title: Simplified assays of hemolytic activity of the classical and alternative complement pathways.
abstract: Simplified hemolytic assays for the classical (CP) and alternative (AP) pathways of complement (C) were developed. The CP function was tested with sensitized sheep erythrocytes in a diluent containing Ca2+ and Mg2+, while AP was tested with unsensitized rabbit erythrocytes in a diluent containing Mg2+-EGTA. In contrast to the commonly used hemolytic titration (CH50) assays, the present techniques tested the activity in reaction mixtures containing C at final dilutions which would not affect its function. These ranges fell between 1/1 and approximately 1/20 for CP and between 1/1 and approximately 1/3 for AP. With the adopted assay techniques single aliquots of serum were tested at single final dilutions of 1/8 for CP and 1/2 for AP, in the presence of excess target cells. Hemolysis was allowed to take place at 37 degrees C for 20 min. The number of cells lysed by CP and AP under these conditions was directly proportional to the dose of serum and unaffected by the presence of a large excess of target cells. Each pathway was tested independently of the other. Serum C levels, measured as described, correlated strongly with those determined by standard hemolytic titration (CH50) assays. The modified assays should offer less laborious alternatives for the functional assay of C than current routine procedures.
mesh_terms: D000818:Animals; D003167:Complement Activation; D003170:Complement Pathway, Alternative; D003171:Complement Pathway, Classical; D003165:Complement System Proteins / Q000276:immunology* / Q000502:physiology; D004306:Dose-Response Relationship, Immunologic; D004912:Erythrocytes / Q000276:immunology; D006461:Hemolysis; D006801:Humans; D007700:Kinetics; D011817:Rabbits; D012756:Sheep
chemical_list: D003165:Complement System Proteins