Standardization of the complement fixation test (CFT) in brucellosis. III. The hemolytic system

: The purpose of this paper was to developed simple and accurate methods for standardization of the particular components of a hemolytic system and to choose optimal amounts of these components in CFT. On the basis of comparative studies and the data from literature the following procedure was chosen: 1. Sheep's blood was conserved with Alsever liquid and used after 5 days of stabilization. A 2% suspension of erythrocytes was standardized by centrifuging in calibrated test tubes at 1000 r for 10 min and spectrophotometrically, assuming the following criterion: the suspension of erythrocytes dissolved in distilled water at 1:10 ratio should show 0.500 extinction (E541), which corresponds to 5 X 10(8) erythrocytes in 1 cm3. 2. Lyophilized hemolysin was dissolved, conserved with glycerol and used after 5 days of stabilization. Hemolysin was titrated with 100% hemolysis in the presence of the excess of complement. The suspension of erythrocytes was sensitized with 5 units of hemolysin directly before using it. 3. Lyophilized complement (C) was dissolved, conserved with boric acid +K2SO4 containing liquid and used after 5 days of stabilization. Titration of C was done by 50% hemolysis; 3C'H50 was used in CFT. 4. A barbital buffer was used as diluent at pH 7.3-7.4, alternatively 0.85% NaCl with an addition of Ca and Mg at optimal concentration. The hemolytic system was incubated at 37 degrees C for 30 min.