Canine Hemolytic Complement: Optimal Conditions for Its Titration

SUMMARY The effects of pH, ionic strength, cation concentration, ethylenediaminetetraacetic acid sodium salt (Na3edta), gelatin, glucose, time, and temperature were studied to determine optimal conditions for the titration of canine hemolytic complement (hcca). The optimal conditions were similar to those for the titration of guinea pig hemolytic complement (hcgp). Ovine erythrocytes (E) sensitized with rabbit antibody were the most sensitive of E tested from 6 species. The highest titers of hcca were detected when glucose-gelatin-barbital buffer (ggbb) was used as diluent, having ionic strength of 0.074, conductance of 6 millimhos/cm., and pH 6.5 and containing Mg2+ to final concentration of 1 × 10−3Μ and Ca2+ to 3 × 10−4Μ. Similar results were obtained when sucrose-barbital buffer (sbb) was used which had ionic strength of 0.065, conductance of 5 millimhos/cm., and pH 6.5 and containing Mg2+ and Ca2+ as for ggbb. Incubation temperature of 37 C. and time of 1 hour were optimal. Heating of serum at 56 C. for 30 minutes destroyed the hccaactivity, which was inhibited by 0.005 Μ Na3edta when guinea pig E were used as target cells and by 0.002 Μ Na3edta when ovine E were used. The hcca titers were not affected when natural complement-fixing antibody against the E used as target cells was present in the canine serum tested. The 50% hemolytic complement (hc) titers in serum from healthy dogs were between 32 and 96, i.e., 0.00312 to 0.00105 ml. of serum was required to lyse 50% of 1 × 107 sensitized E in the test volume of 0.5 ml.