Toxin-Antitoxin Flocculation in Tetanus

Abstract

While the flocculation-test with diphtheria-toxin and antitoxin has given fairly good results, 1 , 2 the method has been found unreliable when applied to toxins and respective antitoxins of tetanus, 3 , 4 , 5 botulinus, 6 , 7 staphylococcus, 8 and Streptococcus scarlatinœ. 9 , 10

Bronfenbrenner and Reichert 6 found that antitoxin obtained by immunizing animals against toxic filtrates of 24-day cultures of B. botulinus precipitated the filtrate while antitoxin prepared against toxic filtrates of 4–day cultures did not precipitate. They concluded that precipitation in the Ramon test may be influenced by the antibacterial antibody in the antitoxic serum. It seemed interesting to see whether these findings might also account for the discrepancies reported 3 , 4 , 5 in the in vitro titration of tetanus-toxin and antitoxin.

Rabbits were immunized with the formolized filtrates of B. tetani grown in 1% dextrose-ground beef-broth for 4 and 20 days, respectively, as well as with filtrates of 4-day and 20-day cultures of an atoxic variant of the same strain of B. tetani. As expected, the antisera prepared against the nontoxic filtrates were found to be entirely devoid of antitoxin as determined by the protection-test on mice, since as much as 0.5 cc. of undiluted serum failed to protect mice against even one m.l.d. of toxin. The sera containing antitoxin and those obtained against the nontoxic filtrates were used in the precipitation-test with the following antigens: (1) filtrates of 4–day cultures of the toxic and atoxic variants; (2) filtrates of 20-day cultures of the toxic and atoxic variants; and (3) 24-day filtrates of Type “A” botulinus-toxin (control). At the same time in vivo neutralization-tests were also carried out, and in the table the letter “N” indicates the neutral point as determined by this method. A wide zone of precipitation occurred when the antitoxic serum prepared against the 20-day formolized filtrate was mixed with either its homologous toxic filtrate or with the 20-day filtrate of the atoxic variant.