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...
