Clin. exp. Immtunol. (1979) 37, 376-380.

Antigen-specific detection of HBsAG-containmg immune complexes in the course of hepatitis B virus infection W. PERNICE, C. P. SODOMANN*, G. LUBEN, F. R. SEILER & H. H. SEDLACEK Research Laboratories of Behringjwerke AG and *Department ofInternal Medicine, University of Marburg, 3550 Marburg/Lahn, W. Germany

(Acceptedfor publication 2 March 1979) SUMMARY

In recent studies extrahepatic manifestations of viral hepatitis have been recognized as immune complex diseases. Hepatitis B surface antigen (HBsAg) has been successfully identified in immune complexes, but the pathogenic role of HBsAg-containing immune complexes (IC) remains questionable. The subject of the present study was the antigen-specific determination of IC in the course of hepatitis B virus infection using a new 1IBsAg-specific IC test (Pernice & Sedlacek, 1978). This test is based on the following principle: rabbit anti-HBs-coated polystyrole test tubes are incubated with the IC-containing test sample. The HBsAg-containing IC bind to the solid phase by their free antigenic determinants. There theye can be quantified using a peroxidaselabelled anti-human IgG antibody. A good correlation was found between the level of HBsAg-containing immune complexes and the clinical state of six patients in a follow-up study. IC could be detected simultaneously with HBsAg and either decreased or disappeared before the occurrence of free anti-HBs. In the sera of an additional twenty-eight patients suffering from chronic active hepatitis, HBsAg-containing immune complexes were detected in 85% of cases. One patient suffering from polyarteritis nodosa was also positive. Occasionally extremely high levels of IC were found in the course of these diseases. IN TRODUCT ION In recent studies, extrahepatic manifestations of hepatitis B, like glomerulonephritis, polyarteritis, polyarthritis, skin rashes, etc. (Duffy et al., 1976) have been recognized as immune complex diseases. Deposited and circulating immune complexes (IC) have frequently been detected using different methods (Brzosko et al., 1974; Gocke, 1975; Michalak, 1978; Myers et al., 1973; Nagy et at., 1978; Nowoslawski et al., 1975; Adler, Takahashi & Wright, 1978; Carella et at., 1977; Dupuy, Lafforet & Dupuy, 1977; Fresco, 1978; Htiteroth et at., 1978; Krafft, Peitrequin & Frei, 1977; Levo et al., 1977; Nydegger et al., 1974; Theophilopopoulos et al., 1976; Thomas et at., 1978; Trepo et at., 1978; Sodomann et al., 1978). The detection of HBsAg and anti-HBs in these immune complexes has been a matter of controversy because their frequency varies greatly (Brzosko et at., 1974; Carella et al., 1977; Fresco, 1978; McIntosh, Koss & Cocke, 1976; Krafft et at., 1977; Levo et at., 1977; Michalak, 1978; Nagy et al., 1978; Prince 1971; Theophilopopoulos et al., 1976; Wands et at., 1975). It is therefore uncertain whether HBsAg-containing IC play any part in the pathogenesis of extrahepatic diseases of hepatitis B. The subject of the present study was, therefore, the antigen-specific determination of IC in the course of hepatitis B virus infection. As the methods used in the past were complicated and time consuming, we applied a new and simplified HBsAg-specific IC test method (Pernice & Sedlacek, 1978) for the determination of antigens in IC. Correspondence: Dr H. H. Sedlacek, Behringwserke AG, Postfach 11 40, 3550 Marburg 'Lahn, WX est GcernanN-. 0099-9104/79/0800-0376$02.00 C, 1979 Blackwell Scientific Publications

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MATERIAL AND METHODS Serial sera from six patients with acute type B hepatitis and single specimens from twenty-eight patients with chronic active hepatitis (CAH) and from one patient with polyarteritis nodosa (PAN) were preserved with sodium azide and stored at 4 C until testing within 2 weeks after bleeding. All patients were positive for HBsAg. Control sera from sixty normal subjects and two healthy HBsAg carriers were collected and stored at -20'C until testing. The antigen-specific IC test was performed as described recently (Pernice & Sedlacek, 1978). Briefly, rabbit anti-HBs were adsorbed to polystyrole test tubes. The tubes were then washed three times with PBS-Tween 20 (01 %), dried and stored at 4 C until use. Patients' sera were diluted 1: 4 in normal rabbit IgG (1",) and added to the test tubes. After an incubation time of 20 hr, the tubes were washed three times with a washing solution and incubated with a peroxidase-labelled rabbit anti-human IgG antibody (Behringwerke AG, No. B 30877) for 1 hr. After washing, the quantification of the label was carried out with an enzyme substrate reaction using ortho-phenylene-diamine-2HCl as a substrate. As a control, all patients' sera were tested simultaneously for non-specific reactions in rabbit normal IgG-coated polystyrene tubes. The difference between both results (test and control values) was evaluated as specific binding of HBsIC and registered. The Raji cell test (Theophilopopoulos et al., 1974) was performed as modified by Sedlacek & Schmidt (1978) using papainFc-fragments to block Fc-receptors; the Clq-polyethylene-glycol test (IC-Clq/p) was carried out according to Nydegger et al. (1974). Both assay systems were used as routine tests for non-specific HBsIC. For HBsAg and anti-HBs determinations, commercial radioimmunoassays (Abbott Laboratories) were used.

RESULTS

The follow-up studies in six patients gave the following results. One patient (case 1) had a normal course of an acute hepatitis B induced by a blood transfusion (Fig. 1). Immediately before the onset of the illness, he became positive for HBsAg. At this time, he was still negative for HBsIC although IC could be detected with the Clq-polvethylene-glycol test. During the acute stage of hepatic illness, the patient wuras positive for HBsAg as well as for HBsIC. Both parameters disappeared within 3 weeks and the patient became asymptomatic. The second patient (case 2) developed a chronic hepatitis after an injury with an injection needle (Fig. 2). Chronic active hepatitis had been proved by repeated histology 3 and 6 months after the onset of acute disease. In the early incubation period, HBsIC and HBsAg were negative. Unfortunately, in the late incubation period the patient could not be examined clinically. During the acute stage of hepatic illness, the patient was positive for HBsAg and for HBsIC. Subsequently, he remained positive for HBsAg and for HBsIC during a further 3 month period of observation. In four other cases, serial sera were tested for anti-HBs, in addition to HBsAg and HBsIC (Fig. 3). One patient (Case 3, Fig. 3a) had a normal course of hepatitis B. HBsIC and HBsAg disappeared simultaneously with clinical recovery, after which free anti-HBs appeared. e e e + + e 500

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The acute hepatitis B of another patient w as characterized by clinical symptoms, an extremely higrh and persistent level of serum GOT and GPT and by a rising concentration of HBsAg and falling concentrations of HBsIC (case 4, Fig. 3b). Anti-HBs where not detectable during the time of observation. Txa o other patients (cases 5 & 6)); Fig. 3c,d), both writh underlying malignant diseases (chronic lymphatic leukaemia and Morbus Hodgkin) remained positive for HBsIc and HBsAg; anti-HBs Were not detectable. The investigation of single specimens of patients w ith chronic active hepatitis gave the followings results: twsent--four sera from twenty--eight patients with HBsAg-p~ositive CAH (850 ) and one serum from a patient writh PAN were positive for HBsIC. Occasionally, extremely high levels of Hl~sIC were detectable not oniy in CAP! but also in the serum of the patient w ith PAN. Sera from sixty normal controls and two sera from twvo healthy HBsAg carriers were negative fo~r HI~sIC. and non-antigen specificallx chronic hepatitis w*Q ere ~tested Fourteen patients with acute *0and~~~~~~~~~Q ~ for~HBsIC ~ ~~ for IC using the Raji cell test as modified by Sedlacek & Schmidt (1978). Non-specific immune complexes were found in all sera, whilst HI3sIC were detected in ten out of fourteen patients studied. No patient fleas found to be positive fo~r HI~sIC alone (Fig. 4).

DIS>

Antigen-specific detection of HBsAG-containing immune complexes in the course of hepatitis B virus infection.

Clin. exp. Immtunol. (1979) 37, 376-380. Antigen-specific detection of HBsAG-containmg immune complexes in the course of hepatitis B virus infection...
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