An antigen detected in the blood during the incubation period of serum hepatitis.

The aim of the following experiments was to provide an objective immunologic criterion for the diagnosis of serum hepatitis, as well as a possible means of screening for carriers of the agent of this disease. An antigen that reacted in the immunodiffusion test with serum from multiply transfused patients was detected in the blood during the incubation period prior to the onset of major chemical or clinical abnormalities. Double blind experiments suggest that this antigen is specific for serum hepatitis virus. Materials and Methods. -Clinical specimens: Sera from cases of transfusion-induced viral hepatitis, which we have collected, were obtained as part of a long-term study involving biweekly follow-up of transfused patients at The New York Hospital. Patients volunteering to participate in this study provided blood samples prior to transfusion and at least biweekly for a period of 6 months or more following transfusion. Test serum: The reference "antiserum" used in the majority of the studies to be described, hereinafter referred to as serum S, was obtained from a 24-year-old male patient with hemophilia who has received more than 10,000 units of blood, fresh-frozen plasma, and cryoprecipitate during the course of treatment for bleeding episodes. He has had no episodes of icteric hepatitis, but it was presumed that he had been multiply exposed to the virus or viruses of serum hepatitis. Serum S was chosen for these studies because the patient's multiple exposure was thought to ensure a hyperimmune status. Subsequently, four other sera from multiply transfused patients have been found to react in a manner similar to serum S. For some experiments, the serum was concentrated by ethanol fractionation. To each milliliter of serum to be concentrated, 8 ml of 30% ethanol in 0.1 Ml NaCl, 0.01 M tris(hydroxymethyl)aminomethane (Tris), 0.001 Ml ethylenediaminetetraacetate (EDTA), (pH 7.0 at -7oC) were added. This mixture was held at -70C and lyophilized. The dried globulin fraction was then rehydrated with distilled water to 0.1 the original volume of serum employed. Immunodiffusion technique: Double diffusion in agar gel was done by a micro-Ouchterlony technique.1 Nonspecific precipitation reactions between adjacent wells were eliminated by the use of 0.9% agarose dissolved in a buffer composed of 0.1 M NaCl, 0.01 M Tris (pH 7.6 at 250C), and 0.001 M EDTA containing 1 mg/ml protamine sulfate. Protamine sulfate has been recently suggested as a means of decreasing virus-agar interaction.2 Plates were incubated in a humid atmosphere at room temperature and read daily for 7 days. Strong reactions were evident after overnight incubation, while weaker reactions required 2 or 3 days' incubation and intensified for several days. Clinical chemical methods: Serum glutamic pyruvic transaminase (SGPT) was assayed by a kinetic spectrophotometric method with the Gilford multiple method sample recording spectrophotometer.3 Serum lactic dehydrogenase (LDH) enzymes were assayed by the method of Amador et al.4 with the same instrument. Serum LDH isoenzymes were separated by thin agar gel electrophoresis and quantitated fluorometrically.5 Results. -Demonstration of an antigen appearing in the blood during the incubation period of serum hepatitis: Failure in the past to isolate a causative virus from serum hepatitis could possibly be attributed to the fact that most isolation attempts have been carried out with specimens obtained early in the clinical course of the disease, at what is actually a late stage of the infection due to the