Clin. exp. Immunol. (1979) 35, 380-389.

Characterization of 'e' antigen associated with hepatitis B R. S. TEDDER & F. G. BULL* Department of Virology, Middlesex Hospital Medical School, London and *Diagnostic Research Unit, Department of Medicine, Southampton University, Southampton

(Received 25 October 1978) SUMMARY

Hepatitis B 'e' antigen (HBe) from the serum of a chronic carrier of HBsAg has been partially purified and characterized. It behaves as an acidic protein, pI 4 5-5-0, which is thermolabile and sulphydryl-sensitive. In serum it usually has a flotation density 1-3 g/cm3, but is sometimes found at density 1 15 g/cm3 because of its association with lipid. HBe from serum is polydisperse on gel filtration although most antigen is recovered with a nominal molecular weight of 3 x 105 Daltons. In contrast, in the presence of chaotropic ions, the bulk of serum HBe is found as a species of 3 x 104 Daltons previously detected in small amounts under non-dissociating conditions. This suggests that the larger material is formed by non-covalent association of the 3 x 104 Dalton species either with itself or other serum components. This would include IgG, although there is no evidence that HBe itself bears immunoglobulin determinants. Analysis of HIBe precipitins by polyacrylamide gel electrophoresis under reducing and dissociating conditions suggests that its component polypeptide chains are about 1-7 x 104 Daltons. INTRODUCTION Three antigen-antibody systems are associated with human infections by the hepatitis B virus (HBV). Hepatitis B surface antigen (HBsAg) and antibody to HBsAg (anti-HBs), hepatitis B core antigen (HBcAg) and antibody to HBcAg (anti-HBc) are well-established markers of past or current infection with HBV. The third and most recently described complex of antigens and antibodies, (Magnius & Espmark, 1972), comprises hepatitis B 'e' antigens (HBe), consisting of at least two determinants, e1 and e2 (Williams & Le Bouvier, 1976), and antibodies to HBe (anti-HBe). This system has attracted much attention since the presence of HBe or anti-HBe may have important clinical implications. HBe in the serum is associated with high infectivity in vertical (Okada et al., 1976) or horizontal transmission (Magnius et al., 1975), numerous Dane particles (Takahashi et al., 1976), high DNA polymerase levels in the serum (Cappel, De Cuyper & Van Beers, 1977) and continuing liver damage (Trepo et al., 1976). Conversely, the presence of anti-HBe may indicate low infectivity and better clinical prognosis in the carrier state. HBe has been described tentatively as the DNA polymerase of HBV (Melnick, Dreesman & Hollinger, 1976), an abnormal idiotype of IgG4 (Neurath & Strick, 1977), and an antigenic marker on the surface of the Dane particle and filamentous forms of HBsAg (Neurath et al., 1976). Further, it may be closely related to an unusual isoenzyme of lactate dehydrogenase (Vyas et al., 1977). With a view to exploring these possibilities we have partially purified and characterized HBeAg from the serum of a single HBsAg carrier. A brief account of this work has already been published (Tedder & Bull, 1978).

Correspondence: Dr R. S. Tedder, Department of Virology, The Middlesex Hospital Medical School, London W.1. 0099-9104/79/0030-0380$02.00 (0 1979 Blackwell Scientific Publications

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Hepatitis B eAg

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MATERIALS AND METHODS The sera used as a source of HBe or anti-HBe were obtained from persistent HBsAg carriers. Sera were stored at - 20'C. Those sera used as reagents in immunodiffusion (ID) analysis were checked for identity with established reference sera (Le Bouvier ox-35573 and ARC 18, Magnius 55667/75 and 3986/77). In this study the source of HBe was a male HBsAg carrier aged 35 (HBsAg titre by Hepatest, 1 in 20,000, subtype ad). Detection of HBe. ID analysis. Fractions from the preparatory procedures described below were concentrated where appropriate and tested for the presence of HBe by immunoprecipitation in an 0.75% agarose gel (Miles Laboratories Ltd., 95-201) in the manner previously described by Tedder (1978). Where it was necessary to distinguish el and e2 antigens, wells of increased diameter were employed. Immunoradiometric assay (e-RIA). Where noted in the text, a sensitive e-RIA was used to detect HBe (either el or e2). This test utilizes a solid-phase anti-HBe sandwich assay with 1251I anti-HBe as the developing radioligand (Mushahwar et al., 1978). The y-globulin fraction from a human serum containing anti-HBe was used to coat polystyrene tubes. After quenching with 0.5% bovine serum albumen (BSA), these were incubated overnight at room temperature with 100 111 test sample, washed and incubated with anti-HBe IgG labelled with 125I imidoester. After 4 hr at room temperature, tubes were washed and the radioactivity determined. Specificity of binding was confirmed by neutralization with human serum containing anti-HBe. Normal human serum or human anti-HBc IgG were without effect. Typically the ratio of radioactivity bound bv an HBe positive serum and a negative serum was between 15 and 30 to 1. HBe was quantified by reference to a calibration curve prepared with dilutions of HBe as either whole serum or serum fractions in the appropriate buffer. HBe was also quantified through its ability to inhibit the uptake of '25I-labelled anti-HBe IgG onto solid-phase HBe. Detection of HBsAg. HBsAg was assayed by either reverse passive haemagglutination (RPHA) using Hepatest (Wellcome Reagents Ltd.), or by an immunoradiometric assay (Heathcote, Cameron & Dane, 1974). Detection of normal serum proteins. Fractions of serum containing HBe were assayed for the presence of normal serum proteins by rabbit anti-human IgG in ID or by immunoelectrophoresis (IEP) using rabbit anti-whole human serum (Wellcome Reagents Ltd.). Iodination of proteins. Proteins were labelled with 1251 using modifications of the chloramine T procedure (Hunter & Greenwood, 1962), or with 125I 3- (4-hydroxyphenyl) propionic acid N-hydroxysuccinimide ester (Bolton & Hunter, 1973) supplied by The Radiochemical Centre Ltd. (Amersham). Autoradiography. Autoradiographs were prepared from ID gels by direct contact of the air-dried gels with Kodirex X-ray sensitive film. Films were developed in alkaline PQUniversal developer at room temperature. Prior to autoradiography, the ID gels were removed from petri dishes and washed for 24 hr in 0-02 M Tris buffered saline pH 7-2, containing 0-1% NaN3 (TBS) to which 100 juM phenyl methyl suphonyl fluoride (PMSF) had been added. PMSF was also included in all buffers used for the preparation of el for electrophoretic analysis. The gels were subsequently air-dried on glass plates at 37°C in contact with filter paper, washed for a further 12 hr and dried as before. Electrophoresis in polyacrylamide gel containing sodium dodedcyl sulphate (SDS-PAGE). Radioactive immunoprecipitins were cut from the dried gels and analysed by SDS-PAGE (Laemmli, 1970). Autoradiographs of SDS-PAGE gels were prepared as described previously by Robinson et al., (1975). Preparation of elAg. The serum used for the preparation of elAg was selected as it gave a prominent 'e' precipitin in ID without concentration. Saturated ammonium sulphate was added to this serum (25-100 ml) at room temperature to give 37 5% saturation, and the mixture centrifuged at 2500 g for 30 min. The supernatant was dialysed overnight at 4°C against TBS, concentrated to one quarter of the original volume by ultrafiltration (Diaflow XM-100), applied to a 5 x 80 cm Sephadex G200 column and chromatographed by upward flow of TBS. 10 ml fractions were assayed for HBsAg and HBe. Those fractions positive for HBe by ID were pooled, concentrated (Diaflow XM-100) and banded by isopycnic centrifugation on a self-forming CsCl gradient, (density 1-05-1-33 g/cm3) in a Beckman rotor SW 50 (72 hr, 4°C at 40,000 rev/min.) 0 5 ml fractions were collected, dialysed against TBS and assayed for HBsAg and HBe. Fractions positive for HBe by ID were pooled, adjusted to 1 0 mm CaCl2 and 0 1 mm MnCl2 and chromatographed on 5 0 ml ConA Sepharose 4B where HBe was not retained. It was then subjected to isoelectric focussing as described previously (Bull & Rosendaal, 1978), in an LKB 8101 110 ml column with 1% Ampholine pH 3 5-7 at 10°C for 24 hr at a final voltage of 1000 V. In some instances as indicated, a delipidation step was included. Samples were shell-frozen in liquid nitrogen, lyophilized and extracted by shaking for 1 hr with chloroform-methanol 3:1 at room temperature. Gel filtration. 500 ,ul of several different sera or serum fractions containing HBe were applied to the top of a 2-6 x 40 cm Sephadex G200 column and eluted with TBS containing 100 gM PMSF. 2-0 ml fractions were collected and assayed for HBsAg and HBe by RIA. Other treatments. Serum containing HBe was diluted 1: 100 in Tris-BSA and treated by one of the following procedures: (a) heating at 56°C for 30 min; (b) reduction with 10 mm dithiothreitol (DTT) at 37°C followed by alkylation with iodoacetamide in 20% molar excess; or (c) treatment with pronase (Type V protease, Sigma, London) or trypsin (Type 1, bovine, Sigma, London). Digestion was for 60 min at 37°C with 2% w/v enzyme. In each case residual HBe was measured by e-RIA and expressed as a percentage of the initial activity.

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RESULTS

Preliminary purificationi of HBe The serum selected for the preparation of HBe gave a prominent line on ID against human anti-HBe without prior concentration. Two reactivities, e1 and e2, were repeatedly demonstrated. In accordance with the convention of Williams & Le Bouvier (1976), the line nearer the antibody well is referred to as e,. Generally, the e2 line predominated although some anti-HBe sera produced a conspicious el line. After treatment with 37.50 0 saturated ammonium sulphate, el activity was retained in the supernatant. Unless stated otherwise this material was used for all subsequent studies of e,. It was polydisperse on filtration through Sephadex G200 and was detected by ID throughout the eluate to the albumen peak. Most HBe eluted just behind the break-through peak w hich corresponded to an approximate molecular Mx eight of 3 x 105 Daltons. HBe positive fractions contained HBsAg and serum proteins as demonstrated by IEP. Some of this contamination could be removed by its preferential binding to Con A Sepharose; the HBsAg fell four-fold for each passage and could be eluted quantitively with 500 a-methyl mannoside. HBe, detected by ID, A-as not significantly bound to lectin-Sepharose adsorbents prepared from either concanavalin A, wheat germ agglutinin or phytohaemagglutinin. HBsAg and HBe were removed by a single passage over the hydrophobic absorbent, phenyl-Sepharose (Pharmacia Ltd.).

'I

2K

K

E

hi3

5 Fraction number

10

LIG. 1. Isopycnic centrifugation of HBe from pooled fractions after Sephadex G200 filtration. HBsAg wGas determined by RPHA (U U) and HBe by ID. Density is shown (0-0).

Isop'cnic density centrij gation and isoelectric Jbcussing of JIBe Following gel filtration on Sephadex G200, pooled and concentrated fractions containing HBe were banded on a self-forming CsCl gradient. Although HBe was detected by ID in all gradient fractions of d 1 3 g/cm3 or less, the bulk of activity associated w ith material of flotation density 1 15 g/cm3; residual HBsAg formed a discrete visible band, d = 1 20 g/cm3 (Fig. 1). On recentrifugation, HBe banded at its original density of 115 g/cm3 which was unaffected by exposure to 1% Triton X-100 either before or during the centrifugation. Its behaviour on isoelectric focussing demonstrated its charge heterogeneity, although most HBe was detected by ID over the range pH 455-50 (Fig. 2). After treatment of this material with choloroform-methanol, HBe activity on ID was recovered at a density 130 g/cm3. IgG was not detected in this material by ID. Three other unfractionated sera containing HBe were examined by isopycnic centrifugation without delipidation. Again, e, and e2 activities were only found by ID in fractions of density 1-30 g/cm3.

Hepatitis B eAg

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64

2

0-

5

10

20

15 Fraction number

FIG. 2. Isoelectric focussing of serum fractions containing HBe was detected by ID.

el.

HBsAg

FIG. 3. Autoradiograph of '251-labelled fraction containing el which and then allowed to form immunoprecipitins with anti-HBe.

25

was

was

determined by RPHA (0) and

added to

an

HBe positive

serum

SDS-PAGE of HBe Partially purified preparations of el (prepared from serum by sequential treatments with ammonium sulphate, gel filtration, isopycnic centrifugation and isoelectric focussing) were labelled with 125I and analysed by ID. Radiolabel was incorporated into both el and e2 precipitin lines (Fig. 3). It was not found in an ovalbumen/anti-ovalbumen precipitin formed concurrently (Fig. 4), indicating that the radioactivity was associated specifically with HBe. Material from these precipitin lines was examined by SDS-PAGE under reducing conditions (Fig. 5) where it was evident that efficient dissociation and recovery of the precipitin had occurred. Thus the major stained material had mobilities corresponding to heavy and light chains of IgG. There was a small quantity of material (mol. wt. 7-7 x 104 and 7 x 104) which may have represented heavy-light chain aggregates and albumen respectively, but no evidence of complete IgG molecules (mol. wt. 1X5 x 105). Radioactivity in the starting preparation of eAg was distributed throughout the gel (Fig. 5). Material of mol. wt 4 -7 x 104 Daltons accounted for most of the staining with Coomassie blue, although this was of

384

R. S. Tedder & F. G. Bull

FIG. 4a

FIG. 4b FIG. 4. Gel diffusion (a) and autoradiograph (b) of 1251-labelled fraction containing el. The labelled material, together with ovalbumen, was added to an HBe positive serum and placed in the central well. Rabbit anti-ovalbumen was placed in well B.

Hepatitis B eAg

I

68 5043

94

215

.25

385

1

.11-7

I

6-5

mol. wt. x 10-3

5

a

.2

a

ca "I

(c)~~~~~~~~~~~~~~~~~~~~~4

0 0

20

_,

40

60

10

14F

80 100 120 Distance migrated (mm)

140

160

180

200

5. SDS-PAGE of l251-HBe precipitins. (a) Photograph of gel after staining with Coomassie blue. Sample stained preparation of 251-antigen. Sample 4, -25I-HBe from HBe anti-HBe precipitin. Samples 2, 3 and 5 are mixtures of reference proteins used as markers of mol. wt. Bottom axis: migration positions of reference proteins: 94, phosphorylase; 68, human serum albumen, 50, human IgG heav y chain; 43, oxalbumen; 25, human IgG light chain; 21-5, trypsin inhibitor; 11-7, cytochrome C; 6 5, aprotinin. (b) and (c) Show the percentage total radioactivity in each gel slice, starting preparation of 125I-antigen and 1251-HBe from precipitin, FIG. 1,

respectix ely.

relatively low specific activity. Only about 10 of the radioactivity in the starting preparation of antigen was incorporated into the 'e' precipitin. Little of this radioactivity was associated with material which could be considered as heavy and light chains derived from immunoglobulin, rather there was a localization of radioactivity at about 104 Daltons. Similar results were obtained irrespective of the method of iodination (chloramine T or imidoester) or the presence of either detergent (0-50% Triton X-100 or 0505 Tween 20) or 200 human serum in the gel during immunodiffusion. This result was not affected by delipidation of the HBe fractions prior to iodination with chloramine T. Radiolabelling of the nondelipidated material with the imidoester, however, apparently led to preferential incorporation of radioactivity into lipid. Under the same conditions of SDS-PAGE HBsAg, purified by isopycnic centri-

R. S. Tedder

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& F.

G. Bull

FIG. 6. Gel diffusion of high (well A) and low (well B) molecular weight HBe reacting with anti-HBe. The two wells on the left contain human anti-HBe sera. Void volume IgG

Albumen

and

cmm

CPl

VL

Q)

I Ir

(c)

a)

a) C m

18 20 22 24 26 28 30 32 34 36 38 40 Froction number

FIG. 7. Elution profile of HBe after filtration of 500 pl of serum through Sephadex G200. Serum (a) was from a chronic carrier; serum (b) was from the carrier studied in this paper; serum (c) was from an acute case evolving into an HBe positive carrier. HBsAg (e-e) and HBe (K-*) were both determined by RIA.

Hepatitis B eAg

387

fugation (Cayzer et al., 1974), gave two major components of nominal mol. At. 2 3 x 104 and 2 6 x 104 Daltons and a minor third component, presumed to be albumen, of 7 x 104 Daltons.

Pkysical heterogeneity and stability of HBe HBe in the range 2 x 105 to 8 x 104 Daltons (pooled fractions) behaved as el, whilst e2 associated with larger material of the break-through peak (Fig. 6). e2, unlike el, was no longer detected by ID after treatment Mwith 1% sodium desoxycholate. The polydisperse nature of HBe was confirmed in experiments where HBe was monitored with the more sensitive e-RIA after gel filtration of sera on Sephadex G200 (Fig. 7). The elution profile of'e' activity was characteristic of the individual serum, although, in each case, the greater proportion of HBe eluted just behind the void volume. It should be noted, however, that HBe also eluted with material smaller than albumen. When serum from an acute case was fractionated on a calibrated Sephadex G100 column, approximately half the HBe, detected by e-RIA, xxas eluted as a discrete peak with an apparent mol. wt. of 3 x 104 Daltons. On chromatography under dissociating conditions (3M KCNS) of a serum where HBe was predominantly of high mol. wt., antigenic activity was largely recovered at 3 x 104 Daltons, suggesting its formation from the larger material. Serum HBe was heat-sensitive and actively was reduced by 720/ after incubation for 30 min at 56'C. Similarly, treatment with trypsin or pronase caused a reduction. Exposure to 10 mM dithiothreitol decreased activity in e-RIA by 730/0 and there xras a parallel loss of HBe measured by the inhibition RIA.

DISCUSSION A striking feature of HBe disclosed by the present experiments is its physiochemical heterogeneity. After gel filtration HBe was detected by ID in fractions from the void volume through to albumen. The relative contribution of HBe of different sizes varied from serum to serum and with the e-RIA HBe was detected in some sera at a particular localization of 3 x 104 Daltons. Hoxxever, the presence of chaotropic ions (3M KCNS) resulted in recovery of most HBe as the 3 x 104 Dalton species. A similar result has been reported (A.R. Neurath, personnal communication). From this it is inferred that the heterogeneity of serum HBe is a consequence of the non-covalent association of a relatively small antigen (mol. wt. 3 x 104 Daltons) with serum constituents and/or perhaps a degree of self-association. HBe has been reported to associate strongly with IgG (Takahashi et a!., 1978) and xxe believe that it is the tenacity of this association which has led some workers (Neurath & Strick, 1977) to suggest that HBe has the identity of an IgG4 idiotype. HBe was sensitive to reduction. This loss of activity evident in e-RIA would not preclude the survival of the HBe antigen as a monovalent hapten. This seems unlikely hoxxev er, as after reduction there is a concomitant loss of activity in the inhibition assay where a monovalent hapten should be active. Substantial losses of activity were also produced either by heating or treatment xith proteolytic enzymes. These results indicate that HBe is a thermolabile sulphydryl-sensitive protein, mol. wt. 3 x 104 Daltons. It is unlikely to contain common sugar determinants in large quantity since it was not absorbed to any degree by several different lectin absorbents. The antigenic relationship between el and e2 remains difficult to define. On gel filtration, e2 appeared to be associated with larger material and unlike el it would no longer form a precipitin after treatment with ionic detergent. '25I-labelled el, on the other hand, appeared to associate with both e, and e2 precipitins. However, it would be necessary to exclude contaminating traces of e2 if one were to comment further on this particular result. It has proved equally difficult to examine the interrelationship of these two antigens using conventional immunodiffusion techniques. Despite the repeated formation of two parallel lines on ID, we have been unable to demonstrate identity satisfactorily by fusion or lack of identity by cross-over. Therefore, especially in viexx of the heterogeneity in size shown by HBe, it must be considered whether such multiplicity of lines could be the result of a single antigen species occurring in different physical forms, rather than a series of distinct antigens (Mulder, Sloots & N erhaar, 1972). Magnius (1975) has reported that HBe has a density of 1 3 g/cm3 and a sedimentation coefficient of

388

R. S. Tedder & F. G. Bull

11-6S suggesting a mol. wt. of 3 4x 105 Daltons. In the present study HBe was heterogeneous with respect to flotation density, whilst e1 and e2 activities were generally detected at 1 3. g/cm3. el from the serum examined in detail in this study had a density of 1 15 g/cm3 which shows that the immunological activity of HBe is maintained in association with lipid. This was a convenient property since it facilitated the removal of the bulk of serum proteins from HBe by centrifugation. It may also confer hydrophobic properties upon HBe which account for its binding to materials such as phenyl-Sepharose. The observation that HBe is of low mol. wt. but has the property of associating with lipid and protein would explain its polydisperse behaviour in sera. It follows that standard physiochemical isolation techniques are likely to give preparations low in yield and purity unless initial steps are taken to dissociate serum HBe into the apparently more homogeneous low mol. wt. form ziz. treatment with 3M KCNS. Radioactive material obtained from the specific 'e' precipitin and presumed to represent HBe is heterogeneous under the reducing and dissociating conditions of SDS-PAGE. Some of this heterogeneity may be a result of limited proteolysis either in zivo or during preparation. Further, HBe of mol. wt. 3 x 104 Daltons is formed from larger material in serum after exposure to 3M KCNS. This suggests that material on SDS-PAGE which is larger than 3 x 104 Daltons represents the non-specific association of serum proteins with HBe and that this occurred before the 'e' precipitin was formed. Despite these cautionary observations the data suggest that the major polypeptide components of HBe have a mol. wt. of about 104 Daltons or less. If these were associated through interchain disulphide bonds it might account for both the material of 3 x 104 Daltons in serum and the loss of antigenic activity caused by reducing agents. The previous estimate for the mol. wt. of 3 4 x 105 Daltons (Magnius, 1975) and the suggestion that IBe is an idiotype of IgG4 (Neurath & Strick, 1977) appeared to preclude the possibility of HBe being a product of the viral genome. However, if HBe can be considered as a low molecular weight hapten which is not derived from IgG, then those restraints need not apply. It has been suggested that HBe is a structural component of the virus, either on the surface of the Dane article (Neurath et al., 1976) or cryptic, only being revealed by chemical treatment of the virus (Lam, Tong & Rakela, 1977), the data, however, is currently contradictory. Nevertheless, there is strong circumstantial evidence that HBe correlates with the phase of active viral replication (Magnius, 1975). Perhaps an alternative explanation is that HBe is a virally coded protein which is a by-product of intact virus replication but not a structural component. ADDENDUM It is now coinsistently found that HBe isolated rapidly from serum previously treated with 3M KCNS has an apparent mol. wt. of 1-7 x 104 under reducing conditions on SDS-PAGE. Tnis implies that the smallest form of HBe in serum may be a dimer of this polypeptide.

We thank Dr D. S. Dane for his support and encouragement, other members of the Department of Virology for their help and Miss Roberta Smith for her technical assistance. Dr R. S. Tedder holds a Wellcome Research Fellowship in Virology. REFERENCES

BOLTON, A.E. & HUNTER, W.M. (1973) The labelling of proteins to high specific activities by conjugation to a 1 251-containing acylating agent. Application to the radioimmunoassay. Biochem. 7. 133, 529. BUI I, F.G. & ROSENDAAL, M. (1978) Macrophage colony development: properties of colony stimulating factors from murine embryo and pregnant uterus. Iimmunology 34, 479. CAPPEL, R., DE CUYPER, F. & VAN BEERS, D. (1977) e Antigen and antibody, DNA polymerase, and inhibitors of DNA polymerase in acute and chronic hepatitis.,7. Iitfect. Dis. 136, 617. CAYZER, I., DANE, D.S., CAMERON, C.H. & DENNING, J.V. (1974) A rapid haemagglutination test for hepatitis-B antigen. Laivet, i, 947.

IIEATIHCOATE, J., CMNIERON, C.H. & DANE D.S. (1974) Hepatitis B antigen in saliva and semen. Laicet, i, 71. HUNTER, W.M. & GREENWOOD, F.C. (1962) Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature (Lonid.) 194, 495. LANi, K., TONG, M., RAKELA, J. (1977) Release of e antigen from Dane particle-rich preparation of Hepatitis B virus. Ihifect. Immunity, 16, 403. LAENIMLI, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage, T4. ANattire (Loutd.), 277, 688. MIAGNIUS, L.O. (1975) Characterisation of a new antigenantibody system associated with Hepatitis B. Cli~i. exp. Immunzol. 20, 209.

Hepatitis B eAg MAGNIUS, L.O. & ESPMARK, J.A. (1972) New specificities in Australia antigen positive sera distinct from the Le Bouvier determinants. I. Immunol. 109, 1017. MAGNIUS, L.O., LINDHOLM, A., LuNDIN, P. & IWARSON, S. (1975) A new antigen-antibody system. Clinical significance in long-term carriers of Hepatitis B surface antigen. J. Amer. Med Assoc. 231, 356. MELNICK, J.L., DREESMAN, G.R. & HOLLINGER, F.B. (1976) Approaching the control of viral hepatitis type B. J. Infect. Dis. 133, 210. MULDER, J., SLOOTS, L.C.E. & VERHAAR, M.A.T. (1972) Molecular heterogeneity affects of IgM immunoglobulins in radial immunodiffusion. J. Immunol. Methods, 2, 89. MUSHAHWAR, I.K., OVERBY, L.R., FROSNER, G., DEINHARDT, F. & LING, C.M. (1978) Prevalence of hepatitis Be antigen and its antibody as detected by radioimmunoassay. J. med. Virol. 2, 77. NEURATH, A.R. & STRIcK, N. (1977) Host specificity of a serum marker for hepatitis B: Evidence that 'e antigen' has the properties of an immunoglobulin. Proc. Nat. Acad. Sci. (Wash.), 74, 1702. NEURATH A.R., TREPO, C., CHEN, M. & PRINCE, A.M. (1976) Identification of additional antigenic sites on Dane particles and the tubular forms of Hepatitis B surface antigen. J. gen. Virol. 30, 277. OKADA, K., KAMIYAMA, I., INOMATA, M., IMAI, M., MIYAKAWA, Y. & MAYUMI, M. (1976) e antigen and anti-e in the serum of asymptomatic carrier mothers as indicators of

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positive and negative transmission of hepatitis B virus to their infants. New Engl. J. Med. 294, 746. ROBINSON, P.H., BULL, F.G., ANDERTON, B.H. & RoITT, I.M. (1975) Direct autoradiographic visualisation in SDS-gels of lectin binding components of the human erythrocyte membrane. FEBS Letters, 58, 330. TAKAHASHI, K., ImAI, M., TSUDA, F., TAKAHASHI, T., MIYAKAWA, Y. & MAYUMI, M. (1976) Association of Dane particles with e antigen in the serum of asymptomatic carriers of Hepatitis B surface antigen. 5. Immunol. 117, 102. TEDDER, R.S. (1978) Testing for e-antigen and e-antibody by immunodiffusion. J. med. Virol. 3, 51. TEDDER, R.S. & BULL, F.G. (1978) Properties of the 'e' antigen system associated with Hepatitis B. Second University of California Symposium on Viral Hepatitis. San Francisco 16-19 March. p. 666. Franklin Institute Press. TREPO, C.G., MAGNIus, L.O., SCHAEFFER, R.A. & PRINCE, A.M. (1976) Detection of e Antigen and Antibody; correlations with hepatitis B surface and hepatitis B core antigens, liver disease and outcome in hepatitis B infections. Gastroenterology, 71, 804. VYAS, G.N., PETERSON, D.L., TOWNSEND, F.M., DAMLE, S.P. & MAGNIUS L.O. (1977) Hepatitis B e antigen: An apparent association with lactate dehydrogenase Isoenzyme-5. Science, 108, 1068. WILLIAMS, A. & LE BoUvIER, G. (1976) Heterogeneity and thermolability of 'e'. Bibl. Haematol. 43, 71.

Characterization of 'e' antigen associated with hepatitis B.

Clin. exp. Immunol. (1979) 35, 380-389. Characterization of 'e' antigen associated with hepatitis B R. S. TEDDER & F. G. BULL* Department of Virology...
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