While using the Kahn Precipitation Test and especially the quantitative procedure, the possibility suggested itself of applying similar principles of concentration to the demonstration of agglutinins. If an immediate agglutination reaction could be obtained by an accurate, quantitative method, the advantages would at once be apparent. Working along these lines, a rapid method for the macroscopic agglutination test has been developed.The method involves concentration of the antigen, the use of small amounts of both antigen and serum, together with thorough mixing. Results read within five minutes after adding serum to suspension correspond to those obtained by a standard method requiring from eighteen to twenty-four hours.
EXPERIMENTALThe first experiments were made with 0.15 cc. of varying dilutions of an antityphoid serum (1:20 to 1:1000) and 0.05 cc. of a very heavy killed suspension of B. typhosus. These were mixed in small test tubes (75 by 13 mm.), shaken vigorously for two minutes, 0.5 cc. saline added and read-thus following the quantities and mechanics of the Kahn Precipitation Test.There was complete agglutination throughout, with no clumping in the control (0.15 cc. saline plus 0.05 cc. suspension). This experiment was repeated with B. typhosus and B. paratyphosu8
Summary
A satisfactory suspension of B. anthracis, for agglutination reactions, has been prepared by the described method. In order to be assured of a homogeneous suspension certain points must be observed. The cultures must be sporeless and must contain vigorous growths free from old organisms. The growths for the suspensions must be young, not more than eighteen hours old. The suspension must be thoroughly shaken; the larger clumps allowed to settle; and then carefully strained.
Agglutinins have been demonstrated in the serum from horses hyper-immunized with B. anthracis. The antianthrax sera from thirteen horses have given agglutination titers of from 1 in 6400 to 1 in 20,000, as against titers of from 1 in 80 to 1 in 200 in normal horses.
The agglutination tests show that certain antibodies have been produced in horses treated with B. anthracis and, in the absence of a satisfactory animal protection test or method of complement fixation the agglutination test may be used as a method for standardizing antianthrax serum.
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