A sensitive and simply calculated complement-fixation test is described in which viral antigen and antibody react to completion for up to 90 min. at 37° before the addition of one 70 % haemolysis unit of complement. The test has been applied to reactions between the components of the virus system of foot-and-mouth disease, tomato bushy stunt virus, bovine plasma albumin and their rabbit or guinea-pig hyperimmune sera. In the restricted region of proportional fixation each μ. of complement represents about 3 × 10 virus particles and 6 × 10 molecules of albumin. In suspensions of foot-and-mouth disease virus of highest infectivity (10 mouse ID50/ml.), probably less than 1 in 30 of the characteristic 25 mμ particles are infective.

Quantitative data define the regions of antibody excess, equivalence and antigen excess, and show that complement is bound as a secondary process to that of antigen/antibody complex formation, that complement is bound only by relatively massive complexes and that the independent formation of unrelated complexes in the same system may be sensitively detected. The location of the optimum reaction and the confirmation of equivalence allow fixation data to be related closely to parallel data on the neutralization of infectivity. Since the concentrations of antigen and of antibody for optimal fixation are almost independent, it is concluded that the maintenance of equivalence in terms of a constant antigen/antibody ratio is not a valid principle for the interpretation of such data.


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