Host specificity of a serum marker for hepatitis B: evidence that "e antigen" has the properties of an immunoglobulin.

Abstract

A family of antigens, referred to collectively as e antigen (eAg), has been detected in sera of some individuals with liver disease who test positive for hepatitis B surface antigen. Studies on eAg partially purified by affinity chromatography on insolubilized antiboies to eAg revealed the following: (i) (eAg). Though distinct from HBsAg in both immunologic and physicochemical properties (8), eAg is also specifically associated with HBV infections and appears to be a marker for the infectivity of HBsAg-positive sera (7, 9, 10); its persistence seems to be associated with chronic liver disease (11). Our attempts to characterize eAg, described here, led to the surprising finding that eAg has the properties of an immunoglobulin. MATERIALS AND METHODSeAg was partially purified by precipitation with polyethylene glycol 6000, followed either by affinity chromatography on columns of insolubilized antibodies to eAg (eAbl (12) or by chromatography on DEAE-Bio-Gel (13). eAb was prepared from human sera by sequential precipitation with Rivanol and Na2SO4 (14). Antibodies to individual human plasma proteins and to IgG subclasses were each linked to Sepharose 4B as described (15). Normal human serum or IgG was insolubilized by the same procedure.Partially purified eAg (total amount of protein: 90-250 mg in 30 mM sodium citrate, pH 7.4) was submitted to isoelectric focusing (16). Fractions after isoelectric focusing were adjusted to pH 7, dialyzed against 0.14 M NaCl/0.01 M Tris-HCI at pH 7.2 (Tris/saline), concentrated about 10-fold, and tested for eAg. Identical results were obtained with either the cathode or the anode positioned in the bottom of the electrofocusing column.To compare the sedimentation rates of eAg and an IgG (18), respectively. The F(ab')2 fragments were isolated by chromatography on Sephadex G-150 (17). The Fab fragments were separated from Fc fragments by affinity chromatography on anti-Fab columns. F(ab")2 fragments from samples containing eAg were prepared by cleavage with CNBr (19), followed by affinity chromatography on anti-Fab columns.eAg purified by affinity chromatography or proteins eluted with 50 mM Na2B407 (pH 10.9) from extensively washed eAg-eAb precipitin lines excised from the agarose gel of rheophoresis plates were each labeled with 1251 (20) or with [125I]-iodinated p-hydroxyphenylpropionic acid N-hydroxysuccinimide ester (Bolton-Hunter reagent; New England Nuclear, Boston, MA). Similar results were obtained by either of the two methods. Labeled eAg was further purified by isoelectric focusing. Fractions collected in the pH range of 4.9-5.7 were pooled, dialyzed against Tris/saline, mixed with 2 ml of lamb serum and with normal human serum proteins, precipitated from 10 ml of serum with polyethylene glycol 6000 (600 mg), and resuspended in 2 ml of Tris/saline. Labeled eAg from this mixture was repurified by affinity chromatography on a 15-ml column of insolubilized eAb.The method for polyacrylamide gel electrophoresis described earlier (16) was used. Samples were dissociated...

Molecular Heterogeneity of Hepatitis B Virus e Antigen in Liver and Serum

“…3c) were also detected in sera. Neurath & Strick (1977) demonstrated that the pI of anti-HBe IgGs were heterogeneous, having the pI range from 5.8 to 10.2. Therefore, the heterogeneity in pI of IgG-associated HBeAg may result from the different number of IgG molecules which bind to HBeAg.…”

“…As for the IgG-associated HBeAgs, the earlier work of Neurath & Strick (1977) indicated that HBeAg, partially purified from the pooled serum of asymptomatic carriers, had the properties of human immunoglobulin, mainly of the IgG4 subclass. Fields et al (1978) and Weft et al (1979) have also shown that HBeAg might be closely associated with human serum IgG.…”

Maternofetal Transmission of IgG-Bound Hepatitis B e Antigen

“…In their study, IgGl and IgGB were equally affinitive to the placenta, but IgG4 was much less reactive. Neurath et al (10) reported that the subclass of IgG in molecules with HBeAg activity was IgG4. Weak affinity of IgG4 for the placenta, a large size of IgG-bound HBeAg, and inability for free HBeAg to pass the placenta would be among the factors responsible for the observed difference of HBeAg activity between maternal and cord sera.…”

Radioimmunoassay and Some Properties of Human Antibodies to Hepatitis B Core Antigen

“…Polystyrene beads (diam. 6 mm) were immersed in a solution of anti-HBc [IOO/~g IgG/ml in o.I M-tris (hydroxymethyl) aminomethane, pH 8.8] prepared from an anti-HBo-positive serum as described before (Neurath & Strick, 1977). After several hours at room temperature, the solution of anti-HBo was removed, the beads were washed several times with TS and then immersed in a solution of bovine haemoglobin (IO mg/ml in TS).…”

“…Isoelectric focusing was performed as described before (Neurath & Strick, 1977). HB~Ag and antibodies to HB~Ag (anti-HB,) were determined by radioimmunoassays using commercial reagents (Abbott Laboratories, North Chicago, Illinois).…”