Measurements of Complement by Agglutination of Human Erythrocytes Reacting in Immune-Adherence

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Abstract
Summary: The number of molecules or particles of antigen and the amount of rabbit antibody giving maximal reactivity of HuC′ or GPC′ in 1.0 ml of reaction mixture containing 2 × 107 human erythrocytes were determined by measurements of IAHA in seven antigen-antibody systems; hen egg albumin, bovine serum albumin, bacteriophage T2, Brucella abortus, Salmonella typhosa, Salmonella enteritidis and sheep erythrocytes. With 5 × 107 Brucella organisms sensitized with 0.314 µg Ab N, fresh human or guinea pig serum showed reactivity in IAHA at a dilution of about 1:4000. The sensitivity of this method exceeds that of assays in immune hemolysis in which the same HuC′ reacted at dilutions of 1:33 to 1:41 and GPC′ at dilutions of 1:181 to 1:235. Complement in high dilutions of human serum combined with sheep E sensitized with rabbit Ab to induce IAHA, but the C′ in the same reaction mixture was unable to induce immune hemolysis of the cells unless 100 to 200 times more concentrated serum was used. On the other hand, the inhibition of IAHA with EDTA, sodium citrate, formaldehyde or Saldox, supports the view that at least four components of the C′ required for immune hemolysis, are similarly necessary to cause IAHA. After the sequence of reaction of IAHA which is analogous to that of immune hemolysis, intermediate complexes, Brucella-Ab-C′1,4,2 were prepared from both GPC′ and HuC′. The reactivity of fresh guinea pig serum or human serum with these complexes in the presence of EDTA is interpreted to represent the reactivity of one or more components of C′3 of the guinea pig serum or the human serum.