Klinische Wochenschrift

Klin. Wochenschr. 57, 1129-1132 (1979)

© Springer-Verlag 1979

Solid-Phase Radioimmunoassay for Hepatitis Be Antigen (HBeAg) H.E. Blum, G. D61ken, and W. Gerok Medizinische Klinik der Universitfit Freiburg i. Br.

Solid-Phase Radioimmunoassay fdr Hepatitis Be Antigen (HBeAg) Zusammenfassung. Ein Radioimmunoassay wurde entwickelt zum Nachweis von HBeAg und anti-HBe in Serum oder Serumfraktionen. HBe/sAg positives Serum, teilweise gereinigtes HBeAg, teilweise gereinigtes HBsAg und HBe/sAg negatives Kontrollserum wurden in Polyacrylamid polymerisiert und auf ihre Bindungsffihigkeit ffir 125I-IgG (anti-HBe) untersucht. Nur Gele mit immobilisiertem HBeAg reagierten spezifisch mit anti-HBe. Die Spezifit/it der Bindung wurde gesichert durch Blockierungs-/Inhibitionsteste mit anti-HBe, HBeAg, HBsAg und negativen Kontrollseren. Der Radioimmunoassay erm6glicht den spezifischen und quantitativen Nachweis von HBeAg und anti-HBe, auch in Gegenwart von Detergentien und hohen Salzkonzentrationen. Schliisselwiirter: Hepatitis B - HBsAg - HBeAg Anti-HBe - Solid-Phase Radioimmunoassay

Summary. A solid-phase radioimmunoassay was developed for the detection o f HBeAg and anti-HBe in sera or serum fractions. HBe/sAg positive sera, partially purified HBeAg, partially purified HBsAg, and HBe/sAg negative sera were polymerized in polyacrylamide and compared for their ability to bind 12sI-IgG (anti-HBe). Only gels containing HBeAg reacted specifically with the iodinated antibody. The Abbreviations: HBsAg hepatitis B surface antigen. HBeAg hepatitis Be antigen. - HBe/sAg hepatitis Be antigen and surface antigen. - anti-HBe antibody to hepatitis Be antigen Offprint requests to." Prof. Dr. W. Gerok (address see page 1132)

specificity of the binding was confirmed by blocking and inhibition tests using anti-HBe, HBeAg, HBsAg, and negative control sera. The radioimmunoassay allows the specific and quantitative detection of HBeAg and anti-HBe even in the presence of detergents and high salt concentrations.

Key words: Hepatitis B - HBsAg - HBeAg - anti-HBe Solid-Phase Radioimmunoassay

In 1972 a new antigen associated with hepatitis B-positive sera was described by Magnius and Espmark [13, 14]. This antigen was demonstrated to be different from the known LeBouvier determinants of hepatitis B surface antigen (HBsAg [12]) and was later termed hepatitis Be antigen (HBeAg [21]). HBeAg is present in serum in the late incubation period and the early phase of acute hepatitis B infection and is followed by the development of anti-HBe [7, 11]. The HBe antigen-antibody system proved to be an excellent marker for the assessment of infectivity and prognosis of hepatitis B [1, 9, 16-19]. Most patients with HBsAg-positive chronic active hepatitis are also positive for HBeAg, whereas HBsAg-positive carriers with no evidence for active liver disease often are positive for anti-HBe [11]. With respect to the biochemical nature of the HBeAg, it is still unknown whether this antigen is an integral part of the virus or whether it reflects some sort of host response to hepatitis B infection. The HBeAg was proposed to be associated or even identical with, among others, DNA-polymerase [1], IgG subclass 4 [6, 15], and lactate dehydrogenase isozyme 5-ex [20], respectively. Since immunodif-

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H.E. Blum et al. : Solid-Phase Radioimmunoassay for Hepatitis Be Antigen (HBeAg)

fusion, r h e o p h o r e s i s , c o u n t e r e l e c t r o p h o r e s i s , a n d passive h e m a g g l u t i n a t i o n are o f low sensitivity a sensitive a s s a y for the d e t e c t i o n o f H B e A g h a d to be d e v e l o p e d in the c o u r s e o f the p u r i f i c a t i o n a n d studies o n the b i o c h e m i c a l p r o p e r t i e s o f H B e A g . This r e p o r t describes a highly sensitive p r o c e d u r e f o r the d e t e c t i o n o f H B e A g a n d a n t i - H B e , a n a l o g o u s to the assay syst e m d e v e l o p e d for E p s t e i n - B a r r virus a s s o c i a t e d m e m b r a n e a n d n u c l e a r a n t i g e n [4, 5] using a n t i g e n i m m o bilized in p o l y a c r y l a m i d e gel a n d 125I-labeled I g G .

increasing amounts of 125i_igG. The test tubes were rotated overnight in the cold. After incubation, the gels were washed three times with 0.05 M Tris HC1 pH 7.5, 0.15 M NaC1, 0.1% Nonidet P 40, 20% calf serum, and the radioactivity bound to the gel was measured by crystal scintillation gamma counting. In antibody blocking tests, the gels were preincubated for 8 h at 4° with serum containing unlabeled antibody before the addition of 125I-IgG. In antigen inhibition tests, the 12sI-IgG was preincubated with soluble antigen for 8 h at 4° before the antigen containing gel suspension was added. Incubation, washing and counting were carried out as described above.

Results Material and Methods

Direct Binding Assay Carrier free Na 125Iodine for protein iodination was purchased from the Radiochemical Centre, Amersham, Buckinghamshire, England. Acrylamide, BIS (N,N'methylene-bisacrylamide) and TEMED (N,N,N',N'-tetramethylene-diamine) were obtained from Serva, Heidelberg, FRG. Calf serum was a product of Seromed, Munich, FRG, and was used after heat inactivation at 56° for 30 min. HBe/sAg positive fractions were prepared from human sera by ammonium sulfate precipitation (0.2-0.4 s). HBeAg was partially purified from human serum by ammonium sulfate fractionation (0.2-0.4 s) and chromatography on DEAE-Sepharose CL-6B, Phenyl-Sepharose CL-4B, and Sephacryl S-200 superfine. HBsAg was partially purified from human serum by affinity chromatography, using anti-HBs covalently linked to Sepharose 4B, the elution being carried out with 3M NaSCN. HBeAg and anti-HBe were detected by Ouchterlony double immunodiffusion [3], HBsAg was tested for by the AUSRIA 11-125 purchased from Abbot GmbH, Diagnostics Division, Langen, FRG. 125I-labeled IgG was prepared from human sera positive for anti-HBe. IgG was purified by ammonium sulfate fractionation and chromatography on DEAE-Sephadex A-50. IgG (50 ~tg) was iodinated with 1 mCi carrier free sodium lZSiodine by use of a modification of the chloramine method described by Hunter and Greenwood [10]. The incubation mixture contained 25 rtl 0.5 M sodium phosphate pH 7.5, 10 g1125iodine (1 mCi), 25 gllgG (50 gg protein), and 10 gl H20. After addition of 25 gl chloramine T (4 mg/ml), the reaction was stopped after 1 min by the addition of 50 ~tl sodium metabisulfite (2 mg/ml) and 50 gl 100 mM NaI. Protein-bound and free iodine were separated by chromatography on Sephadex G-25. The specific radioactivity of the labeled IgG was about 1 x 104 cpm per ng protein. Fractions containing HBeAg, and/or HBsAg as well as HBe/ sag negative sera were immobilized in polyacrylamide gel according to the procedure described by D61ken and Klein [4, 5]. To 1.5 ml antigen solution (HBe/sAg positive or negative serum, partially purified HBeAg or HBsAg) 4 ml 10% acrylamide containing 4% BIS, 100 gl riboflavin (3 mg/ml), 500 gl calf serum and finally 2 gl TEMED were added. After mixing, the gel was allowed to polymerize at room temperature for 1 h. The gel was then homogenized with a tightly fitting Dounce homogenizer and washed 5-6 times with 0.05 M Tris HC1 pH 7.5, 0.15 M NaC1, 0.1% Nonidet P 40, until the supernatant OD 280 was less than 0.005. The gel suspension was stored at 4° in the presence of 0.1% NaN 3. The solid-phase radioimmunoassay was carried out as follows : The homogenized gel was suspended in 0.05 M Tris HCI pH 7.5, 0.15 M NaC1, 0.1% Nonidet P 40, 50% calf serum, to give a volume of 1.0 ml suspension per 0.2 ml packed gel. One milliliter of this suspension was used per test. Three different types of assays were carried out: direct binding, antibody blocking and antigen inhibition. In direct binding tests the gels were incubated with

Fractions containing human HBe/sAg, partially p u r i f i e d H B e A g o r H B s A g as well as H B e / s A g negative c o n t r o l sera were p o l y m e r i z e d in p o l y a c r y l a m i d e gel. The h o m o g e n i z e d gel was used as the source o f i m m o b i l i z e d antigen in the r a d i o i m m u n o a s s a y . F i g u r e 1 shows the b i n d i n g o f 125i.ig G p r e p a r e d f r o m a n t i - H B e p o s i t i v e s e r u m to the p o l y a c r y l a m i d e gel cont a i n i n g the f r a c t i o n s d e s c r i b e d above. T h e m e a n perc e n t a g e o f c o u n t s b o u n d to p o l y a c r y l a m i d e gel was 0.41% a n d 0.34% for gels c o n t a i n i n g H B e / s A g a n d H B e A g , respectively. F o r the H B e / s A g n e g a t i v e cont r o l s e r u m the b i n d i n g was 0.07% a n d 0.05°,/0 for the gel c o n t a i n i n g p a r t i a l l y purified H B s A g . F r o m these d a t a a specificity r a t i o o f 5-8 was calculated. The v a r i a t i o n s w i t h i n triplicate s a m p l e s were less t h a n 10%. U n d e r the e x p e r i m e n t a l c o n d i t i o n s d e s c r i b e d , the b i n d i n g o f l a b e l e d I g G i n c r e a s e d linearly with the a m o u n t o f c p m a d d e d , i n d i c a t i n g t h a t the i m m o bilized a n t i g e n was n o t limiting.

Antibody Blocking Assay T o establish the specificity o f the b i n d i n g b e t w e e n H B e A g a n d l a b e l e d I g G , a b l o c k i n g assay was c a r r i e d o u t using a n t i - H B e c o n t a i n i n g s e r u m d i l u t e d 1:5 to 1:1,000. A s d e m o n s t r a t e d in Fig. 2, the b i n d i n g o f 125I-IgG is b l o c k e d b y a different a n t i - H B e positive serum, w h e r e a s a n t i - H B e negative s e r u m gave no b l o c k i n g over the w h o l e r a n g e o f dilutions. This result shows t h a t this assay system is also useful for the detection and quantitation of anti-HBe.

Antigen Inhibition Assay F u r t h e r evidence for the specificity o f the r a d i o i m m u n o a s s a y for the d e t e c t i o n o f H B e A g is d e r i v e d f r o m the a n t i g e n i n h i b i t i o n assay. A s s h o w n in Fig. 3, p r e i n c u b a t i o n s o f s o l u b l e H B e A g d i l u t e d 1:2.5 to

H.E. Blum et al. : Solid-Phase Radioimmunoassay for Hepatitis Be Antigen (HBeAg)

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1:1,000 resulted in an inhibition of the binding of labeled antibody to the antigen containing polyacrylamide gel, whereas preincubation with HBe/sAg negative samples or with partially purified HBsAg did not influence the binding over the whole range of dilutions. Apart from being specific for the detection of HBeAg, the antigen inhibiton assay offers the possibility to quantitate HBeAg in individual sera. Corn-

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Fig. 3. Antigen Inhibition Assay. Binding of ~25I-IgG (anti-HBe) to polyacrylamide gel containing HBe/sAg after preincubation with a HBe/sAg positive (o) and with HBe/sAg negative serum or partially purified HBsAg ( i ) and polyacrylamide gel containing partially purified HBeAg after preincubation with partially purified HBeAg (o) and with HBe/sAg negative serum or partially purified HBsAg (D)

pared with the Ouchterlony double immunodiffusion test where the fractions investigated were positive for HBeAg only when incubated undiluted, in the radioimmunoassay HBeAg could be detected up to dilutions of 1:500 to 1 : 750. Therefore, this assay system is 500-750 times more sensitive for the detection of HBeAg than the Ouchterlony double immunodiffusion test.

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H.E. Blum et al. : Solid-Phase Radioimmunoassay for Hepatitis Be Antigen (HBeAg)

Discussion

The method of protein immobilization in polyacrylamide gel was introduced by Carrel and Barandun [2], initially designed for immunoadsorbent procedures. Subsequently, protein fixed in polyacrylamide turned out to be useful as antigen source in solidphase radioimmunoassays for membrane and nuclear antigens [4, 5, 8]. This study describes a radioimmunoassay for HBeAg and anti-HBe based on this technique. The assay system proved to be specific for the detection of HBeAg and its antibody with a sensitivity 500-750 times greater than that of the Ouchterlony double immunodiffusion test. Different from the test system recently reported by Fr6sner et al. [7] the radioimmunoassay with antigen immobilized in polyacrylamide described here is useful not only for the detection of HBeAg and anti-HBe in serum samples but also in dilute solutions, e.g., effluents from column chromatographies, even in the presence of high salt concentrations or detergents. For this reason and due to its high sensitivity the radioimmunoassay can also be applied for the detection of HBeAg in biological material other than sera, e.g., liver biopsy specimens. References 1. Alter, H.J., Seeff, L.B., Kaplan, P.M., McAuliffe, V.J., Wright, E.C., Gerin, J.L., Purcell, R.H., Holland, P.V., Zimmerman, H.J. : Type B Hepatitis: The infectivity of blood positive for e antigen and DNA polymerase after accidental needlestick exposure. N. Engl. J. Med. 295, 909-913 (1976) 2. Carrel, S., Barandun, S.: Protein-containing polyacrylamide gels : their use as immunoadsorbents of high capacity. Immunochemistry 8, 39~48 (1971) 3. Clausen, J. : Immunochemieal techniques for the identification and estimation of macromolecules. In: Work, T.S., Work, E. (eds.), Laboratory Techniques in Biochemistry and Molecular Biology, pp. 399-556. Amsterdam, London: North-Holland 1972 4. D61ken, G., Klein, G.: A solid-phase radioimmunoassay for Epstein-Barr virus-associated membrane antigen prepared from B95-8 cell culture supernatants. J. Natl. Cancer Inst. 58, 1239-1245 (1977) 5. Dflken, G., Klein, G. : Radioimmunoassay for Epstein-Barr Virus (EBV)-associated nuclear antigen (EBNA). Binding of iodinated antibodies to antigen immobilized in polyacrylamide gel. Eur. J. Cancer 13, 1277-1286 (1977) 6. Fields, H.A., Bradley, D.W., Davis, C.L., Maynard, J.E. : Purification and partial characterization of hepatitis e antigen (HBeAg). Infect. Immun. 20, 792-803 (1978) 7. Fr6sner, G.G., Brodersen, M., Papaevangelou, G., Sugg, U., Haas, H., Mushahwar, I.K., Overby, L.R., Deinhardt, F.: Detection of HBeAg and anti-HBe in acute hepatitis by a sensitive radioimmunoassay. J. Med. Virol. 3, 67-76 (1978) 8. Gorsky, Y., Sulitzeanu, D. : A radioactive antibody binding-

inhibition assay for the detection of cell membrane related antigens in body fluids. J. Immunol. Methods 6, 291-300 (1975) 9. Grady, G.D., Gitnick, G.L., Prince, A.M., Kaplan, M.M., Fawaz, K.A., Vyas, G.N., Schmid, R., Levitt, M.D., Galambos, J.T., Bynum, T.E., Senior, J.R., Akdamar, K., Singleton, J.W., Clowdus, B.F., Steigmann, F., Aach, R.D., Schiff, G.M., Winkelman, E.I., Hersh, T., Murphy, B.L., Hindman, S.H., Maynard, J.E. : Relation of e antigen to infectivity of HBsAg-positive inoculations among medical personnel. Lancet 2, 492-494 (1976) 10. Hunter, W.M., Greenwood, F.C.: Preparation of iodine T M labelled human growth hormone of high specific activity. Nature 194, 495-496 (1962) 11. Krugman, S., Overby, L.R., Mushahwar, I.K., Ling, C.-M., Fr6sner, G.G., Deinhardt, F. : Viral Hepatitis, Type B. Studies on natural history and prevention re-examined. N. Eng. J. Med. 300, 101-106 (1979) 12. LeBouvier, G. : Seroanalysis by immunodiffusion : the subtypes of type B hepatitis virus. In: Vyas, G.N., Perkins, H.A., Schmid, R. (eds.), Hepatitis and Blood Transfusion, pp. 97 109. New York: Grune and Stratton 1972 13. Magnius, L.O., Espmark, J.A.: A new antigen complex cooccurring with Australia-antigen. Acta Pathol. Microbiol. Scand. [B] 80, 335-337 (1972) 14. Magnius, L.O., Espmark, J.A.: New specificities in Australia antigen positive sera distinct from LeBouvier determinants. J. Immunol. 109, 101%1021 (1972) 15. Neurath, A.R., Strick, N. : Host specificity of a serum marker for hepatitis B: evidence that e antigen has the properties of an immunoglobulin. Proc. Natl. Acad. Sci. USA 74, 1702~1706 (1977) 16. Nielsen, J.O., Dietrichson, O., Juhl, E. : Incidence and meaning of the e determinant among hepatitis B antigen positive patients with acute and chronic liver diseases. Lancet 2, 913-915 (1974) 17. Nordenfelt, E., Kjellen, L. : Dane particles, DNA polymerase, and e antigen in two different categories of hepatitis B antigen carriers. Intervirology 5, 225-232 (1975) 18. Nordenfelt, E., Adren-Sandberg, M. : Dane-particle associated DNA-polymerase and e antigen. Relation to chronic hepatitis among carriers of hepatitis B surface antigen. J. Infect. Dis. 134, 85 89 (1976) 19. Okada, K., Kamiyama, I., Inomata, M., Imai, M., Miyakawa, Y., Mayumi, M. : e antigen and anti-e in the serum of asymptomatic carrier mothers as indicators of positive and negative transmission of hepatitis B virus to their infants. N. Engl. J. Med. 294, 746-749 (1976) 20. Vyas, G.N., Peterson, D.L., Townsend, R.M., Damle, S.R., Magnius, L.O. : Hepatitis B e antigen: an apparent association with lactate dehydrogenase isozyme-5. Science 198, 1068-1070 (1977) 21. World Health Organization Expert Committee on Viral Hepatitis: Terminology of hepatitis virus and antigens. Weekly Epidemiological Records 51, 365-366 (1976)

Received May 7, 1979 Accepted May 21, 1979

Prof. Dr. W. Gerok Direktor der Med. Univ.-Klinik Hugstetter Str. 55 D-7800 Freiburg i.Br. Federal Republic of Germar~y