Serum, in concentrations greater than 1:25, causes a marked inhibition of complement fixation in general and of the Wassermann reaction in particular. The serum protein is probably adsorbed by the colloidally dispersed lipoid-reagin complexes, forming a protective film which prevents the fixation (adsorption) of complement. This inhibition explains the zone phenomenon in complement fixation: a weakly positive serum may give a completely positive reaction in e.g., 1:5 dilution, and yet, because of this serum inhibition, may appear completely negative when tested as whole serum.
The greater sensitivity of the ice box test is due (1) to the fact that the serum inhibition just described is less marked at lower temperature; (2) to the prolonged incubation time, making for greater specific fixation; (3) to a more marked non-specific destruction of complement by antigen; and (4) a spontaneous deterioration in the longer ice box test.
Because of the inhibition by serum protein in high concentration, a quantitative Wassermann technique involving the use of graded quantities of serum is worthless when carried out at 37°C. Even the ice box test, which is less susceptible to this inhibiting effect, will yield a positive reaction with whole serum only when the circulating reagin exceeds a surprisingly high threshold (six to ten times the quantity which could be detected in dilute serum). It is well known that a negative Wassermann, even by a very sensitive test, does not exclude syphilis: it now appears that a negative Wassermann does not exclude circulating reagin.