Modified hemolytic plaque technique for the detection of bluetongue virus antibody-forming cells

A hemolytic plaque assay was developed for the detection of antibody-forming cells to bluetongue virus (BTV). Sheep erythrocytes (SRBC), onto which BTV had been absorbed, served as the indicator of lysis due to the presence of BTV antibody-forming cells. The ratio of BTV to SRBC was found to be critical for optimum hemolytic plaque formation. For routine use, 50 mul of 12% BTV SRBC, 0.1 ml of a spleen cell suspension, and 0.5 ml of 0.5% agarose in a balanced salt solution were mixed and plated on a microscope slide precoated with 0.1% aqueous agarose. Slides were incubated for 1 h at 37 C in a humidified incubator and subsequently flooded with 0.4 ml of a 1:15 dilution of complement. Incubation was continued for a further 2 h before the hemolytic plaques were scored. It was not possible to establish BTV serotype specificity by this technique.