Evaluation of Enzyme Immunoassay for Diagnosis of Hepatitis

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1981, p /81/ $02.00/0 Vol. 13, No. 4 Immunoglobulin M Antibodies to Hepatitis B Core Antigen: Evaluation of Enzyme Immunoassay for Diagnosis of Hepatitis B Virus Infection M. ROGGENDORF, F. DEINHARDT,* G. G. FROSNER, R. SCHEID, B. BAYERL, AND R. ZACHOVAL Max von Pettenkofer-Institut fur Hygiene und Medizinische Mikrobiologie, University ofmunich, 8000 Munich 2, Federal Republic of Germany Received 1 October 1980/Accepted 12 January 1981 In acute and chronic hepatitis B, antibodies of the immunoglobulin M (IgM) class against the hepatitis B core antigen (anti-hbc IgM) have been demonstrated. For the determination of anti-hbc IgM, a sensitive enzyme immunoassay with anti-,u-coated flat-bottomed microtiter plates is described and evaluated. The specificity of the anti-hbc IgM test system was proven by pretreatment of presumed anti-hbc IgM-positive samples with anti-m to block anti-hbc IgM. The test system was highly sensitive. In the acute stage of hepatitis B, anti-hbc IgM could be demonstrated in serum dilutions up to 10-7 (mean titer, 10-5), and in sera from patients with chronic hepatitis B, the mean titer was In a study of unselected patients whose sera were sent at irregular intervals for testing, anti- HBc IgM persisted in a high percentage (52%) for at least 13 to 18 months after onset of illness despite the fact that these patients eliminated hepatitis B surface antigen (HBsAg) and produced antibodies to HBsAg (anti-hbs). By using the anti-hbc IgM test as an additional aid in the diagnosis of acute HBsAg-negative hepatitis, the hepatitis B etiology could be established in 13 of 42 patients (31.4%). Investigations of the prevalence of anti-hbc IgM in different groups of patients with chronic hepatitis B infection showed 89.4% anti-hbc IgM-positive results in patients with chronic active hepatitis B, 60% in patients with HBsAg-negative chronic active hepatitis, 58.2% in patients with primary liver carcinoma and markers of hepatitis B infection, and 34.9% in healthy carriers of HBsAg. Detection of antibodies of the immunoglobulin M (IgM) class has become important for diagnosing infection with several viruses, such as hepatitis A virus (3, 4, 12), rubella virus (10), cytomegalovirus (12a), and hepatitis B virus (1, 7, 9). In the early acute phase of infection with hepatitis B virus, for example, antibodies to hepatitis B core antigen (anti-hbc) develop, and these have been shown to belong mainly to the IgM class (anti-hbc IgM). Such IgM antibodies have been detected by the use of anti-,u-coated, solid-phase techniques, and recently a highly sensitive enzyme immunoassay specifically designed to demonstrate anti-hbc IgM was described (7). Identification of anti-hbc IgM is particularly useful in distinguishing between acute or past infections with hepatitis B virus, particularly in those patients who are already negative for hepatitis B surface antigen (HBsAg) in the late acute phase of the disease. The tests described for anti-hbc IgM are highly sensitive and specific, but for each system, the optimal dilutions of sera and reagents need to be determined; the purposes of this study were to establish the conditions favoring greatest specificity and sensitivity of the anti-hbc IgM test system, to investigate the persistence of anti-hbc IgM after the acute stage of hepatitis B virus infection, and to evaluate the significance of anti-hbc IgM titers in acute and chronic infections. Also, groups of patients with acute hepatitis who were negative for HBsAg in the acute stage of illness, patients with chronic active hepatitis, patients who were carriers of HBsAg, and patients with primary hepatocellular carcinoma were tested for anti-hbc IgM as well as for the other known markers of hepatitis B virus infections. MATERIALS AND METHODS Sera. The following sera were used. (i) Sequential sera drawn at different times up to 18 months after onset of illness from 57 patients with acute hepatitis 618

2 VOL. 13, 1981 B were sent to us for routine diagnostic screening. Ail patients eliminated HBsAg and developed antibodies to HBsAg (anti-hbs). (ii) Sera from 19 patients with serologically and histologically proven chronic active hepatitis B were supplied in 1979 by J. Eisenburg, Department of Internal Medicine, University of Munich, Germany. (iii) Sera from 42 patients with a clinical diagnosis of acute hepatitis who were HBsAg negative but anti-hbc positive were sent to us for routine diagnostic screening. (iv) Sera from 18 patients with a histological diagnosis of chronic active hepatitis and who were HBsAg negative were collected in 1978 by H. Bannaski, Privatklinik Kempfenhausen, Germany. (v) Sera from 91 patients with primary hepatocellular carcinoma and with markers for past or ongoing hepatitis B infection and 62 sera from a control group were kindly supplied by J. J. Alexander, National Institute of Virology, and M. C. Kew, University of Witwatersrand Medical School, Johannesburg, South Africa. (vi) Sera from 106 healthy carriers of HBsAg (blood donors) were obtained from the Bavarian Red Cross Blood Bank. (vii) Sera from the staff of the Max von Pettenkofer-Institute were used as negative controls. Preparation of peroxidase-coupled anti-hbc. The anti-hbc IgG fraction was obtained from the serum of a blood donor who was anti-hbc positive, with an anti-hbc titer of io-5 as determined by radioimmunoassay (CORAB; AbbottLaboratories, North Chicago, III.). The IgG fraction was precipitated three times in 45% saturated ammonium sulfate solution and dialyzed overnight against phosphatebuffered saline. The fraction was purified further by chromatography on diethylaminoethyl cellulose. Coupling of the anti-hbc IgG fractions with horseradish peroxidase (Sigma Chemical Co., St. Louis, Mo.) was performed as described previously (15). Anti-HBc IgM test. Anti-HBc IgM was determined by the Gerlich and Luer technique (7) with the following modifications. Flat-bottomed microtiter plates (Cooke Engineering, Alexandria, Va.) were incubated overnight at 4 C with 50 pl of an anti-g IgG fraction of rabbit serum (DAKO Immunoglobulins, Copenhagen, Denmark) diluted 10-3 in 0.1 M carbonate buffer (ph 9.4). Thereafter, the plates were washed three times with a semiautomatic microtiter plate washer (Dynatech, Alexandria, Va.) with phosphatebuffered saline (ph 7.4) containing 0.05% Tween 20 (Serva, Heidelberg, Germany). A 50-,l amount of the patient's serum diluted in phosphate-buffered saline containing 3% Tween 20 and 1 mg of aggregated anti-hbc-negative IgG per ml (2) was added. For screening purposes, sera were diluted routinely 1:100 (dilutions used for titration are shown below). After incubation for 2 h at 37 C, the plates were washed, and 50 pl of a hepatitis B core antigen (HBcAg) preparation diluted 1:20 in phosphate-buffered saline containing 1% anti-hbc-negative human serum was added and incubated overnight at 4 C. HBcAg was prepared from the liver of a patient with chronic hepatitis B as described previously (7). IgM ANTIBODIES TO HEPATITIS B CORE ANTIGEN 619 After a further washing procedure, 50 Ail of a horseradish peroxidase-conjugated IgG fraction of anti-hbc was added. The conjugate was diluted 10-3 in phosphate-buffered saline containing 1% human serum negative for anti-hbc and incubated for 2 h at 37 C. After a final washing, orthophenylenediamine (Merck, Darmstadt, Germany) (1 mg/ml) and H202 (1 pl/ml in phosphate buffer, ph 6.0) were added. After 30 min, the enzymatic reaction was stopped with 50 pl of 2 N H2SO4. The optical density at 492 nm (OD492) of the yellow color was measured with an eight-channel photometer (Multiscan; Flow Laboratories, Inc., Rockville, Md.). Samples showing an OD times higher than those of negative controls were considered positive. Determination of hepatitis B markers other than anti-hbc IgM. HBsAg, anti-hbs, and anti-hbc (IgG plus IgM) were determined by commercially available tests (AUSRIA Il, AUSAB, and CORAB; Abbott Laboratories) Hepatitis B e antigen (HBeAg) and anti-hbe were determined by radioimmunoassay as described previously (6). RESULTS Specificity and sensitivity of the anti- HBc IgM test system. To determine the optimal dilution of anti-g for coating the solid phase, decreasing concentrations of anti-g were added to the microtiter plates. Up to a dilution of 1:5,000 of the rabbit anti-g immunoglobulin fraction, the test measured a high OD492 with serum from a patient in the beginning phase of acute hepatitis B. For all further tests, anti-pu was diluted 1:2,000 for coating the solid phase. The specificity of the reaction of the rabbit anti-p with human IgM, but not with human IgG, was shown by coating microtiter plates with 25 gl of serial dilutions of purified human IgG (15 mg/ml) and human IgM (3.4 mg/ml), incubating the plates at room temperature for 6 h, washing the plates, and then adding horseradish peroxidase-coupled anti-,u (the same anti-g as used in the anti-hbc IgM test). Anti-,u reacted with the IgM coated to the solid phase up to a dilution of 1:164,000, from which it may be calculated that the minimal amount of IgM detectable by this method was 0.2,.tg/ml. No reaction was observed with purified human IgG. The optimal concentration of HBcAg was tested as follows. The solid phase was coated with a 10-3-diluted human anti-hbc IgG fraction; after washing, various concentrations of HBcAg were added, and the plates were incubated overnight at 4 C. After an additional washing cycle, peroxidase-coupled human anti- HBc was added, and the test was completed as described for the routine test of anti-hbc IgM. With this technique, binding of HBcAg could be detected in dilutions up to 1:80 of the HBcAg preparation used in this study (Fig. la). Titration of HBcAg in the anti-hbc IgM test is shown in Fig. lb. To maintain a background OD492 equal to or lower than 0.2, in ail subsequent

3 620 ROGGENDORF ET AL. O.D. 1,2 e 1,1 1,0 o9 0,8 a O.D. 1,2 1,1 1,0 0,9 0,8 b * serum anti HBc bgg positive * and anti HBc lgm positive A serum anti HBeC IgG positive and anti HBc IgM negative negative serum J. CLIN. MICROBIOL. 0,7 0,6 0,5 04 0,3 02 c' 0,l vi ut-off ahie e :80 dituon of HBc Ag 0,7 o,6 os 0,4 0,3 0,2 0,1_ \es 1, "00 dilution of HBc Ag FIG. 1. Titration of HBcAg. (a) Sandwich technique with an anti-hbc-coated solid-phase HBcAg and peroxidase-coupled anti-hbc IgG fraction; (b) anti-hbc IgM test with a serum from the acute stage of hepatitis B, a serum negative for anti-hbc (IgM plus IgG), and a serum which was anti-hbc IgG positive but anti-hbc IgM negative. tests, HBcAg was diluted to 1:20. Blocking with anti-,i was used to establish the specificity of the system. Sera from patients with acute and chronic hepatitis B were diluted to 10-2; samples were divided, and one-half of the samples were preincubated with anti-m (diluted 1:10) for 1 h at 37 C. The OD492 of sera after preincubation fell below the positive level. Sera containing rheumatoid factor produced no falsepositive reactions when the sera were diluted 1:100 in phosphate-buffered saline containing 3% Tween 20 and 1 mg of aggregated human IgG per ml. A total of 20 sera without markers of previous hepatitis B infections but which were positive for rheumatoid factor (latex test; Behringwerke, Marburg, Germany) gave negative results in the anti-hbc IgM test when examined under these conditions. The sensitivity of the method was tested by titration of sera drawn at the acute, convalescent, and later stages of illness for anti-hbc IgM (Fig. 2). In week 1 after onset of illness, anti- HBc IgM was demonstrated up to a dilution of 10-6 to 10-7 and decreased to 10-3 to months later. Of interest and importance is the striking prozone seen in the sera drawn 1 week and 2 months after onset of illness and the minor prozone observed only at the dilution of 10-1 in sera drawn at 3.5 months or later. Anti-HBc IgM in patients with acute and chronic hepatitis, in patients with primary hepatocellular carcinoma, and in healthy carriers of HBsAg. (i) HBsAg-positive acute hepatitis B. Figure 3 shows the persistence of anti-hbc IgM in the sera of 57 patients drawn at different times up to 18 months after onset of illness. Within the first 2 months after onset of disease, all 65 sera were positive for anti-hbc IgM. At 3 and 4 months after onset of illness, 32 of 36 sera (88%) were still anti-hbc IgM positive, and the positive results decreased to 60% (23 of 38 sera) at 7 to 12 months and to 52% (10 of 19 sera) at 13 to 18 months after onset of illness. Sera of the 10 patients positive for anti-hbc IgM drawn 13 to 18 months after onset of illness had a mean titer of 10-3 (data not shown), a titer similar to that seen in patients with chronic hepatitis. The sample OD492/negative control OD492 ratio correlated to some extent with the time at which the serum was drawn after onset of illness. The mean ratio of positive sera in the anti-hbc IgM test was about 8.6 during the first 2 months and declined with increasing time after onset of disease to 3.7 at 13 to 18 months after onset of illness. The low OD492 ratios in some sera in the 1 to 2- and 3 to 4-month groups probably were due to prozone effects, but no more serum was available for retesting at a 10- dilution. These sera were sent to us for routine diagnostic evaluation so there may have been some unintentional selection because physicians continued to submit samples

4 VOL. 13, 1981 IgM ANTIBODIES TO HEPATITIS B CORE ANTIGEN 621 QO 1- O,8 0,l o o --- o -o _ o I», O OX 1O 7 dition f FIG. 2. Titration of anti-hbc IgM in sera from a patient drawn at 1 week (@), 2 months (-), 3.5 months (*), 7 months (U), 9 months (A), and 14 months (*) after onset of hepatitis B. Sera were diluted in phosphate-buffered saline containing 1 mg of aggregated IgG per ml and 3% Tween 20. 0, Negative control. from patients with slow recoveries, but all patients eliminated HBsAg and developed anti- HBs within the 18-month observation period. (il) HBsAg-negative acute hepatitis B. To test whether HBV was the cause of disease, a group of 42 patients with acute hepatitis who were anti-hbc positive (IgG plus IgM, CORAB test) but negative for HBsAg when serum was first drawn were examined for anti-hbc IgM. Of the 42 sera, 13 (31.4%) were anti-hbc IgM positive. Table 1 shows the titers of anti-hbc (IgG plus IgM, CORAB test) and the titers of anti- HBc IgM. In five of six patients with a second serum sample available, HBV etiology could be verified by a seroconversion to anti-hbs 6 to 12 months after onset of illness. The serum from the one patient who did not develop anti-hbs was obtained 6 months after onset of the disease, and a later seroconversion may have occurred. In general the anti-hbc IgM titer in these cases were somewhat lower than those observed in HBsAg-positive cases which may be due to less virus activity in these patients, but the number of cases evaluated is too small for conclusions to be drawn. o O.D. sample 0.0. negative control il E L0 L. t L I s. s s 0 0 r j:... s s i 3. F i r -t--- - e. ee0 s _e_ month months montts imono months 100%D 88% 59% 60% 52% * %O positive for ant - HBc -IgM FIG. 3. Sample OD492/negative control OD492 ratios for 180 sera from 57 patients drawn at different' times after onset of hepatitis B. Ail sera were tested in dilutions of (iii) HBsAg-positive chronic active hepatitis B. The prevalence of anti-hbc IgM was studied in chronic hepatitis B patients with different patterns of expression of infection. In Table 2, data from 19 patients with HBsAgpositive chronic active hepatitis are summarized; 13 patients were positive for HBeAg, 6 were positive for anti-hbe, and 17 (89%) were anti- HBc IgM positive. The two sera negative for anti-hbc IgM were anti-hbe positive. Most patients had high titers (in 16 of 19 patients, equal to or higher than i(-5) of anti-hbc (IgG plus IgM, CORAB test). The titers of anti-hbc IgM were i0-o in eight sera, 1O-' in six sera, and 1i-5 in three sera. (iv) HBsAg-negative chronic active hepatitis B. A total of 18 patients with a histological

5 622 ROGGENDORF ET AL. TABLE 1. Determination of anti-hbc (IgG plus IgM, CORAB test) and anti-hbc IgM in sera of 13 patients with HBsAg-negative acute hepatitis Time Anti- Anti- Anti-HBs 6 Pa- Age onset of titer HBc to 12 mo tent (yr) illness (IgG + IgM ti- after onset (dys).igm ter of illness (days) Igm) " 10-2 NT' NT " 10-2 NT , 10- Positive " 10-4 Positive " 10-2 Negativeb 7 28 il NT ' NT >- lo-1 Positive NT il ' NT e5 Positive Io-0 Positive 'NT, Not tested. b Tested 6 months after onset of illness. TABLE 2. Anti-HBc titer (IgG plus IgM, CORAB test) and anti-hbc IgM in patients with chronic HBsAg-positive hepatitis B (n = 19)a Titer of anti- No. of anti-hbc HBc (IgG + Total no. of sera.eb IgM, CORAB) IgM-positive sera O 1-3_ (1) 1 (1) 1(-4_1(--5 2 [2] 1 [1] 1(-5_ (12) [4] 15 (12) [3] a Number of HBeAg-positive sera is shown within parentheses; number of anti-hbe-positive sera is shown within brackets. b Total positive, 89%. TABLE 3. Anti-HBc titer (IgG plus IgM, CORAB test) and anti-hbc IgM titer in patients with HBsAg-negative chronic active hepatitis (n = 18) Anti-HBc titer Total patients no. of No. of anti-hbc IgM-positive serah 10o î_ (10-4) 10-2_1O (-3_ (10-) (10-3, 10-4) ; 2 2 (10-4, 10-4) a Eight were b anti-hbc negative. Anti-HBc IgM titers are shown within parentheses; total positive, 60%. diagnosis of chronic active hepatitis who were negative for HBsAg were tested for anti-hbc (IgG plus IgM, CORAB test) and anti-hbc IgM (Table 3). Of the 18 patients, 10 were anti-hbc (IgG plus IgM) positive, 5 had low titers (equal to or lower than 10-2), and the other half had titers from 10-3 to Of these 10 patients, 6 J. CLIN. MICROBIOL. (60%) were positive for anti-hbc IgM. The titers of the anti-hbc IgM in the sera of these six patients varied from 10-3 to (v) Primary hepatocellular carcinoma. A total of 91 sera of patients with primary hepatocellular carcinoma and 62 sera of a control group collected by M. C. Kew (group 1) and J. J. Alexander (group 2) were tested for anti-hbc IgM (Table 4). (a) Group 1. Of the 10 HBsAg-negative hepatoma patients, who all showed other markers of past hepatitis B infection (anti-hbc or anti- HBs or both), only 1 serum was positive for anti- HBc IgM, whereas of 31 HBsAg-positive hepatoma patients, 15 sera were positive for anti- HBc IgM (48.3%). In contrast to the hepatocellular carcinoma patients, only 4.1% of the HBsAg-negative and 15.3% of the HBsAg-positive sera of the control group were positive for anti-hbc IgM. The percentage of anti-hbc IgM positive results was significantly higher in patients with primary hepatocellular carcinoma (39%) than in sera of the control group (6.5%). A more detailed comparison of other markers of hepatitis B within these two groups will be published elsewhere (J. Desmyter, M. C. Kew, and F. Deinhardt, Prog. Med. Virol., in press). (b) Group 2. Of 50 sera from patients with primary hepatocellular carcinoma collected by J. J. Alexander, a higher prevalence of anti-hbc IgM was found. Of the 50 sera, 37 were positive for anti-hbc IgM (74%). Of 33 HBsAg-positive hepatocellular carcinoma patients, 32 were anti- HBc IgM-positive (96.9%), whereas only 29.4% of the HBsAg-negative hepatocellular carcinoma patients were positive. Most of the HBsAg-positive patients had higher titers of anti-hbc (IgG plus IgM, CORAB test) than did the HBsAg-negative hepatocellular carcinoma patients (data not shown). (vi) Carriers of HBsAg. A group of 106 healthy carriers of HBsAg, all blood donors from the Bavarian Red Cross Blood Bank with normal transaminases, were tested for anti-hbc IgM and other markers for hepatitis B infection (HBeAg, anti-hbe, anti-hbc). Thirty-seven of these carriers (34.9%) were positive for anti-hbc IgM. The titer of anti-hbc IgM ranged from 10-2 to Details on the correlation of titers of anti-hbc (IgG plus IgM, CORAB test) and the presence of anti-hbc IgM will be published separately (G. G. Frosner, M. Roggendorf, B. Bayerl, R. Scheid, R. Zachoval, and F. Deinhardt, manuscript in preparation). Comparison of titers and frequencies of anti-hbc IgM in different patient groups. The titers of anti-hbc IgM in patients with acute and chronic hepatitis were compared (Fig.

6 VOL. 13, 1981 TABLE 4. IgM ANTIBODIES TO HEPATITIS B CORE ANTIGEN 623 Anti-HBc IgM in sera of 91 patients with primary hepatocellular carcinoma and of 62 control patients Group la Group 2b Group 1 + group 2 Patient Serum response No. positive/total % No. positive/total % No. posi- tive/total % no. no. no. Hepatoma HBsAg negative 1/ / / HBsAg positive 15/ / / Total 16/ / / Control HBsAg negative 2/ / HBsAg positive 2/ / Total 4/ / a Obtained from M. C. Kew. b Obtained from J. J. Alexander. dbloedof mm diuion of ser FIG. 4. Titration of anti-hbc IgM with 10 sera ofpatients with acute hepatitis B (a) and 10 sera patients with chronic hepatitis B (b). 4a and 4b). Anti-HBc IgM titers were, on the average, higher in patients from whom blood was drawn during the first 4 weeks of illness, and prozones were observed in most of these sera. Anti-HBc IgM titers of 14 of 20 (70%) randomly selected patients with acute hepatitis B had titers equal to or higher than i0-5, whereas 18 of 20 (80%) randomly selected patients with chronic active hepatitis had titers equal to or lower than 10-'. In Table 5, the prevalence of anti-hbc IgM in different groups of patients is shown. Anti-HBc IgM was always found in acute HBsAg-positive hepatitis B but only in 31.4% of HBsAg-negative acute hepatitis. Anti-HBc IgM was found in a high frequency in chronic hepatitis (chronic active HBsAg-positive hepatitis, 89.4%; chronic active HBsAg-negative hepatitis, 60%). A significant difference in the prevalence of anti-hbc IgM was found in patients with HBsAg-positive hepatocellular carcinoma (73.4%) and with HBsAg-negative hepatocellular carcinoma (22.2%). In healthy carriers of HBsAg, the prevalence of anti-hbc IgM was about 34.9%. DISCUSSION In the present investigation, each step of the test procedure was studied separately to deter-

7 624 ROGGENDORF ET AL. TABLE 5. Prevalence of anti-hbc IgM in different population groups' Patient and serum response No. positive/ % total no. Acute hepatitis B, HBsAg 57/ positive Acute anti-hbc positive, 13/ HBsAg negative hepatitis Chronic active hepatitis, 17/ HBsAg positive Chronic active hepatitis, 6/ HBsAg negative Hepatoma, HBsAg positive 47/ Hepatoma, HBsAg negative 6/ Healthy carriers of HBsAg 37/ a All sera were tested at a dilution of mine the optimal conditions. It was shown that the anti-.i IgG fraction used in our test for coating the microtiter plates had a high antibody titer and could therefore be diluted in carbonate buffer up to 1:5,000 without loss of activity. It was also demonstrated that the anti-i IgG fraction reacted specifically only with immunoglobulins of the IgM class. There was no reaction of the peroxidase-coupled anti-m with the IgGcoated solid phase. To avoid nonspecific reactions caused by rheumatoid factor and nonspecific binding of anti-hbc IgG molecules to the solid phase, it was necessary to add 1 mg of aggregated IgG and 3% Tween 20 to the diluent used for serum dilutions. A further critical point in the test system was the optimal dilution of HBcAg, which was prepared as described by Gerlich and Lüer (7). It has been shown that high concentrations of semipurified antigens can lead to nonspecific reactions with negative sera (12) due to impurities in the antigen preparations. Therefore, it is necessary to use antigen dilutions which give a low reaction with negative sera but sufficiently high reactions with positive sera to give a high positive serum OD492/negative control OD492 ratio. As already shown for anti-hepatitis A virus IgM (12), the anti-,u-coated solid-phase test system was very sensitive. The test for anti-hbc IgM revealed similar sensitivity with anti-hbc IgM titers of up to 10-' in the acute phase of illness. However, in both methods, there was often a prozone effect in sera drawn during the first 2 months after onset of the disease (Fig. 2). The low sample OD492/negative control OD492 ratio seen in some of the sera drawn at an early stage after onset of illness and tested in a dilution of 10-2, as shown in Fig. 3, may be explained by this prozone effect. As a prozone effect was never demonstrated at a 10-3 dilution, both 10-2 and 10-3 dilutions should be tested if one wants to J. CLIN. MICROBIOL. exclude this effect. Comparison of anti-hbc IgM titers in acute and chronic hepatitis B. Calculation of the amount of anti-hbc IgM in acute and chronic hepatitis B by measuring the OD492 of only one serum dilution as described by Gerlich et al. (8) was not entirely satisfactory in our test system because of the prozone effect. Therefore, titrations of anti-hbc IgM were done for this comparison. In acute hepatitis B, anti-hbc IgM had a mean titer of about i0-5, whereas in chronic hepatitis B, the mean titer of anti-hbc IgM was about 10-k. The titers of anti-hbc IgM in chronic hepatitis corresponded to titers of anti-hbc IgM in sera from patients with acute hepatitis B drawn 13 to 18 months after onset of illness and lower anti-hbc IgM titers (10-2 to 10-3) were also observed in a number of HBsAgnegative acute hepatitis B infections. Clinical application of the anti-hbc IgM test. It has been demonstrated (J. Aldersvhile, M. Roggendorf, P. Kryger, U. Tage-Jensen, F. Deinhardt, G. G. Frosner, F. Hardt, and J. O. Nielsen, The Liver, in press) that patients with acute hepatitis B who showed elimination of HBsAg and complete recovery had a short persistence (less than 6 months) of anti-hbc IgM, whereas patients with elimination of HBsAg but no clinical recovery had a much longer persistence (up to several years) of anti-hbc IgM. In our study of 57 patients with acute HBsAg-positive hepatitis (Fig. 3), we found in spite of an elimination of HBsAg and the appearance of anti-hbs a long persistence of anti-hbc IgM (52% still positive after 13 to 18 months). As we have no clinical data regarding the recovery of these patients from the acute disease, we cannot exclude an overrepresentation of patients with a slow recovery. This may actually be the case, as these were unselected patients evaluated routinely by our diagnostic service, and it is likely that late sera were submitted predominantly from patients with persisting disease. These data suggest that the disappearance of anti-hbc IgM in addition to the elimination of HBsAg and the appearance of anti-hbs may be an additional indication of a complete recovery from hepatitis B. The relatively long persistence of anti-hbc IgM even in patients who eliminated HBsAg within 1 to 3 months after onset and developed anti-hbs may have been caused by an otherwise undetectable continued virus multiplication or, less likely, by an exceptionally efficient stimulation of antibodies of the IgM class by HBcAg. As anti-hbc IgM is found in acute and chronic hepatitis B infection, its significance as a diagnostic tool is restricted. However, in acute hepatitis B, which is negative for HBsAg when the

8 VOL. 13, 1981 first serum is drawn, the demonstration of anti- HBc IgM, especially at high titers, is an important additional diagnostic parameter for defining a hepatitis B etiology and for separating these forms of hepatitis from non-a-non-b forms of hepatitis. In this study, a diagnosis of hepatitis B infection could be made with the help of demonstration of anti-hbc IgM in 13 of 42 patients with acute HBsAg-negative hepatitis. Of similar diagnostic importance was the demonstration of anti-hbc IgM in patients with HBsAg-negative chronic active hepatitis. Of 10 patients with HBsAg-negative chronic active hepatitis, 6 were anti-hbc IgM positive, and all but 1 had high titers of anti-hbc (IgG plus IgM, CORAB test) as found in patients with HBsAg-positive chronic active hepatitis (5). Demonstration of both anti-hbc IgM and high titers of anti-hbc (IgG plus IgM) can further strengthen the diagnosis of a hepatitis B infection in such instances because presence of anti-hbc IgM probably indicates persistent hepatitis B virus activity, and this combination of antibodies is similar to that observed in HBsAg-positive chronic hepatitis. Previous studies documented a close association of hepatocellular carcinoma and markers of hepatitis B (14). In our investigation of sera from patients with hepatoma and a control group, a significant difference of prevalence of anti-hbc IgM was found. Of importance seems to be the difference of prevalence of anti-hbc IgM in HBsAg-positive hepatoma patients (48.3%) and the HBsAg-positive control group (15.3%), as other hepatitis B markers, for example, HBeAg, revealed no significant differences between these two groups (J. Desmyter, M. C. Kew, and F. Deinhardt, Prog. Med. Virol., in press). The frequency of anti-hbc IgM in different population groups (Table 5) suggests that the demonstration of anti-hbc IgM indeed might be correlated with the persistence of HBcAg in the blood or liver. This is likely because 13 of the 17 patients with chronic active hepatitis and positive for anti-hbc IgM (Table 2) were also positive for HBeAg, and as the presence of HBeAg in the serum correlates with the presence of Dane particles and with polymerase activity (11, 13), HBcAg must also be produced. Demonstration of HBcAg by the fluorescent-antibody technique in liver biopsies of patients with chronic hepatitis would provide more direct evidence in this regard, especially in patients without circulating HBeAg, but such studies were not possible in this retrospective investigation. Anti-HBc IgM was less frequent (34.9%) in healthy carriers of HBsAg and perhaps reflected only a partial IgM ANTIBODIES TO HEPATITIS B CORE ANTIGEN 625 expression of the hepatitis B virus in anti-hbc IgM-negative cases with the production of HBsAg only. In summary, presence of anti-hbc IgM seems to indicate current or recent hepatitis B virus multiplication, although further studies are needed to correlate more precisely the presence of HBcAg, other hepatitis B virus components, and complete virus in the tissues and the circulation of patients, convalescents, and carriers. Nevertheless, measurement of anti-hbc IgM titers provides an additional parameter for diagnosing acute cases of hepatitis and for follow-up studies on healthy hepatitis B virus carriers or patients with chronic disease. ACKNOWLEDGMENTS We thank Brunhilde Bohm for excellent technical assistance. These studies were supported in part by grant Fr. 400/8 from the Deutsche Forschungsgemeinschaft. ADDENDUM IN PROOF and low anti-hbc IgM titers the theoretical possibility of an occasional nonspecific cross-reaction of anti-hbc IgG in the anti-hbc IgM should be taken into consideration. This was not observed in representative sera used in this study in additional blocking experiments with anti-g (as described in the text, Results section), but it is recommended that low anti-hbc IgM in sera with very high anti-hbc IgG titers should be verified. In sera with very high anti-hbc IgG (>10-a) LITERATURE CITED 1. Cohnen, B. J The IgM antibody responses to the core antigen of hepatitis B virus. J. Med. Virol. 3: Dissanayake, S., F. C. Hay, and I. M. Roitt The binding constants of IgM rheumatoid factors and their univalent fragments for native and aggregated human IgG. Immunology 32: Duermeyer, W., and J. van der Veen Specific detection of IgM antibodies by ELISA applied in hepatitis A. Lancet ii: Flehmig, B Laboratoriumsdiagnose der Hepatitis A-Infektion. 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