Critical Variables of the Jerne Plaque Technique As Applied to Rodent Antibody-Forming Systems Responding to Heterologous Red Cell Antigens

Abstract

Summary Improved conditions were developed for the Jerne plaque technique; a simple test (“hemolytic spot-testing”) allows a study of interspecies antigen-antibody systems and interspecies complement effects as well as ionic requirements for hemolysis in agar. These optimal conditions were applied to the Jerne plaque test with parallel results. The Jerne technique can easily be applied to study the hemolytic antibody response of C3BF1 mice, Golden Syrian hamsters, and Wistar rats immunized with heterologous RBC. Optimal results depend on the type and dilution of complement used for a certain heterologous combination: guinea pig complement gives best results in the mouse anti-sheep system; rabbit complement is superior in the mouse anti-rat system; and human complement is hemolytically most active in the mouse anti-hamster, hamster anti-mouse, and rat anti-mouse combination. Each C′ requires different ionic conditions of the reaction medium. It is demonstrated that optimal results of the Jerne plaque test depend on adequate concentrations of Ca++ and Mg++ in the agar. The data suggest also that the anticomplementary effect of agar is ionic. This hemolysis-reducing or inhibiting factor is overcome by DEAE dextran and higher cation concentrations of the agar. The mechanism of action of the polycation and Ca++ and Mg++ cations is discussed. The hemolytic activity of complement of different species is not only reflected in variation of the number of plaques but also in average plaque size. A quantitative complement-antibody correlation is considered as a possible explanation for this observation, and suggests that this method may provide a model for the study of the dynamics of cell lysis.