used in assays containing agarose. These assays gave a threefold increase in plaque numbers when compared with assays without agarose. It is evident from the above that the most sensitive and reproducible hemolytic plaque assay to be used by an investigator will depend primarily upon the antigen, and on reagents selected for the test.
D000818:Animals; D000941:Antigens; D004912:Erythrocytes / Q000276:immunology; D005260:Female; D006462:Hemolytic Plaque Technique; D008297:Male; D051379:Mice / Q000276:immunology; D051381:Rats; D012756:Sheep / Q000276:immunology; D013154:Spleen / Q000276:immunology
D000941:Antigens