An Improved Benzene Extracted Complement Fixing Antigen Applied to the Diagnosis of the Arthropod-Borne Virus Encephalitides
Authors: Españna Carlos , Hammon W. McD. 
Published: January 2023

Summary and Conclusions
An improved, lyophilized and benzene extracted mouse-brain complement fixing antigen for the diagnosis of the arthropod-borne virus encephalitides is described. The procedure employed in its preparation is shorter, simpler and less dangerous than the original described by DeBoer and Cox. The antigens are of higher titer and more specific, when used in the optimal dilution for sensitivity, than those prepared in our laboratory by technics previously described. Benzene extraction appears to remove the fractions usually responsible for anti-complementary and non-specific reactions. Wassermann positive sera and sera from certain other non-syphilitic, but febrile patients, do not fix complement in the presence of this antigen even when the sera are inactivated at only 60 C. 56 C was found to be inadequate for syphilitic sera. Antigens so prepared can be lyophilized a second time with no change in their antigenic activity and such lyophilized products can be used after rehydration without further centrifugation. This product after lyophilization the second time is suitable for commercial preparation and distribution. It appears to be very stable, at least at 5 C. When this antigen is used in dilutions lower than the “optimal,” certain immunological overlapping can be demonstrated between Eastern and Western equine encephalomyelitis viruses as well as with St. Louis and Japanese B encephalitis viruses. California virus reacts very slightly, one way only, to Japanese B and St. Louis immune guinea pig sera. No immunological relationship was observed between the equine viruses and the St. Louis-Japanese B complex or the California virus. Overlapping between related viruses can be entirely eliminated by determining the optimal range of activity of each antigen with its homologous immune guinea pig serum and using the highest dilution of antigen in this range. In this dilution the antigen has maximal sensitivity and specificity. This usually represents a 1 to 5 per cent concentration by weight, of the original mouse brain. This same dilution is usually that most suitable for tests on human sera. Antigens, although slightly infectious (105 or 106 fold loss of original infectivity) lose their remaining infectivity with no loss in their antigenicity, after a few weeks storage in the liquid state at 5 C. Results are presented of tests with several antigens on nine series of multiple blood sera from Western equine, St. Louis and Japanese B encephalitis patients. The development of an excellent amniotic sac (chick embryo) antigen for the equine viruses is described and discussed. The preparation and limitations of a Japanese B chick embryo antigen and failure to produce a similar antigen for the St. Louis virus are also presented. Both Western equine and Japanese B chick embryo antigens were tested extensively on human encephalitis sera. None of the whole embryo antigens were of practical value because many normal human sera reacted with normal embryo.