6. 7. 8. 9.
Education and Welfare: Public health implications of the presence of hepatitis B antigen in human serum. Morbid Mortal Wkly Rep 21: no 16, 1972 IRwIN GR, ALLEN AM, BANCROFT WH, et al: Hepatitis B antigen in saliva, urine and stool. Inject Immun 11: 142, 1975 MAZZUR S: Menstrual blood as a vehicle of Australia-antigen transmission. Lancet 1: 749, 1973 DIcK SJ, TAMBURRO CH, LEEvY CM: Hepatitis B antigen in urban-caught mosquitoes. JAMA 229: 1627, 1975 MACQUARRIE MB, FOROHANI B, WOLOCHOW DA: Hepatitis B transmitted by a human bite. JAMA 230: 723, 1974
10. SzMUNEss W, PRICE AM, HIRsCH RL, et al:
Familial clustering of hepatitis B infection. N Engi J Med 289: 1162, 1973 II. ADAM E, HOLLINGER FB, MELNICK JL, et al: Type B hepatitis antigen and antibody among prostitutes and nuns: a study of possible venereal transmission. I Inject Dis 129: 317, 1974 12. FAss RJ: Sexual transmission of viral hepatitis? JAMA 230: 861, 1974 13. Committee on viral hepatitis, division of medical sciences, US Department of Health,
14.
15. 16. 17.
Education and Welfare: Public health implications of hepatitis B antigen in human blood. Revised statement. Morbid Mortal Wkly Rep 23: no 14, 1974 KoURoUPIs GM, LEERS W-D: Radioimmunoassay, complement fixation and counterimmunoelectrophoresis in the laboratory diagnosis of hepatitis B. Arch Gesamte Virusjorsch 43: 112, 1973 LEERS W-D, KouRouPIs GM: Comparison of the reversed passive haemagglutination with radioimmunoassay methods for hepatitis B antigen. J Clin Microbiol 2: 8, 1975 Idem: Prevalence of hepatitis B antibodies in hospital personnel. Can Med Assoc 1 113: 844, 1975 POLAKOFF S: Decrease of the incidence of
hepatitis in dialysis units associated with prevention program. Br Med 1 4: 751, 1974 18. WANDS JR, WALKER JA, DAVIS TT, et al: Hepatitis in an oncology unit. N Engi I Med
26: 1371, 1974 et al: Hospital-acquired serum hepatitis. JAMA 219: 1577, 1972 20. RIMLAND D, PARKIN WE: An outbreak of hepatitia B traced to an oral surgeon. Gastroenterology 67: 822, 1974 19. GARIsALDI RA, RASMUS5EN CM, HOLMES AW,
21. LEVIN ML, MADDREY WC, WANDS JR: Hepa-
titis B transmission by dentists. JAMA 228: 1139, 1974 22. KouRoupis GM, LEERS W-D: A rapid radioimmunoassay method for the detection of
hepatitis B antigen. Arch Gesamte Viruslorsch
44: 370, 1974 23. Idem: Prevalence of anti-hepatitis B. antigen
in hospital staff with respect to ethnic origin. Presented at 43rd annual meeting of the laboratory division, Canadian Public Health Association, Joint Meeting of Infectious Diseases, Toronto, Nov. 26-28, 1975
24. FEINMAN SV, KRAS5NITZKY 0, SINCLAIR JC, et al: Prevalence and significance of hepatitis B surface antigen in a general hospital. Can
Med Assoc / 112: 43, 1975 al: Acquisition of antibody to hepatitis B antigen in three socioeconomically different medical populations. Lancet 2: 149, 1972
25. CHERUBIN CE, PURCELL RH, LANDER JJ, et
26. HADZIYANNI5 5, MERIKAS G, PANETSOS 5: Hepatitis associated antigen carriers among
blood donors in Greece. Am J Dis Child 123: 381, 1972 27. PLOTZ PH, ALTER HI, HOLLAND PV: Exposure to a food-handler with hepatitis B (C). Lancet 2: 333, 1972
Detection of core antibody in hepatitis B infection L. SPENCE, FRCP[C]; M. FAUVEL, M Sc
Hepatitis B core antigen (HB.Ag) is found on the decoated Dane particle and on a morphologically similar particle detected mainly in the nucleus of hepatocytes of patients with hepatitis B. HB.Ag prepared from the liver of a chimpanzee infected with hepatitis B virus was used to test human serum for core antibody (anti-HB.) by complement fixation. Anti-HB. was found in serum collected from patients with hepatitis B in both the acute and convalescent stages, from carriers of hepatitis B surface antigen (HB,Ag) and from patients with chronic liver or renal disease who were carriers of HB,Ag: It was not found in patients with hepatitis A or infectious mononucleosis, or in healthy persons who were not carriers of HB5Ag. La nucleocapside du virus de l'hepatite de type B (HB.Ag) est retrouve sur Ia particule Dane debarrassee de sa game et sur une particule morphologiquement semblable decelee principalement dans le noyau des hepatocytes des patients souffrant d'hepatite de type B. L'HB.Ag prepare a partir du foie d'un chimpanze infect6 avec le virus de l'hepatite de type B a et6 utilis6 afin de d.tecter Ia pr6sence de I'anticorps dirig6 contre I'HB.Ag (anti-HB.) dans le serum humain par fixation du compl6ment. L'anti-HB. a .te retrouve dans les s6rums preleves chez des patients atteints de I'h6patite de type B, From the department of medical microbiology, faculty of medicine, University of Toronto and the department of microbiology, Toronto General Hospital Reprint requests to: Dr. L. Spence, Department of medical microbiology, Banting Institute, 100 College St., Toronto, Ont. M5G 1L5
998
aussi bien durant Ia phase aigue que durant Ia convalescence, chez des porteurs de l'antigene de surface de l'hepatite de type B (HB,Ag) et chez des patients souffrant de maladies hepatiques ou r6nales chroniques et porteurs de l'HBAg. L'anti-HB. n'a pas ete decel6 chez des patients souffrant d'hepatite A ou de mononucleose infectieuse, ou chez des personnes saines qui n'etaient pas porteurs de l'HBAg.
In 1970 Dane, Cameron and Briggs1 described in the blood of hepatitis B patients a particle 42 nm in diameter with a complex structure. By negative staining and examination under the electron microscope it was found to have an outer coat 7 nm in thickness surrounding an inner core 28 nm in diameter. The particle shared an antigen, hepatitis B surface antigen (HB.Ag), with the small spherical and tubular particles previously described in association with Australia antigen.1'2 In 1971 Almeida, Rubenstein and Stott3 reported that treatment of the Dane particle with a detergent, polysorbate (Tween) 80, disrupted the particle and released an internal component (the core), 27 nm in diameter, that resembled a particle Almeida and colleagues4 had found previously in homogenates of liver prepared from two patients who had died from hepatitis. Immune electron microscopy (IEM) showed that the internal component reacted with posthepatitis but not prehepatitis serum and that this represented a new antigen-antibody reaction in hepatitis B infection. Similar results were reported by Deutsch and Spence5 in 1972. In this paper we report the results
CMA JOURNAL/NOVEMBER 20, 1976/VOL. 115
of further studies with hepatitis B core antigen (HB.Ag) on serum of patients with hepatitis B. Patients and methods Patients The patients in the study, all adults, included the following: (a) healthy women admitted to the maternity ward for delivery and found to be HB5Agnegative by routine screening (not matched for age and sex with the other patients); (b) healthy carriers of HB8Ag, including Red Cross blood donors and healthy maternity patients found to be HB8Ag-positive by routine screening; (c) patients with hepatitis B; (d) HB8Agpositive patients with chronic liver or renal disease; (e) patients with hepatitis A, diagnosed from clinical and epidemiologic history, physical examination and biochemical and virologic tests; and (f) patients with infectious mononucleosis. Serum collection Serum was collected from the patients admitted to or visiting a general hospital for the investigation and treatment of acute hepatitis B and again after* HB.Ag was no longer detectable in the blood. Usually the first HB9Agnegative sample obtained was tested for antibodies to HB8Ag (anti-HB.) and to HB.Ag (anti-HB.). Serum was also collected from all other categories of patients. Preparation of HB.Ag The HB.Ag used in these studies was prepared from the liver of a chimpanzee infected with hepatitis B virus at Connaught Laboratories, Toronto.6
Evidence of infection was obtained by demonstrating HB.Ag in the animal's blood and virus-like particles in the nuclei of hepatocytes. When the animal was sacrificed a portion of the liver was given to us. HB.Ag was prepared in the following manner: Approximately 2.5 g of liver was homogenized in a tissue grinder and the homogenate was reconstituted as a 12% suspension of phosphate-buffered saline (PBS), 0.01 M. Electron microscopy of the homogenate revealed spherical virus-like particles 27 nm in diameter (Fig. 1), similar to the particles described by Almeida and colleagues.4 The homogenate was then centrifuged at 500 x g for 30 minutes. The supernatant was removed and centrifuged at 78 800 x g for 2 hours; this last procedure was repeated twice. The cores detected in the pellet by electron microscopy were found to be associated with cellular material. For further purification of the cores, the pellet was resuspended in PBS, treated with an equal volume of pronase (0.1% w/v aqueous suspension, pH 7.4) for 1 hour at 37 0C and then centrifuged at 200 000 x g for S hours. The pellet was resuspended in approximately 2.0 ml of PBS and treated with . detergent (Nonidet-P40, Shell, 10 .d/ml) at room
temperature for 1 hour to decoat the Dane particles, then 0.5 ml of each preparation was layered on a continuous sucrose gradient (25 to 60% in PBS) and centrifuged at 90000 x g for 29 hours. This final centrifugation separated HWAg from HB.Ag. Fractions of the gradients were collected and electron microscopy showed cores only to be present in fractions 17 to 21. These fractions were pooled and constituted the purified HB.Ag. All the procedures described were carried out in glassware and plasticware coated with 1 % bovalbumin in PBS to prevent adsorption of the antigen. Since liver from a noninfected chimpanzee was not available a control antigen was prepared by the methods described above from a continuous tissueculture cell line derived from chinipanzee liver (supplied by Flow Laboratories, Rockville, Maryland). Tests performed Testing for anti-HB. was carried out with the prepared HB.Ag by the complement fixation microtechnique described by Sever,7 modified slightly: 2 U of complement, 2 U of hemolysin and 4 U of antigen were used and the sheep cell concentration was reduced to 0.4%. Testing for HB8Ag was performed
by radioimmunoassay with the Ausria test (Abbott Laboratories, North Chicago), and anti-HB, was detected by the passive hemagglutination assay described by Vyas and Shulman.8 TEM was carried out to detect antiHB. as described by Fauvel and colleagues.9 Results
Five months after preparation the core particles had retained their morphology (Fig. 2) and the preparation was free from HB,Ag, as the negative results of the Ausria test indicated. HB.Ag did not fix complement in the presence of rabbit anti-HB, but did fix complement with a reference serum containing anti-HB. (supplied by the United States National Institutes of Health). Of the human serum samples collected for testing 95 (50%) were found to be anticomplementary and unsuitable for use in the complement fixation test. One of these samples was found to be suitable for use after treatment with 25% kaolin borate saline buffer for 20 minutes. Of the 96 serum samples tested (including the one kaolin-
FIG. 1-Aggregate of core particles (hepatitis B core antigen) in homogenate from liver
FIG. 2-Purified core particles after 5
of chimpanzee infected with hepatitis B virus (reduced 40% from x306 850).
months' storage (x300 300).
CMA JOURNAL/NOVEMBER 20, 1976/VOL. 115 999
Table I-Distribution of core (anti-HB0) and surface (anti-HB,) hepatitis B antibodies in Canadian patients
Patient category
Tested
No. of sera With anti-HB.
Healthy 19 0 HB5Ag-negative 13 13 HB8Ag-positive Hepatitis B 6 iserum sample 11 > 1 serum sample Acute phase 9 5 Convalescent phaset 21 17 Chronic liver disease1 6 6 HB8Ag-positive Chronic renal disease, 4 4 HB.Ag-positive 5 0 Hepatitis A Infectious mononucleosis 8 0 96 51 Total . by passive hemagglutination. tNine patients, but more than one serum sample collected from some patients.
treated sample) 51 contained anti-HB., 41 did not, and in 4 the results were considered inconclusive because the serum reacted with the control antigen. The distribution of anti-HB. and of anti-HB. in the groups of patients is shown in Table I. Serum from a nurse was studied for anti-HB. before and after development of acute hepatitis B infection. The results are shown in Fig. 3. Before development of hepatitis B, anti-HB. was not detected by IBM. Development of jaundice was accompanied by the transient presence of HB.Ag in the patient's blood. During convalescence IBM was carried out at first; later, complement fixation tests were done. High concentrations of anti-HB. persisted for several months but anti-HB5 was not detectable. Discussion There is now considerable evidence
FIG. 3-Results of study of nurse's serum for hepatitis B surface antigen (HB3Ag) and antibody (anti-JIB,) and core antibody (anti-HB.) before and after development of acute hepatitis B In May 1973. CF = complement fixation test; IEM = immune electron microscopy; PHA = passive hemagglutination assay.
With anti.HB5* 0 1 0 0 1 0 0 1 0 3
that the Dane particle is the virus of hepatitis B. The core of this particle is identical in size and appearance to the particle found in the nucleus of hepatocytes of patients with hepatitis B. A DNA polymerase has been found to be associated with the core,10 as have double-stranded DNA molecules.'1 The particle carries two distinct antigens, one on the core and one on the surface. Studies of these antigens and the immune response to them may be important in understanding the pathogenesis of hepatitis B infection. These studies may also contribute to prophylaxis of hepatitis B and may lead to development of more sensitive tests for detection of hepatitis B virus in human plasma. Krugman and colleagues'2 noted that anti-HB. was detected 3 to 4 months after exposure to hepatitis B, during the period of antigenemia and at the height of liver dysfunction. This antibody appeared several months before anti-HB8 and was detected consistently in the serum of chronic carriers of HB5Ag. Our results confirm these observations. Our failure to detect antiHB5 in many of the serum samples collected during convalescence from hepatitis B probably results from the fact that the samples were collected prior to the development of this antibody. The presence of anti-HB. in the serum of the patient with hepatitis A may represent past exposure to hepatitis B virus. The complement fixation test is not entirely satisfactory for detection of anti-HB.. Many serum samples from hepatitis patients are unsuitable for use in this test because they are anticoniplementary, a feature that may be due to storage of serum with frequent freezing and thawing or to the presence
1000 CMA JOURNAL/NOVEMBER 20, 1976/VOL. 115
of antigen-antibody complexes in HB5Ag-positive serum. Shulman and Barker13 reported that serum samples from hepatitis patients were often anticomplementary. Serum collected by us from healthy persons and tested before freezing was seldom found to be anticomplementary. Thus in routine testing of healthy persons for anti-HB. anticomplementary serum should not be a problem. Recently a radioimmunossay for antiHB. has been reported by Purcell and colleagues.14 This test may have advantages over the complement fixation test because it may be more sensitive and unlikely to be affected when serum is anticomplementary. Difficulty in obtaining HB.Ag has been a factor in limiting the amount of testing for antiHB.. This may be overcome by preparing HB.Ag from HB,Ag-positive plasma by the method described by Fauvel and associates.9 We thank Dr. S. Wilson of Connaught Laboratories for supplying the infected liver and for helpful comments on this mansucript, the Canadian Red Cross and the US National Institutes of Health for supplying serum, Dr. Susan Ritchie for carrying out electron microscopic studies on the chimpanzee liver, and Jane Pagel for technical assistance. This investigation was supported by national health grant no. 605-7-766. References 1. DANE DS, CAMERON CH, BRIGOS M: Virus like particles in serum of patients with Australia-antigen-associated hepatitis. Lancet 1: 695, 1970 2. BAYER ME, BLUMBERO BS, WERNER B: Par-
ticles associated with Australia antigen in sera of patients with Ieukaemia, Down s syndrome and hepatitis. Nature 218: 1057, 1969 3. ALMEIDA JD, RUBENSTEIN D, STOTY El: New antigen-antibody system in Australia-antigenpositive hepatitis. Lancet 2: 1225, 1971
4. ALMEIDA 3D, WAmsoN AP, TROWELL 3M,
5. 6.
7. 8. 9. 10. 11. 12. 13.
et al: The finding of virus-like particles in two Australia-antigen-positive human livers. Macrobios 2: 145, 1970 Daurscs G, SPENCE L: Virus-like particles in the liver and their relationship to Australia antigen. Lancet 1: 447, 1972 WILsON 5, LOGAN LM: Hepatitis B core antigen in the immunosuppressed chimpanzee. International symposium on viral hepatitis, Milan, Dec. 1974. Dev Blol Stand 30: 240, 1975 SEVER JL: Application of a microtechnique to viral serological investigations. J Immunol 77: 320, 1962 VYAs GN, SHULMAN NR: Hemagglutinatlon assay for antigen and antibody associated with viral hepatitis. ScIence 170: 332, 1970 FAUvEL M, BABsuic L, SHEAFF ET, et al: Preparation of a Dane core (hepatitis B) antigen for use in the immune electron microscopy test. Can I Mlcrobiol 21: 905, 1975 RosINsoN WS, GREENMAN RL: DNA polymerase in the core of the human hepatitis B virus candidate. / VIrol 13: 1231, 1974 RossNso.i WS, CLAYTON DA, GREENMAN RL: DNA of a human hepatitis B virus candidate. I VIrol 14: 384, 1974 KRUGMAN 5, HOOFNAGLE JH GERILFY RI, et al: Viral hepatitis, type i DNA polymerase activity and antibody to hepatitis B core antigen. N Engi J Med 290: 1331, 1974 SHULMAN NR, BAsucaa LF: Virus-like antigen, antibody, and antigen-antibody complexes in hepatitis measured by complement fixation. Science 165: 304, 1969
14. PURCELL RH, GERIN JL, ALMEIDA JB, et al:
Radioimmunoassay for the detection of the core of the Dane particle and antibody to it. Intervirology 2: 231, 1973/74
Education and Welfare: Public health implications of the presence of hepatitis B antigen in human serum. Morbid Mortal Wkly Rep 21: no 16, 1972 IRwIN GR, ALLEN AM, BANCROFT WH, et al: Hepatitis B antigen in saliva, urine and stool. Inject Immun 11: 142, 1975 MAZZUR S: Menstrual blood as a vehicle of Australia-antigen transmission. Lancet 1: 749, 1973 DIcK SJ, TAMBURRO CH, LEEvY CM: Hepatitis B antigen in urban-caught mosquitoes. JAMA 229: 1627, 1975 MACQUARRIE MB, FOROHANI B, WOLOCHOW DA: Hepatitis B transmitted by a human bite. JAMA 230: 723, 1974
10. SzMUNEss W, PRICE AM, HIRsCH RL, et al:
Familial clustering of hepatitis B infection. N Engi J Med 289: 1162, 1973 II. ADAM E, HOLLINGER FB, MELNICK JL, et al: Type B hepatitis antigen and antibody among prostitutes and nuns: a study of possible venereal transmission. I Inject Dis 129: 317, 1974 12. FAss RJ: Sexual transmission of viral hepatitis? JAMA 230: 861, 1974 13. Committee on viral hepatitis, division of medical sciences, US Department of Health,
14.
15. 16. 17.
Education and Welfare: Public health implications of hepatitis B antigen in human blood. Revised statement. Morbid Mortal Wkly Rep 23: no 14, 1974 KoURoUPIs GM, LEERS W-D: Radioimmunoassay, complement fixation and counterimmunoelectrophoresis in the laboratory diagnosis of hepatitis B. Arch Gesamte Virusjorsch 43: 112, 1973 LEERS W-D, KouRouPIs GM: Comparison of the reversed passive haemagglutination with radioimmunoassay methods for hepatitis B antigen. J Clin Microbiol 2: 8, 1975 Idem: Prevalence of hepatitis B antibodies in hospital personnel. Can Med Assoc 1 113: 844, 1975 POLAKOFF S: Decrease of the incidence of
hepatitis in dialysis units associated with prevention program. Br Med 1 4: 751, 1974 18. WANDS JR, WALKER JA, DAVIS TT, et al: Hepatitis in an oncology unit. N Engi I Med
26: 1371, 1974 et al: Hospital-acquired serum hepatitis. JAMA 219: 1577, 1972 20. RIMLAND D, PARKIN WE: An outbreak of hepatitia B traced to an oral surgeon. Gastroenterology 67: 822, 1974 19. GARIsALDI RA, RASMUS5EN CM, HOLMES AW,
21. LEVIN ML, MADDREY WC, WANDS JR: Hepa-
titis B transmission by dentists. JAMA 228: 1139, 1974 22. KouRoupis GM, LEERS W-D: A rapid radioimmunoassay method for the detection of
hepatitis B antigen. Arch Gesamte Viruslorsch
44: 370, 1974 23. Idem: Prevalence of anti-hepatitis B. antigen
in hospital staff with respect to ethnic origin. Presented at 43rd annual meeting of the laboratory division, Canadian Public Health Association, Joint Meeting of Infectious Diseases, Toronto, Nov. 26-28, 1975
24. FEINMAN SV, KRAS5NITZKY 0, SINCLAIR JC, et al: Prevalence and significance of hepatitis B surface antigen in a general hospital. Can
Med Assoc / 112: 43, 1975 al: Acquisition of antibody to hepatitis B antigen in three socioeconomically different medical populations. Lancet 2: 149, 1972
25. CHERUBIN CE, PURCELL RH, LANDER JJ, et
26. HADZIYANNI5 5, MERIKAS G, PANETSOS 5: Hepatitis associated antigen carriers among
blood donors in Greece. Am J Dis Child 123: 381, 1972 27. PLOTZ PH, ALTER HI, HOLLAND PV: Exposure to a food-handler with hepatitis B (C). Lancet 2: 333, 1972
Detection of core antibody in hepatitis B infection L. SPENCE, FRCP[C]; M. FAUVEL, M Sc
Hepatitis B core antigen (HB.Ag) is found on the decoated Dane particle and on a morphologically similar particle detected mainly in the nucleus of hepatocytes of patients with hepatitis B. HB.Ag prepared from the liver of a chimpanzee infected with hepatitis B virus was used to test human serum for core antibody (anti-HB.) by complement fixation. Anti-HB. was found in serum collected from patients with hepatitis B in both the acute and convalescent stages, from carriers of hepatitis B surface antigen (HB,Ag) and from patients with chronic liver or renal disease who were carriers of HB,Ag: It was not found in patients with hepatitis A or infectious mononucleosis, or in healthy persons who were not carriers of HB5Ag. La nucleocapside du virus de l'hepatite de type B (HB.Ag) est retrouve sur Ia particule Dane debarrassee de sa game et sur une particule morphologiquement semblable decelee principalement dans le noyau des hepatocytes des patients souffrant d'hepatite de type B. L'HB.Ag prepare a partir du foie d'un chimpanze infect6 avec le virus de l'hepatite de type B a et6 utilis6 afin de d.tecter Ia pr6sence de I'anticorps dirig6 contre I'HB.Ag (anti-HB.) dans le serum humain par fixation du compl6ment. L'anti-HB. a .te retrouve dans les s6rums preleves chez des patients atteints de I'h6patite de type B, From the department of medical microbiology, faculty of medicine, University of Toronto and the department of microbiology, Toronto General Hospital Reprint requests to: Dr. L. Spence, Department of medical microbiology, Banting Institute, 100 College St., Toronto, Ont. M5G 1L5
998
aussi bien durant Ia phase aigue que durant Ia convalescence, chez des porteurs de l'antigene de surface de l'hepatite de type B (HB,Ag) et chez des patients souffrant de maladies hepatiques ou r6nales chroniques et porteurs de l'HBAg. L'anti-HB. n'a pas ete decel6 chez des patients souffrant d'hepatite A ou de mononucleose infectieuse, ou chez des personnes saines qui n'etaient pas porteurs de l'HBAg.
In 1970 Dane, Cameron and Briggs1 described in the blood of hepatitis B patients a particle 42 nm in diameter with a complex structure. By negative staining and examination under the electron microscope it was found to have an outer coat 7 nm in thickness surrounding an inner core 28 nm in diameter. The particle shared an antigen, hepatitis B surface antigen (HB.Ag), with the small spherical and tubular particles previously described in association with Australia antigen.1'2 In 1971 Almeida, Rubenstein and Stott3 reported that treatment of the Dane particle with a detergent, polysorbate (Tween) 80, disrupted the particle and released an internal component (the core), 27 nm in diameter, that resembled a particle Almeida and colleagues4 had found previously in homogenates of liver prepared from two patients who had died from hepatitis. Immune electron microscopy (IEM) showed that the internal component reacted with posthepatitis but not prehepatitis serum and that this represented a new antigen-antibody reaction in hepatitis B infection. Similar results were reported by Deutsch and Spence5 in 1972. In this paper we report the results
CMA JOURNAL/NOVEMBER 20, 1976/VOL. 115
of further studies with hepatitis B core antigen (HB.Ag) on serum of patients with hepatitis B. Patients and methods Patients The patients in the study, all adults, included the following: (a) healthy women admitted to the maternity ward for delivery and found to be HB5Agnegative by routine screening (not matched for age and sex with the other patients); (b) healthy carriers of HB8Ag, including Red Cross blood donors and healthy maternity patients found to be HB8Ag-positive by routine screening; (c) patients with hepatitis B; (d) HB8Agpositive patients with chronic liver or renal disease; (e) patients with hepatitis A, diagnosed from clinical and epidemiologic history, physical examination and biochemical and virologic tests; and (f) patients with infectious mononucleosis. Serum collection Serum was collected from the patients admitted to or visiting a general hospital for the investigation and treatment of acute hepatitis B and again after* HB.Ag was no longer detectable in the blood. Usually the first HB9Agnegative sample obtained was tested for antibodies to HB8Ag (anti-HB.) and to HB.Ag (anti-HB.). Serum was also collected from all other categories of patients. Preparation of HB.Ag The HB.Ag used in these studies was prepared from the liver of a chimpanzee infected with hepatitis B virus at Connaught Laboratories, Toronto.6
Evidence of infection was obtained by demonstrating HB.Ag in the animal's blood and virus-like particles in the nuclei of hepatocytes. When the animal was sacrificed a portion of the liver was given to us. HB.Ag was prepared in the following manner: Approximately 2.5 g of liver was homogenized in a tissue grinder and the homogenate was reconstituted as a 12% suspension of phosphate-buffered saline (PBS), 0.01 M. Electron microscopy of the homogenate revealed spherical virus-like particles 27 nm in diameter (Fig. 1), similar to the particles described by Almeida and colleagues.4 The homogenate was then centrifuged at 500 x g for 30 minutes. The supernatant was removed and centrifuged at 78 800 x g for 2 hours; this last procedure was repeated twice. The cores detected in the pellet by electron microscopy were found to be associated with cellular material. For further purification of the cores, the pellet was resuspended in PBS, treated with an equal volume of pronase (0.1% w/v aqueous suspension, pH 7.4) for 1 hour at 37 0C and then centrifuged at 200 000 x g for S hours. The pellet was resuspended in approximately 2.0 ml of PBS and treated with . detergent (Nonidet-P40, Shell, 10 .d/ml) at room
temperature for 1 hour to decoat the Dane particles, then 0.5 ml of each preparation was layered on a continuous sucrose gradient (25 to 60% in PBS) and centrifuged at 90000 x g for 29 hours. This final centrifugation separated HWAg from HB.Ag. Fractions of the gradients were collected and electron microscopy showed cores only to be present in fractions 17 to 21. These fractions were pooled and constituted the purified HB.Ag. All the procedures described were carried out in glassware and plasticware coated with 1 % bovalbumin in PBS to prevent adsorption of the antigen. Since liver from a noninfected chimpanzee was not available a control antigen was prepared by the methods described above from a continuous tissueculture cell line derived from chinipanzee liver (supplied by Flow Laboratories, Rockville, Maryland). Tests performed Testing for anti-HB. was carried out with the prepared HB.Ag by the complement fixation microtechnique described by Sever,7 modified slightly: 2 U of complement, 2 U of hemolysin and 4 U of antigen were used and the sheep cell concentration was reduced to 0.4%. Testing for HB8Ag was performed
by radioimmunoassay with the Ausria test (Abbott Laboratories, North Chicago), and anti-HB, was detected by the passive hemagglutination assay described by Vyas and Shulman.8 TEM was carried out to detect antiHB. as described by Fauvel and colleagues.9 Results
Five months after preparation the core particles had retained their morphology (Fig. 2) and the preparation was free from HB,Ag, as the negative results of the Ausria test indicated. HB.Ag did not fix complement in the presence of rabbit anti-HB, but did fix complement with a reference serum containing anti-HB. (supplied by the United States National Institutes of Health). Of the human serum samples collected for testing 95 (50%) were found to be anticomplementary and unsuitable for use in the complement fixation test. One of these samples was found to be suitable for use after treatment with 25% kaolin borate saline buffer for 20 minutes. Of the 96 serum samples tested (including the one kaolin-
FIG. 1-Aggregate of core particles (hepatitis B core antigen) in homogenate from liver
FIG. 2-Purified core particles after 5
of chimpanzee infected with hepatitis B virus (reduced 40% from x306 850).
months' storage (x300 300).
CMA JOURNAL/NOVEMBER 20, 1976/VOL. 115 999
Table I-Distribution of core (anti-HB0) and surface (anti-HB,) hepatitis B antibodies in Canadian patients
Patient category
Tested
No. of sera With anti-HB.
Healthy 19 0 HB5Ag-negative 13 13 HB8Ag-positive Hepatitis B 6 iserum sample 11 > 1 serum sample Acute phase 9 5 Convalescent phaset 21 17 Chronic liver disease1 6 6 HB8Ag-positive Chronic renal disease, 4 4 HB.Ag-positive 5 0 Hepatitis A Infectious mononucleosis 8 0 96 51 Total . by passive hemagglutination. tNine patients, but more than one serum sample collected from some patients.
treated sample) 51 contained anti-HB., 41 did not, and in 4 the results were considered inconclusive because the serum reacted with the control antigen. The distribution of anti-HB. and of anti-HB. in the groups of patients is shown in Table I. Serum from a nurse was studied for anti-HB. before and after development of acute hepatitis B infection. The results are shown in Fig. 3. Before development of hepatitis B, anti-HB. was not detected by IBM. Development of jaundice was accompanied by the transient presence of HB.Ag in the patient's blood. During convalescence IBM was carried out at first; later, complement fixation tests were done. High concentrations of anti-HB. persisted for several months but anti-HB5 was not detectable. Discussion There is now considerable evidence
FIG. 3-Results of study of nurse's serum for hepatitis B surface antigen (HB3Ag) and antibody (anti-JIB,) and core antibody (anti-HB.) before and after development of acute hepatitis B In May 1973. CF = complement fixation test; IEM = immune electron microscopy; PHA = passive hemagglutination assay.
With anti.HB5* 0 1 0 0 1 0 0 1 0 3
that the Dane particle is the virus of hepatitis B. The core of this particle is identical in size and appearance to the particle found in the nucleus of hepatocytes of patients with hepatitis B. A DNA polymerase has been found to be associated with the core,10 as have double-stranded DNA molecules.'1 The particle carries two distinct antigens, one on the core and one on the surface. Studies of these antigens and the immune response to them may be important in understanding the pathogenesis of hepatitis B infection. These studies may also contribute to prophylaxis of hepatitis B and may lead to development of more sensitive tests for detection of hepatitis B virus in human plasma. Krugman and colleagues'2 noted that anti-HB. was detected 3 to 4 months after exposure to hepatitis B, during the period of antigenemia and at the height of liver dysfunction. This antibody appeared several months before anti-HB8 and was detected consistently in the serum of chronic carriers of HB5Ag. Our results confirm these observations. Our failure to detect antiHB5 in many of the serum samples collected during convalescence from hepatitis B probably results from the fact that the samples were collected prior to the development of this antibody. The presence of anti-HB. in the serum of the patient with hepatitis A may represent past exposure to hepatitis B virus. The complement fixation test is not entirely satisfactory for detection of anti-HB.. Many serum samples from hepatitis patients are unsuitable for use in this test because they are anticoniplementary, a feature that may be due to storage of serum with frequent freezing and thawing or to the presence
1000 CMA JOURNAL/NOVEMBER 20, 1976/VOL. 115
of antigen-antibody complexes in HB5Ag-positive serum. Shulman and Barker13 reported that serum samples from hepatitis patients were often anticomplementary. Serum collected by us from healthy persons and tested before freezing was seldom found to be anticomplementary. Thus in routine testing of healthy persons for anti-HB. anticomplementary serum should not be a problem. Recently a radioimmunossay for antiHB. has been reported by Purcell and colleagues.14 This test may have advantages over the complement fixation test because it may be more sensitive and unlikely to be affected when serum is anticomplementary. Difficulty in obtaining HB.Ag has been a factor in limiting the amount of testing for antiHB.. This may be overcome by preparing HB.Ag from HB,Ag-positive plasma by the method described by Fauvel and associates.9 We thank Dr. S. Wilson of Connaught Laboratories for supplying the infected liver and for helpful comments on this mansucript, the Canadian Red Cross and the US National Institutes of Health for supplying serum, Dr. Susan Ritchie for carrying out electron microscopic studies on the chimpanzee liver, and Jane Pagel for technical assistance. This investigation was supported by national health grant no. 605-7-766. References 1. DANE DS, CAMERON CH, BRIGOS M: Virus like particles in serum of patients with Australia-antigen-associated hepatitis. Lancet 1: 695, 1970 2. BAYER ME, BLUMBERO BS, WERNER B: Par-
ticles associated with Australia antigen in sera of patients with Ieukaemia, Down s syndrome and hepatitis. Nature 218: 1057, 1969 3. ALMEIDA JD, RUBENSTEIN D, STOTY El: New antigen-antibody system in Australia-antigenpositive hepatitis. Lancet 2: 1225, 1971
4. ALMEIDA 3D, WAmsoN AP, TROWELL 3M,
5. 6.
7. 8. 9. 10. 11. 12. 13.
et al: The finding of virus-like particles in two Australia-antigen-positive human livers. Macrobios 2: 145, 1970 Daurscs G, SPENCE L: Virus-like particles in the liver and their relationship to Australia antigen. Lancet 1: 447, 1972 WILsON 5, LOGAN LM: Hepatitis B core antigen in the immunosuppressed chimpanzee. International symposium on viral hepatitis, Milan, Dec. 1974. Dev Blol Stand 30: 240, 1975 SEVER JL: Application of a microtechnique to viral serological investigations. J Immunol 77: 320, 1962 VYAs GN, SHULMAN NR: Hemagglutinatlon assay for antigen and antibody associated with viral hepatitis. ScIence 170: 332, 1970 FAUvEL M, BABsuic L, SHEAFF ET, et al: Preparation of a Dane core (hepatitis B) antigen for use in the immune electron microscopy test. Can I Mlcrobiol 21: 905, 1975 RosINsoN WS, GREENMAN RL: DNA polymerase in the core of the human hepatitis B virus candidate. / VIrol 13: 1231, 1974 RossNso.i WS, CLAYTON DA, GREENMAN RL: DNA of a human hepatitis B virus candidate. I VIrol 14: 384, 1974 KRUGMAN 5, HOOFNAGLE JH GERILFY RI, et al: Viral hepatitis, type i DNA polymerase activity and antibody to hepatitis B core antigen. N Engi J Med 290: 1331, 1974 SHULMAN NR, BAsucaa LF: Virus-like antigen, antibody, and antigen-antibody complexes in hepatitis measured by complement fixation. Science 165: 304, 1969
14. PURCELL RH, GERIN JL, ALMEIDA JB, et al:
Radioimmunoassay for the detection of the core of the Dane particle and antibody to it. Intervirology 2: 231, 1973/74
