Complement fixation has been successfully employed in identifying unknown enteroviruses by their reactions with poliomyelitis ( 1-3 ) , ECHO Types 1-14(4-6), and Coxsackie Group B, Type l ( 5 ) antisera. This report extends the experience to identifications with specific monkey antisera of ECHO Types 15-25, Coxsackie Group B, Types 2-5, and Group A, Type 9. One hundred and fortyeight freshly isolated enterovirus samples grown in monkey kidney tissue culture were classified and the results correlated with neutralization tests. The diagnostic reliability and efficiency of a simple, 1-step complementfixation technic using pooled ECHO antisera were demonstrated. Materials and methods. Typing antiserawere obtained from monkeys immunized respectively against poliomyelitis Types I-III,* ECHO Types 1-25,t Coxsackie Group B, Types 1-5,t and Coxsackie Group A, Type 9 ; t they were stored a t -25°C. Just prior to testing they were heated 30 minutes a t 60°C. When they proved anticomplementary, as did Coxsackie Group B, Types 1 and 2, Coxsackie Group A, Type 9, and ECHO Types 1-3, 5, 7, and 10, they were inactivated one hour undiluted, then diluted and reheated 30 minutes before use. When pools of the ECHO antisera were made, the anticomplementary specimens were distributed as equally as possible among them. Viral antigens. Monkey kidney tissue cultures were maintained in tubes with 1.0 ml medium containing 85% 199H, 15% tryptose-phosphate broth, and antibiotics. At inoculation time the medium was replaced with 1 ml 10% or 20% fecal (or other) suspension, and the tubes incubated 90 minutes a t 35°C. The inoculum was then replaced with 1 ml maintenance medium, and the cultures * Obtained from Communicable Disease Center, t Obtained from National Foundation, New York. U.S.P.H.S., Chamblee, Ga.reincubated. They were observed daily for cytopathic effects, and the fluids harvested when cell degeneration was complete. Onetenth ml infected fluid was then inoculated into each of 3 tubes containing monkey kidney cells in 1 ml maintenance medium consisting of 400 mg glucose plus 2 ml calf serum added to 100 ml 80% 199H and 20% tryptose-phosphate broth. These cultures were incubated and the fluids harvested as soon as cell degeneration was complete. They were clarified by centrifuging 15 minutes in the cold (4OC) at 1000 g , and the undiluted, unheated supernates typed by complement fixation with monkey antisera. CornpZement fixation. Following standard procedure(7,8) total volume was 0.5 ml, the reagents being used in 0.1 ml aliquots. The standard reagent diluent was 0.85% NaCl solution. In practice, 0.1 ml of optimally diluted antiserum was tested with 0.1 ml each of viral antigen and three 50% units (C'HSO) of complement (C'); 18-24 hours a t 3"-6°C were allowed €or fixation. Antisera and antigens were tested for nonspecific reactivity with uninoculated tissue culture fluid and with normal monkey serum, respectively. They were examined for anticomplementary activity with 1, 2 and 3 CIHB07 and C' stability was tested by incubating th...

Methods for the removal of anticomplementary (AC) qualities from sera(1-11) are generally too complex or inconvenient for frequent use in complement-fixation (CF) tests. Since Coons and his colleagues have found that absorption of fluorochrome-labeled antibody with tissue powder reduces or removes nonspecific staining activity( 12,13) the effect of tissue powder on the AC properties of human * Based on a thesis submitted by F. Rapp in partial fulfillment of the requirements for the degree of Master

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A number of workers have shown that antibodies to the influenza A viruses which are demonstrable in the sera of normal adults undergo periodic fluctuations in titre related to epidemic occurrence of the disease. The rise in antibodies accompanying infection and demonstrable by neutralization, agglutination-inhibition and complement-fixation tests is used as a method of diagnosis which is admitted to be of greater sensitivity than actual recovery of virus from the throat. Studies of the population during an epidemic have also revealed the existence of subclinical infection with rise in antibodies comparable to that occurring in those suffering clinical illnesses. But surveys of sera from large samples of the population show no general upward shift in antibody levels unless an actual epidemic occurs (Martin, 1940). The studies made by Francis, Magill, Rickard & Beck (1937), Hoyle & Fairbrother (1937), Rickard, Lennette & Horsfall (1940) and Martin (1940) also indicate the relative impermanence of the enhanced antibody levels consequent upon an epidemic. A relatively rapid decrease in antibody is shown both by the neutralization and complement-fixation tests during the 3–6 months after an epidemic and then a slower fall occurs until the next epidemic again causes a rise in titres. There have, however, been relatively few studies on large samples of populations situated in different geographical areas before and during an outbreak.