CA2186338A1 - Method for diagnosing chronic hepatitis b virus infection - Google Patents

Abstract

Human antibodies that bind to woodchuck hepatitis virus core antigen are elevated in chronic hepatitis B patients in comparison to acute hepatitis B patients. Immunoassays for detection of the level of anti-WHV core antigen antibodies is used to distinguish chronic from acute hepatitis B patients.

Description

w0 9~/~7083 , 2 ~ ~ 6 3 3 8 I ~

METHOD FOR DIAGNOSING CHRONIC
HEPATITIS B VllRUS INFECTION
This invention was made with Government support under Grant No. Al-20720 awardedby the National Institute of Health. The Government has certain rights in this invention.

1. Field oJfhe Invention The present invention relates generally to the hepatitis B virus (HBV) and specifically to a method for diagnosing chronic hepatitis B virus infection.

2. Description of Related Art 10 Infection with hepatitis B virus (HBV) is a worldwide public health problem, with chronic carriers accoumting for ~ v~u~._~ly 10 percent of the population of Asia and Africa.
Major causes of HBV-associated mortality include chronic active hepatitis, liver cirrhosis and l , l~ ~ carcinoma. Both chronic carriers and newly infected individuals areat risk Of ~ , - g to such ...",.~ One important i route is the 15 infection of newborn infants at parturition by mothers who have active infections or are chronic carriers. Other routes for l,,."~.,.: - -", include . ' blood or blood products used to treat other health problems.
Infection with HBV often results in subclinical or acute self-limited liver disease or can result in chronic long-term infection. Chronic HBV infection elicits a spectrum of disease 20 entities ranging from the most severe form of chronic active hepatits (CAH) to less severe chronic persistent hepatitis (CPH) to the 4a~ A J1114liC calrier (ASC) state. An array of diagnostic assays have recently been developed to aid the clinician in dilrc~c~LiaLulg hepatitis B virus infections from other forms of viral hepatitis (i.e., HAV, HEV, HCV).

W095/27083 2~ ." L ~l However, the ability to distinguish between an acute hepatitis B (AH-B) infection and D,y~ wll~Li~: chronic hepatitis B (CH-B) infection is still 1 ", .1,1 -' ;~ This is especially true since CAH and CPH patients often .1~..,....~1..'` a cyclic pattem of hepatitis by æute "A~ ,,bAALiul~., (A.E.) of liver injury altemating wjth nommal liver 5 function.
After infection with HBV, large quantities of the virus and associated particles are present in the serum. During the Dylll,ululll_Lc phases of infection, both æute and chronic HBV
patients have elevated liver enzyme levels, possess the hepatitis B surface antigen (HBsAg) in their seram, and produce antibodies to the ",.. ~ ;.1 antigen (HBcAg).
10 Antibodies specific for the HBsAg or the hepatitis B e antigen (HBeAg) are not detected.
The appearance of antibody to HBsAg is usually not observed until ~ two months following ~" ,, of circulating HBsAg. The vjral particles present in the serum are known to shed their surfæe coat exposing the "... lf v ~ known as the core antigen (BcAg). Antibody production to HBcAg occurs early in the course ofthe æute phase of HBV infection and can persist for many years, and chronically infected patients produce high titers of anti-Bc amtibodies.
Previous studies have indicated that the ~ of the HBV and the woodchuck hepatitis virus (WHV) share a ~IU ~ Live epitope (Wemer, ef aL, J Virol. 1:314, 1979;
Millman, ef aL, Infecfion and Immunify ~:752, 1982; Stannard, ef aL. ~ Gen. Virol.
~L:975, 1983; Ponzetto, ef aL, Virus Research ~:301, 1985). Prior to the presentinvention, it was not known whether human anti-HBc antibodies recogluzed woodchuck hepatitis ,. lf~ ;.1 antigen (WHcAg) or if sera from patients at different stages of BV infection could be ,~ , J based on differential reactivity to WHcAg.
In contrast to most viral infections, æute and chronic BV infected patients often produce both IgM and IgG anti-Bc antibodies, therefore, the mere presence of IgM anti-Bc is not diagnostic of an æute infection. However, higher levels of IgM anti-HBc are generally produced during the acute phase as compared to chronic infection, and this ~lu~lLiL_iiv~ difference has become the only serologic means of diL~ Lil-g an acute .

wo 95r27083 2 1 8 6 3 3 8 HBV infection from an A.E. of a chronic infection. The usefulness of IgM anti-HBc assays in the differentiation of acute from chronic HBV infection has also been The distinction betvveen æute and chronic HBV infection is important in terms of prognosis and possible treatment modalities.
5 Thus, there eAists a need for a simple, ill.~A~ ;VC: and reliable assay to distinguish patients ~-vith acute E~3V infection from those with chronic HBV infection. The present invention provides such an assay.

wo ssmo83 r~ s ~ ~36~3 SUMMARY OF TIIE INVENTION
The present invention is based on the unexpected discovery that subjects with acute and chronic hepatitis B virus infection can be easily .~ 1,. d based on the level ofantibody to HBcAgW.

5 In one ~ ' the invention provides an assay for diagnosing chronic HBV infection in a subject by detecting IgG anti ~ oo(l~h~lrl~ hepatitis core antigen antibody in a subject.
In another ~".l,o~ , the above-described assay is perforrned in ~ ;oll with detection of IgM HBcAg antibodies and the ratio of IgM HBcAg:lgG HBcAgW antibodies is ~ t~
10 In yet another ~ -, the invention provides a series of assays which include detection of serum levels of anti-HBe anfibody, HBeAg/anti-HBe imrnune complex (IC) and HBsAg/anti-HBs ICs, all of which are S l~fir~ ly elevated in chronic versus acute hepatitis B patients.

W0 95127083 . 2 1 ~ ~ 3 3~ r~ .^61 BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE I is a comparison between acute hepatitis B (AH-B) and chronic hepatitis B
(CH-B) patient setum levels of anti-HBe (panel A), HBeAg/anti-HBe ICs (panel B) and HBsAg/arlti-HBs ICs (panel C).
5 FIGURE 2 is a comparison between acute hepatitis B (AH-B) and chronic hepatitis B
(CH-B) patient serum levels of anti-HBc, IgM anti-HBc, IgG anti-HBcW, and IgM anti-HBc/lgG anti-HBcW ratio. (Panel A = total anti-HBc; Panel B = IgM anti-HBc; Panel C
= IgG amti-HBcW; and Panel D = ratio of IgM anti- HBc/lgG anti HBCW.) FIGURB 3 is an endpoint titration of anti-HBcW IgG in AH-B (~-~) and CH-B (o-o) 10 patient sera (.-- = normal human serum ~NHS)).
FIGUPE 4 is the kinetics of amtibody p}oduction to the BcAg in AH-B and CH-B
patients. Sequential serum samples from AH-B (panel A) and CH-B (panel B) were analyzed for IgM anti-HBc ( --), IgG anti-HBc (---), and IgG anti-HBcW (A-~
FIGURE 5 is the serologic profile of a CH-B patient. The top panel shows standard 15 serologic parameters.
FIGURE 6 is antigen inhibition analysis of CH-B patient sera. Panel A is anti-HBcW and panel B is anti-HBc assayed by direct EIA. (HBeAg, --, HBcAg, o-o; and WHcAg, ~-~) FlGURE7showsthesensitivityofanti-HBcWreactivityto.~. AI...,.I;...,ofwoodchuck 20 Hc~g (Mab2221,~ -WHC,~ :Ml-WHC,----- H~lgc~,-O) WO95127083 ~ 1 ~36338 r~ Cl .

DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a sensitive new assay which detects antibody to w~ ' hepatitis core antigen (WHcAg) and ~ chronic from acute hepatitis B infection. The assay is based on the unexpected finding that patients with 5 chronic hepatrtis B infection have elevated serum levels of a species of anti-HBc antibody that crossreadts with woodchuck hepatitis core antigen (i.e., anti-HBcW). In another .. , . l ,o.~;.. l the rnvention provides a series of assays which include detection of serum levels of anti-HBe antibody, HBeAglanti-HBe immune complexes (IC) and HBsAg/anti-HBs ICs, all of which are shown in the present invention to be significantly elevated in 10 chronic versus acute hepatitis B patients.
The invention provides a method for diagnosing chronic hepatitis B virus (HBV) infection in a subject comprising contacting a sample from a subject suspected of having chronic HBV with an epitope that contains the amino acid sequence of a ~. - ' ' ' hepatitis virus antigen wherern the epitope binds with antibody to woodchuck hepatitis B core anti-15 gen and detecting the presence of antibody that reacts with woodchuck hepatitis B coreantigen in the sample. The subject can be any animal and preferably is a human.
As used rn this inventdon, the term "epitope" means any antigenic .l. ~ lll;IIA .~ on an antigen to which the paratope of an antibody bmds. Epitopic ~L -.. - - -- .: - usually consist of chemically active surface groupings of molecules such as atnino acids or sugar side 20 chains and usually have specific three l;.. ..~ structural ..l. -- ~. r.. ;~ as well as specific charge ..l. - A. . . ;~l;. ~
The method of the invention for detection of anti v~ co~ Ak hepatitis B core antigen AAntibodies (anti-BcW) in a sample is performed in vilro, for example, in ;, ." ,.. ,. l.~ ~Ay~
in which the antibodies can be identified in liquid phAse or bound to an antigen or epitope 25 that is bound to a solid phase carrier. In addition, an antibody for detection of anti-woodchuck Bc antibodies in these; " .., . ~ Ay, can be detectably labeled in various wo 95/27083 2 1 8 6 3 3 8 r~ c - I

ways. EAamples of types of ,"".,., ~ which can be utiliAed to detect amti-woodchuck HBc antibodies in a sample, include uu~ iLive and non-~;ulll~ ive , in either a direct or indirect format. Examples of such ;.. , .. ~ are the, A~ (RIA) and the sandwich (;., . . . ~ ) assay. Detection of the 5 antibodies using the method of the invention can be done utili~ing; .... "... " ~ which are rlm in either the forward, reverse, or ' modes, including rnmr~titinn and ' ' ' assayson~,Ily~;olo~;.,lsamples. Thoseofskill irl the art will know, or c_n readily discern, other ;... - ~ A,Y formats without umdue ..1...;..,...- :;....
10 The woodchuck HBc antigen (WHcAg), or epitopes thereof, cAn be bound to many different carriers and used to detect arlti-woodchuck HBc antibodies in a sample.
Examples of well-known carriers include glass, polystyrene, polypropylene, pol ~ .,;I~.r lc.lc, dextran, nylon, amyloses, natural and modified celluloses, polyA~ idc~, agaroses, and magnetite. The nature of the carrier can be either soluble or insoluble for purposes of the 15 invention. Those skilled in the art will know of other suitable carriers for binding antigen or epitopes, or will be able to ascertain such, using routine ~ ~l,.. ;... .:- ;..
There are many different labels and methods of labeling known to those of ordinary skill irl the art. Examples of the types of labels which can be used in the present invention include enzymes, ,~ vl,.~ fluorescent Aomro~ A, colloidal metals, . l.. ,.l.. ;, .. ~ . ' amd bio-l . ~ ' Those of ordinaty skill in tbe art will know of other suitable labels for binding to the ~ I l. ,.., ,l. .. IAI antibodies of the invention, or will be able to ascertain such, using routine CA~)~ '-Fu~ llllulc, the binding of these labels to the antibodies used in the method of the invention c~m be done usmg stAAndard techniques common to those of ordinary skill in the 25 art.
rul ~J~JJu~_~ ofthe invention, antibodies that bind to woodchuck HE~cAg may be detected using amti-human IgG antibodies. Any sample containing a detectable amount of wo gs/27~83 ~ 3 ~ ~ c~ ~
.

woodchuck HBcAg antibody can be used. A sample can be a liquid such as urine, saliva c~,lcbl~la~ lal fiuid, blood, serum and the like. Preferably, the sample is serum.
The invention also provides a series of assays which include detection of serum levels of anti-HBe antibody, HBeAg/anti-HBe immune complexes (IC) and HBsAg/anti-HBs ICs, 5 all of which are ~ .lirn~ ly elevated in chronic versus acute hepatitis B patients. The detection of these antigens and amtibodies is performed according to methods described above for the detection of amti-woodchuck hepatitis virus antibodies. For example, to detect, anti-HBe antibody, HBe antigen or peptide containing an epitope can be fixed to a solid support and a species specific antibody which binds to tbe anti-HBe antibody 10 added after the antigen and antibody in the sample react. Similarly, the immune complexes can be detected in a sample by reaction with a second antibody that binds to the antigen in the complex and a third antibody which binds to the antibody in the complex. For example, a norl-cc,lll~,.,.iL~., .""..~I. .,.~ antibody specific for HBsAg or HBeAg is fixed to a solid support and a serum sample is added followed by the addition 15 of a labeled . ,.~,. ,. ~I. ., . ~' antibody to human IgG.
The P/N ratio, which represents the level of antibody and ICs in the sample (P) as compared to the level of antibody and ICs irl control sera, is preferably greater than about 2 as a lower limit or cut-off, and most preferably greater than 3. Preferably, the detection is performed by d .`..., . :..;..~ the absorbance (OD492) value of the sample and the control 20 sera. Thoseofordinaryskillmtheartwillknowofothertechniquesfor~L~ .. ,.:,.~the level of antibody or IC's in a sample.
Tbe materials for use in the assay of the invention are ideally suited for the preparation of a kit. Such a kit may comprise a carrier means bemg: , " ' to receive in close ~..,.1~.... ,...1 one or more container means such as vials, tubes, and the like, eæh of 25 the container means comprising one of the separate elements to be used in the method.
For example, one of the container means may comprise a woodchuck HBcAg or w095127083 2l 8~ 3 r~"~
woodchuck HBcAg epitope, and another container may comprise an anti-human antibody which is, or c~m be, detectably labelled.
The types of assays which can be iUl~.UI,UUl_'~,d in kit form are many, and irclude, for example, competitive and non-competitive assays. Typical examples of assays which can 5 utilize the antibodies of the invention are ~ Ay;~ (RIA), enzyme (EIA), enzyme-linked ;.,..,.--,..~-..l- -~ assays (ELISA), and or sandwich i ", .., .. .A~AY
The term " - assay" or "sandwich; ,..", ..IA~AY", includes ~iullulkul~,u~
sandwich, forward sandwich and reverse sandwich ;lll.ll~ Ay,. These terms are well 10 understood by those skilled in the a~t. Those of skill will also appreciate that antibodies according to the present invention ~vill be useful in other variations and forms of assays which are presently knowrl or which may be developed in the future. These are intended to be included within the scope of the present invention.
In performing the assays, it may be desirable to include certain "blockers" in the 15 incubation medium (usually added with the labeled soluble antibody). The "blockers" are added to assure that non-specific proteins, proteases, or ~u.~ u~llillic p,lul.-l; . to anti-HBcW immlln~A~globlllin~ for example, present in the I~ . j",. ..~_l sample do not cross-lirk or destroy the antibodies on the solid phase support, or the lA~1;n~ 1 indicator antibody, to yield false positive or false negative 20 reslllts. The selection of '`blockers" therefore may add aub~ullidlly to the specificity of the assays described in the present invention.
It has been found that a number of nulu~L, ~ . (i.e., nonspecific antibodies of the same class or subclass (isotype) as those used in the assays (e.g., IgGI, IgG2a, IgM, etc.) can be used as "blockers". The ~ , of the "blockers" (normally 1-100 ~.g/~,l) is 25 important, in order to maintain the proper sensitivity yet ir~hibit unwanted i~ f~ by mutually occurring cross reactive protems in the specimen.
.

WO 95n7083 . ~
2~86338 The assays of the invention include using antibodies illllllUllVlCOUliV~ with WHcAg, HBcAg~, HBcAg, HBeAg/anti-HBe ICs ~md HBsAg/anti-ABsAg ICs or fragments thereof. Monoclonal amtibodies, such as tbose used m the present Exarnples, cam be made by ~ an appropriate host, such as a mouse, with antigen containing fragments of the whole protein by methods well known to those skilled in the art (Kohler, et al., Nat~lre, ~:495,1975). Antibodies with reactivity to ICs described herein are available commercially or cam be produced as described in Maruyama, et aL, (~ Immunol Meth., 155:65, 199~). The term antibody, as used in this invention, is meant to include intact molecules as well as fragments thereof, such as Fab and F(ab')2, which are capable of binding an epitopic ~ on the different HBV or WHV amtigens.
The following examples are intended to illustrate but not limit the invention. While they are typical of those that might be used, other procedures known to those skilled in the art may alt~ ,l.y be adopted without resort to ur due .
FXAMPl,h`. 1 MATF.RIAI ~ ANI) M~.THODS
1. ~rrnnnhin~.lt Antu~rn~ :3n~1 Syr~thrtir Pl',3~ti~
R. ' HBcAg of the ayw subtype, . ~ HBeAg of the ay~v subtype, amd ' WHcAg were derived from ~ coli expression vectors as previously described (Schodel, er aL, Vaccines 90, Cold Spring Harbor Laboratory, pp. 193-198, 1990; Schodel, et al, J. BioL Che~2., ~:1332-1337, 1993, Schodel, et al, Yaccine, ~i:624-628, 1993 (shows the lu~.lcvLi~c,'r...ulo acid sequence for WHcAg)). Synthetic peptides derived from the WHcAg sequence were synthesized by the Merrifield solid-phase method, and were subjected to HPLC on a Ci 8 reverse phase column. All peptides used eluted as a single major peak ()90%). Synthetic peptides were produced in the -w09S/~7083 21 863~ r~l,o~
peptide laboratory of the R.W. Johnson Pl~ Research Institute by G.B.
Thornton (La Jolla, CA).
2. ~i~
Murine polyclonal anti-Bc, anti-WHc and rabbit polyclonal anti-WHc were produced5 by; " .. ., ~ ;. ,., with l r~ .~., .l ,; " . ,l HBcAg or WHcAg. Polyclonal rabbit anti-HBc was purchased from DAKO (CA). Mf~nnrl~n:~l (Mab) anti-HBc (3105, 3120), anti-HBe (904, 905) and anti-HBeAg peptide (2221) antibodies were provided by M. Mayumi aichi Medical College, Japan) (Takahashi, et aL, ~ ImmunoL, 130:2903-2907, 1983; Imai, et aL, J.ImmunoL, 128:69-72,1982;Takahashi, etaL, J.ImmunoL, 147:3156-3160,1991).
10 These antibodies can be purchased from the Institute of I~ lo~ y, Japan. M~-n~rl r,n "1 anti-HBc (440, 442), and anti-HBe (420, 422, 426) antibodies were obtained from Green Cross (Osaka, Japan). Peroxidase conjugated Mab anti-human IgG was p}ovided by Ortho Diagnostics (NJ). rt,lu~ -conjugated polyclonal goat anti-mouse or rabbit Ig was purchased from Boeh~inger Marmheim aN).
15 3. Direct Solid-Ph~c~ Fn7;ylne T",., .. ~
Solid-phase erayme y~ (EIA) were used to measure serum anti-HBcW, IgG
anti-HBc, IgM arlti-HBc or anti-HBe levels. R.~.. l.. .l WHcAg, HBcAg or HBeAg were coated onto microtiter plates (50 ng/well) overnight at 4C, the plates were IJIC~ ' ' ' for I hour at 37C with 50~1 of PBS contairfmg 1% BSA, 0.005% Tween 20, and 5% heat inactivated goat serum (blocking buffer). Human seM were diluted 1/500 in blocking buffer and added to the plates and incubated for 2 hours. The plates were incubated with 50~1 of peroxidase conjugated anti-human IgG (1:3000) or anti-human IgM (1:1000) for 2 hours. The plates were developed by a final incubation for 10 min.
with 50~1 of ul alu~ll.,l..~l..lc diamine (OPD). The absorbance was read on an automatic 25 microtiter plate reader. The data are expressed as a P/N ratio, which represents the W0 95/27083 ~ 1 8 `~ F~ a.. _ ~41 absorbance (OD49~) value of the sample (P) as compared to the mean absorbance value of at least 15 control sera.
Quantitation of serum IgM anti-HBc was also performed by using the Abbott Corzyme-M
kit (Abbott Labs, Chicago, IL) æcording to the " A- 111 r- ~. rl ~ procedure. A cut-offvalue 5 of 0.25 tirnes the positive control mear~ (PC) plus the negative control mean (NC) was calculated (0.25 x PC+NC), and the sample/cut-off ratio was defined as the cut-off index (C.l.) according to the Ill~ IIICI. At the same time, 'l~ ;.... of serum IgM anti-HBc was performed using the direct EIA. There was a high correlation in IgM anti-HBc values in sera tested irl the Corzyme-M kit versus the direct EIA (p(0.001, r-0.62, n=86).
10 Detection of total serum anti-Bc was also pe~formed by using the Abbott Corzyme kit (Abbott Labs, Chicago, IL) æcording to the " . --, -. l l ,. ~. ~ procedure. In this assay, anti-HBc was expressed as a percent inhibition, and inhibition of more than 50% is considered positive for anti-HBc.
4 Q~ nfjt6~tjve~JReA~ orT~eA~-~pPrifirImm~mpCrln~,nlPvpcAe~ vs 15 To detect HBsAg or HBeAg-specific immune complexes in human serum, a solid phase EL~ has been developed (Maruyama, et a~., J immunol. Meth., 155:65-75, 1992).
Briefly, plates were coated with mr,nnrlrln~l antibody (0.5~g/ml) specific for either HBsAg (IBIE7F7) (Ortho Diagnostic, N.J.) or specific for HBe Ag-specific peptide(2221) as the capture reagent~c. These solid phase Mabs were chosen because they were previously d.. ,.. ~ l to bind immune-complexed as well as free antigen. Test sera diluted 1/25 in blocking buffer were added to the wells coated with solid-phase Mabs and the plates were washed after a 2 hr. incubation. The serum anti-HBs or anti-HBe antibody component of the IC was then detected with peroxidase-labeled mnnflrir n^l antibody to human IgG. The data are expressed as a P/N ratio, which represents the absorbance 25 (OD~9~) value ofthe sample (P) as compared to the mean absorbance value of at least 15 control sera.

WO 95127083 r~ ' IC'I
~ ~ ~633~

5. Patients Twenty-six patients with æute hepatitis type B (AH-B), fifty-tbree patients with chronic hepatitis-B (CH-B) including twelve patients with chronic persistent hepatitis (CPH) and forty-one patients with chronic active hepatitis (CAH) were studied. The diagnosis of 5 AH-B was bæed on the findirlgs of elevated values of serum alarline ~,.,;.,~.l.,."~F~
(ALT) (at least 10 times the upper limit of normal), associated with the detection of HBsAg and IgM anti-HBc antibodies in the serum together with the recent onset ofjaundice and other typical symptoms of acute hepatitis. All AH-B patients were knovin to be HBsAg negative prior to liver injury. Fu~lL.,~ ul~;, all AH-B patients recovered 10 completely from illness, with 11I ~ ;I II I of ALT and clearance of BsAg from the serum. All CH-B patients in this study who were persistently positive for HBsAg and BeAg in their serum for more than I year, were diagnosed as CPH or CAH by histology. All AH-B patients were followed at the First Department of Internal Medicine, Tokyo University (Tokyo, Japan). All sera were tested for BsAglanti-Bs, 15 HBeAg/anti-Be, anti-Bc, IgM anti-Bc, IgM anti-hepatitis A virus, anti-HDV andanti-HCV by using commercial enzyme ;". ~ (ElAs; Abbott T slhhr~thn~) in addition to the ~A~ assays for IgM anti-Bc, IgG arlti-Bc, anti-HBe, anti-BcW, and HBsAg or BeAg-specific immume complexes measured by direct solid-phase EIAs. All AH-B and CH-B patients were negative for IgM-anti-HAV, anti-HDV and 20 anti-HCV. As controls for the novel anti-HBcW assay, sera were collected from an additional 26 healthy controls, 7 acute hepatitis A, 8 acute hepatitis C, 30 chronic hepatitis C, and 10 BsAg-positive and BeAg-negative chronic hepatitis B patients.
BV-DNA was determined using a dot blot procedure (Lieberman, e~ aL, ~epatology, 3:285-291, 1983). Briefly, 101,1 of serum was denatured and applied to a nitrocellulose 25 membrane under vacuum. Hybl;d;~i~ll was perFormed using 32p labeled cloned BV-DNA (specific activities; 2 ~ x 108 cpml,~lg). The membranes were ~ h~ d for 96 hours on X-ray films. Serially diluted, known quantities of cloned BV-DNA served as positive controls, and 0.5 pg of HBV-DNA was detectable in the assay.

WO 951270~ J ~.,5 o t ~ 33~ ~
Comparison between mean values in all assays was determined by Student's t-test. A p value (0.05 was corlsidered sigmficant.
F.XAMPI.F, 2 CO~IPARI~ON QF T~TF, PRTrVAT.T~.NCT~ QF ANTI-~RP
TTR_~/ANTI TTR_ 1(~ AND EIRC~lAl~TI-~TRC l~c IN ACUTE ~NI) (~TRONIC ~RV INFl~ CTION
SerlAm samples from 26 acute hepatitis type B (AH-B) patients and 53 chronic hepatitis type B (CH-B) patients collected during period of peak alanine .~ (ALT) elevations (mean ALT in AH-B patients 1790+ 1351 UA; in CH-B patients 385 + 229 UA) were analy~ed for the presence of amti-HBe, HBeAg/amti-HBe immune complexes (ICs) and HBsAg/anti-HBs ICs by the . I ... ;., ... ,l -l ;l l l .. l. I~A~A,I~ described. Note that all AH-B and CH-B patient sera were negative for amti-HBe antibody when tested m a commercial assay (Abbott T.-AhA,rr~AriP~). In contrast, when sera were analy~ed by the direct EIA method all CH-B patients ~1...,.... 1....1 sigluficant anti-HBe antibody 15 production (FIGURE l,A).
FIGURE 1 shows a IA.An~rAri~An between AH-B and CH-B patient serum levels of anti-HBe, HBeAg/anti-HBe ICs, and HBsAg/anti-HBs ICs. Serum samples from 26 AH-B
and 53 CH-B patients were analyAed for (A) anti-Be at a 1/500 dilution; (B) HBeAg/anti-HBe ICs at a 1/25 dilution; and (C) HBsAg/anti-HBs ICs at a 1125 dilution 20 in direct ElAs as described. Single serum samples were collected from each patient at the peak of ALT elevation IC~JIC " _ the period of most severe liver injury. Results are expressed as a PAN (PositivelNegative) ratio and the mean + s.d. for each patient group is also shown. N=0.0 16 + 0.0002 (anti-HBe assay); N=0.08 1 0.028 (HBeAg/anti-HBe ICs assay); and N=0.093 + 0.034 (HBsAg/anti-HBs ICs assay).

w0 ss/27083 2 1 8 6 3 3 8 r~l/o Although most AH-B patient sera were positive for anti-HBe, the sera of AH-B patients contained s;~lirl~all~ly less anti-HBe antibody as compared to CH-B patients. However, 13% of CH-B and 46% of AH-B patients exhibited u~,.lau~ lg levels of anti-HBe between 7.9 and 24.1 P/N values. This degree of overlap ~,UIIII~IUll~ .S the usefulness of 5 this assây m terms of ~ ; " ,; " ~ between AH-B and CH-B infection. Because the sera of AH-B and CH-B patients taken during periods of liver injury were also positive for the HBeAg, sera were analyæd for the presence of HBeAg/anti-HBe ICs (FIGURE
I ,B). The sera of CH-B patients contained ~ , greater levels of HBeAg/anti-HBe ICs as compared to AH-B patient sera and the degree of overlap between the two 10 patient groups was quite minimal (7% of CH-B and 19% Of AH-B patients exhibited Uv~ ,U,U;II~ P/N values between 2.2 and 3.5). Similarly, CH-B patient sera contained si~ llirl.,all~ly bigher levels of HBsAg/arlti-HBs ICs as compared to AH-B patient sera (FIGURE l,C). However, there was a significamt overlap in the HBsAg/anti-HBs IC
assay between P/N values of 1.1 and 5.4 amongst CH-B patients (18%) and AH-B
15 patients (92/0).
CUIIIUIa~ ,lY, the ~ assays for the lll."w~ of anti-HBe, HBeAg/amti-Be ICs, and HBsAg/anti-HBs ICs illustrate very significant differences in the mean levels of these parameters between AH-B and CH-B patient sera, however, complete .1;~ ;,, ,;, -' ;I~. . based on any single assay would be difficult due to u v~la~,uillg values at 20 the margins (E~IGURES IA-C). However, all AH-B and CH-B patient sera were analyzed at the same single dilution (i.e., 1/500 for anti-HBe analysis; and 1/25 for IC analysis).
It is possible that ., ~ in assay design may yield results more useful in .l;~. . ;.,.;..- ;.,P between acute and cbronic BV patient groups.

wo95127083 2~ 8633~ P~I,. o,1~1 E~MPLT~. 3 CQMPAR~TIVI~ T,~VF.T.C ~F Tu~G ANTI-}~Rr ~M ANTI-JTRr ANn AI~TTI-~RrW IN AClITE ANn ('~RONIC I~KV INFECTION
A commercial anti-HBc assay (Corzyme, Abbon Labs) modified by using 1/500 dilutiorls of sera was used to compare AH-B and CH-B patient sera for total anti-HBc. FIGTJRE
2 shows a c~nnr~nC~m between AH-B and CH-B patient serum levels of anti-HBc, IgManti-HBc, IgG anti-HBcW, and IgM anti-HBc/IgG amti-HBcW ratio. Serum samples from 26 AH-B and 53 CH-B patients were analyzed for (A) total anti-HBc at a dilution of 1/500 in the Corzyme assay (Abbott T .:~h~ q); (B) IgM anti-HBc at a 1/1000 dilution in the Corzyme-M assay (Abbon T.l ' ); (C) IgG anti-HBcW at a dilution of 1/500 in a direct EIA using WHcAg as the solid-phase ligand; and (D) the ratio of IgM anti-HBc/IgG anti-HBcW. Single serum samples were collected from each patient at the peak of ALT elevation. Results are expressed as % inhibition in the anti-HBc assay, as a Cut-Of ~ Index (C.I.) and as a P/N ratio in the IgM anti-HBc assay, and as a P/N ratio in the IgG anti-anti-HBcW assay. The mean + s.d. for each patient group is also shown. N=4.6 + 9.5% (IgG anti-HBc assay); N=0.075 _ 0.012 and I C.I. was 0.298 (IgM anti-HBc assay)l and N=0.014 _ 0.002 (IgG anti-HBcW assay).
As previously shown, both AH-B and CH-B patients produce anti-HBc efficiently although CH-B patient sera ~'~- ~ ' higher levels (101 + 3% inhibition) as compared to AH-B patient sera (89 _ 15% inhibition) (p (0.001). A direct EIA was also used to measure IgG anti-HBc in AH-B and CH-B patient sera and the results were similar to the commercial assay. Even using 1/500 serum dilutions in both anti-HBc assays there was very significant overlap between AH-B and CH-B patients in terms of IgG anti-HBc values (FIGTJRE 2, panel A). In contrast, use of the Corzvme-M kit (Abbott T 5Ih~ ) to measure IgM anti-HBc antibody illustrates the preferential production of IgM anti-HBc antibodies in AH-B patients as compared to CH-B patients (FIGURE 2, panel B). The mean IgM anti-HBc level expressed as a Cutoff Index (C.I.) vas 9.2 _ 3 .0 (or 3 7.0 _ 12.1 P/N ratio) in AH-B patient sera and I . I + 0.9 C.l. (or 4.6 +

Wo9sl27083 21 8 6338 r~
.

4.0 P/N ratio) in CH-B patients (p (0.001). Nu~ a~ 20% of CH-B and 23% of AH-B
patients exhibited IgM anti-HBc levels that oYerlapped between the 1.6 Cl. (6.6 P/N) and 6.3 C.I. (25.4 P/N) values.
Previous studies have indicated that the ~ of the HBV amd the WHV share a ~luaalca~.~ive epitope(s) (Wemer, et al., J. ViroL, 1:314-322, 1979; Millmam, et aL, Infection and lmmunit,v, 2:752-757, 1982; Stannard, et aL, J. Gen. ViroL, 64:975-980, 1983; Ponzetto, et aL, Virus Research, ?:301-315, 1985). Therefore, it was of interest to detemline if human amti-HBc antibodies recogluzed WHcAg, and if AH-B and CH-B
patient sera could be .~ 1 based on differential reactivity to WHcAg. For this purpose, a direct EIA was developed using, r- ~ WHcAg as solid-phase ligand amd a ,.. f~ AI amtibody (Mab) specific for human IgG as the probe.
FIGURE 3 illustrates the end-point titrations of pools of 5 AH-B patient sera, 5 CH-B
patient sera and S nommal human sera (NHS) assayed for IgG binding to solid-phase WHcAg. The d2ta are expressed as meam + s.d. absorbance values (OD492). The end-pointtiteroftheCH-Bpatientserawas 1/62,500ascomparedto l/lOOinAH-Bpatient sera. This result suggested that at least a proportion of human anti-HBc amtibodies can crossreact on WHcAg, and that chronically infected HBV patients may preferentially produce this ~lV~ iV~ specificity designated as anti-HBcW.
Based on the end-point titration analysis, a 1/500 serum dilution was chosen to screen the 26 AH-B and 53 CH-B patient sera for IgG binding to solid-phase WHcAg (FIGURE 2,panel C). The CH-B patient sera exhibited a;~5.lirlualltly higher levels of IgG anti-HBcW
(P/N 37.5 _ 10.5) as compared to AH-B patient sera (P/N 2.5 + 1.4) (p(O.OOI).
~ ~L~,. IIIUI L~ there was no overlap between the IgG anti-HBcW values of AH-B and CH-B
- patient sera (FIGURE 2, pamel C). IgM anti-HBcW amtibody production was also examined and no sigluficant difference was observed between the low levels of IgM anti-HBcW produced in AH-B patients (P/N 2.1 _ 1.6) and CH-B patients (P/N 2.4 + 4.2).

wo 95r27083 r l"~
21 ~338 --/~
The IgM anti-HBc assay and the IgG anti-HBcW assays performed singly were the two most useful assays to ~' between acute and ay~ Lulll~Liu chronic HBV
infection. However, performing both of these assays and expressmg the results as a ratio of IgM anti-HBc/IgG anti-HBcW provided a very powerful serologic method of 5 .~;~-, ;, . ,;, .~: ;. .~ between AH-B and ~ Ullld~i-, CH-B infections.
MPL~ 4 PRODUCTION OF ANTI-~RrW J~ UNIOUE TO EIRV INFECTIONS
Before proposing the utilization of the anti-HBcW assay to distinguish between acute and chronic HBV infections, it was necessary to establish tbat anti-HBcW antibody production 10 is specific to HBV infection and not a non-specific result of æute or chronic liver injury.
In addition to the ~3 HBeAg-positive CH-B patient serapreviously described, lû HBeAg negative CH-B patient sera, 7 acute hepatitis A patient sera, 10 acute hepatitis C patient sera, 30 chronic hepatitis C patient sera, amd 26 normal control sera were evaluated in the anti-HBcW assay (Table 1). Only HBsAg-positive, CH-B patients positive or negative for 15 the HBeAg produced anti-HBcW antibody, and these patient groups produced anti-HBcW
~It~freq~lencyof ~0~/.

w09s/27083 2 ~ ~6338 r l,~, c 1~ 1 TABLE; 1 pRODUCTlON OF ANTl'~TRrw I~G
T~ UNIOUE TO ITRV INFECTION
¦Anti-HBW
Patients No. Mean + s.d. Range ~requency (%) 5HBsAg (+) HBeAg (+) CH-B 53 0.556 + 0.156 0.141 - 0.957 100 HBeAg (-) CH-B 10 0.494 + 0.266 0.131-1.003 100 HBsAg (-) Acute hepatitis-A 7 0.012 + 0.002 0.009 - 0.015 0 10 Acutehepatitis-C 10 0.008+0.003 0.005-0.016 0 Chronic hepatitis-C 30 0.008 + 0.002 0.004 - 0.017 0 Normal Controls 26 0.007 + 0.003 0.006 - 0.014 0 Serum samples taken during periods of elevated ALT values from patients with the15 indicated diagnosis were diluted 1/500 and analyzed for IgG anti-HBcW reactivity by direct EIA. Data are expressed as mean + s.d. absorbance (OD~g~) values.
F.XAMPl.li. S
llCINETICS OF ~M ANTI-lTRc. I~G ANTI-ITR.~ AND ANTI-~TT~cW
~NTTT~OPY PRODUCTION IN ~CUTE AND CIIRONIC ITT~V INFECTION
20 To examine the kinetics of anti-HBc antibody production, temporal serum samples from 14 AH-B and 21 CH-B (4 CPH and 17 CAH) patients taken during a period of 4-5 months aroumd the time of peak ALT elevations were amalyzed for IgM anti-HBc, IgG
anti-HBc and IgG anti-HBcW. FIGURE 4 shows the kinetics of arltibody production to the HBcAg in AH-B and CH-B patients. Sequential serum samples from 14 AH-B (A) 25 and 21 CH-B (B) patients were analyzed for IgM anti-HBc (.-.), IgG anti-HBc (.-~), and IgG anti-HBcW (~-~) at the indicated time points relative to peak elevations irl ALT
values~ All antibodies were measured by direct EIA. The results are expressed as mean w095/27083 ~ 3.~ P .,~, o~

+ s.d. PIN ratios. N=0.015 + 0.00~ (IgM anti-HBc assay); N=0.011 + 0.00~1 (IgG anti-HBc assay); and N=0.011 + O.OOS (IgG amti-HBcW assay).
In AH-B patients, IgM anti-HBc levels were initially high before the peak ALT elevation and declined thereafter (from a mean of 9.8 to 4.1 P/N), conversely, the production of IgG
5 anti-HBc was initially low and increased during resolution of the hepatitis (from a mean of 9 to 27.5 P/N), and IgG anti-HBcW production remained low to negative throughout the observation period (FIGURE 4,A). In CH-B patients, the level of IgM anti-HBc was relatively low S-8 weeks prior to liver mjury, but increased in parallel with ALT elevation and declined thereafter (from a mean of 3.0 to 6.2 PIN) (FIGURE 4B). The levels of IgG
10 anti-HBc in CH-B patients remained quite high throughout the observation period (from a mean of 32.3 to 39.0 P/N), as did IgG anti-HBcW production (from a mean of 43.0 to 50.3 PIN) (FIGURE 4,B). These data indicate that during the course of liver injury a single serum sample taken from an AH-B or CH-B patient may reflect fluctuating IgM
and IgG anti-HBc antibody levels that may not clearly distinguish between an acute 15 infection and an acute ~ ;. of a chronic infection. In contra3t, the anti-HBcW
levels remain ,u~ LIy low in AH-B patients and Cu~ ..ly high in CH-B patients before, during and after periods of liver injury (FIGURE 4A, B).
The consistency and usefulness of the anti-HBcW assay is ~' - ' in the serological profile of a single CH-B patient shown in FIGURE 5. A CAH patient (H,M. a ~2 year-20 old female) was monitored for a 6 year period during which two A.E. of liver injury (4/88and 4190) were recorded. The standard serologic parameters shown (top) were measured by commercial a3says. IgM anti-HBc, IgG-anti-HBc, and IgG anti-HBcW were measured by direct EIA a3 described. The data are expressed as P/N ratios for each time point (---= IgM Anti-HBc; .-~ = ALT; ~-~ = IgG anti-HBc; o-o = IgM anti-HBcW).
25 This CH-B patient ~ . .,. Fd two A.E. of liver injury in April of 1988 and in April of 1990. If this patient appeared in the clinic for the first time in April of 1990 without a prior history of HBV infection, the only serological marker that would indicate an A.E.

Wog5/270s3 2 18 63~8 r~l,. o~
.

of a chronic HBV infection rather than an acute HBV infection is the elevated anti-HBcW
value. Note that the IgM anti-HBc value is ai~lirl~,~lLly elevated during the period of maximum liver injury when the patient is most likely to seek medical attention.
.
h~AMPT.F, 6 lMl~lONOLOGlCAl. Cl~A~AcTFRl7~TIoN OF Tll~ RcW FPlTOPE
Although the production of IgG antibody reactive with WHcAg correlated with IgG anti-HBc antibody production in chronic HBV infection, it was necessary to confirm the antigenic relationship between the WHcAg and the HBcAg directly. For that purpose, a pool of sera obtained from 6 CH-B patients positive for anti-HBcW was analyæd in an 10 antigen ~ assay. FIGURE 6 shows an antigen inhibition analysis of CH-B
patient sera Sera from 6 CH-B patients positive for anti-HBc and anti-HBcW antibodies were ~,.. ' ' with the indicated c.~.. IIIAlI.. ~ of the soluble inhibitors HBcAg, WHcAg, or HBeAg overnight at 4C. The residual anti-HBcW (A) or anti-HBc (B) reactivity was tben assayed by direct EIA using solid-phase WHcAg or HBcAg, 15 I-a~ ,Li~,ly,assolid-phaseligands. Thedataareexpressedaspercentbindirlgcompared to tbe absorbance value (OD~92) obtained without soluble inhibitor.
The CH-B patient sera was 1~ ' ' with soluble HBcAg, HBeAg or WHcAg prior to addition to either solid-phase WHcAg (FIGURE 6A) or HBcAg (FIGURE 6,B), and the percent of IgG binding to the solid-phase ligands as compared to unabsorbed CH-B
20 patient sera was ~' ' The binding of CH-B patient IgG to solid-phase WHcAg was ~ ,ly inhibited by WHcAg and HBcAg to the same degree but was not inhibited by HBeAg (FIGURE 6,A). This result indicates that the HBcW epitope(s) recogniæd by CH-B patient IgG is present on both the HBcAg and the WHcAg but noton the HBeAg.

Wo 9s/27083 ;~

In the reciprocal assay, the binding of CH-B patient IgG to solid-phase HBcAg was ~lu~LiL~ y inhibited only by HBcAg and not by WHcAg or HBeAg (FIGURE 6,B).
The inability of soluble WHcAg to inhibit the binding of CH-B patient IgG to BcAg irldicates that the HBcW epitope(s) is ~IU~ iVG with only a minor proportion of 5 HBcAg epitopes, and that the HBcW-specific antibody detected irl CH-B patient sera represents only a minûr compûnerlt ofthe total IgG anti-HBc response. This is consistent with the observation that no HBcAg-specific Mab recogrlized the WHcAg (Table 2).

WO 95127083 2 1 ~ ~ ~ 3 ~ . t ~

~LE~
T~, ElRrW ~PITOPE 1~; NOT l?~.CO~N-7.~n BY
T~, l~OWN ~Rr~G ANn l:lRr~G ~PECTIilC
- MONOCLONAI ANT-RODl~
Antibody WHcAG HBcAG HBeAg 5 Mab Arlti-HBc 3105 0.01 1.29 0.16 3120 0.04 1.99 0.08 440 0.03 1.65 0.35 442 0.01 0.58 0.1 1 Mab Anti-HBe 904 0.01 0.08 0.92 905 0.02 0.10 1.39 420 0.01 0.12 1.93 4æ 0.01 0.09 1.53 . 426 0.01 0.03 1.43 Mab Anti-HBe peptide (2221) 0.69 0.63 2.00 Poly Arlti-HBc/e 0.12 2.0 2.00 10 PolyAnti-WHc 1.90 0.16 0.11 nmh;~ t WHcAg, HBcAg and HBeAg were coated on the solid-phase (50ng/well), and the panel of indicated arltibodies were assayed for bindirlg in direct EIA. Mabs were assayed at 0.5 llg/rnl arld polyclonal sera was diluted 1/8000. The data are expressed as 15 absorbance (OD 492) values.

wo ssr27083 r~"
21 8~33~ ~

Similarly, a panel of HBeAg-specific Mabs also failed to recognize the WhcAg. Only Mab2221 specificforresidues 129-1400ftheBc/BeantigensboundWHcAg(Table 2). The 129-140 sequence is highly corlserved between WHcAg and BcAg.
Next, the IC~IUil~ll.~lL for natiYe WHcAg structure for binding by human anti-BcW
5 antibody was examirled. R~ ' WHcAg was treated with a range of cl..... l.~l;.... ~ of SDS/2ME (0.006-6.4%), and human anti-HBcW IgG, derived from a group of CH-B patient sera, and mouse anti-WHc and rabbit anti-WHc IgG, produced by ;l.. l.. ;~_ ;~.. with rWHcAg, were analyzed for binding to solid-phase denatured WHcAg. FIGURE 7 shows sensitivity of anti-gCW reactivity to A..,"l~..,.l;"" of WHcAg. R.-- ' WHcAg(I mg/ml)wastreatedwiththeindicatedf.. ,l.,.l;.,,,~
of SDS/2ME for 2 hours at 37C, and WHcAg (diluted 1000 X) was coated on the solid-phase (50 ng/well). Polyclonal mouse 3nti-WHc (Ma-WHc, .--.) polyclonal rabbit anti-WHc (Ra-WHc) (---), monoclonal anti-Be peptide (Mab 2221) (A-A), or humam anti-BcW-positive antisera from 4 CH-B patients (Ha-BcW) (o-o) were added to the wells, 15 and bound antibodies were detected by peroxidase-labeled anti-murine, rabbit or human Ig. The data are expressed as percent binding compared to the absorbance value (OD492) obtained without SDS/2ME treatment.
The Mab 2æl, which birlds tbe linear peptidic epitope 129-140, served as a positive control to insure that adequate denatured WHcAg a&ered to the solid-phase well.
20 Binding of human anti-HBcW to WHcAg was very sensitive to ~ ,a ;~ of WHcAg, and was reduced 80% by the treatment of WHcAg with 0.1% SDS/2ME. Similarly, the binding of polyclonal murine and rabbit antisera raised against native WHcAg were equally sensitive to the ~ of WHcAg. The .. ~ f~ of human anti-HBcW was futther fl .... ~ J by the failure to detect binding of human 25 anti-HBcW to a panel of WHcAg-derived u .~ iUlg peptides.
Lastly, to determine if the epitope(s) on WHcAg recognized by human anti-HBcW IgG
w~ species-specific, antibody f f~nrf titif n assays were performed between human anti-WO 95~27083 2 1 8 6 3 3 8 HBcW antisera and muline and rabbit polyclonal antibody raised against rWHcAg (Table 3). The binding to WHcAg of a panel of 4 CH-B patient sera, positive for anti-BcW
reactivity, was ~ Li~ii~ ,y inhibited by competitor rabbit and murine polyclonal anti-WHc antisera Reciprocally, the binding to WHcAg of both rabbit and murine anti-WHc 5 antisera were irlhibited by a panel of 6 human amti-HBcW-positive antisera used as c.~ (Table 3). Therefore, the WHcAg . I~aal~Liv~: epitope(s) recogniæd by human anti-BcW-positive antisera is also recognized by mice and rabbits immunized with WHcAg.

ANTI-FlRrW ANTIT~ODY PRODUCTION l!;; NOT SPI ('TT~ SPE('Tli'IC
Primary Competitor Inhibition (%) Antibody Antibody 1:100 1:500 1:2500 H anti-HBcW R anti-WHc 96.7 + 0.9 96.0 + 0.8 91.7 + 2.7 (N=4) M anti-WHc 91.2 + 1.8 87.2 + 5.3 77.2 + 8.5 Ranti-WHc Hanti-HBcW 40.7+11.6 23.2+17.0 17.2+14.4 (N=6) Manti-WHc Hanti-BcW 36.2+10.1 23.2+12.0 14.5+8.8 (N=6) The imdicated competitor antibodies polyclonal rabbit (R) or murine (M) amti-WHc, or human (H) anti-BcW derived from CH-B patient sera were l " . :. ,. ', ~. . I with solid-phase WHcAg (50 nglwell) for 2 hours at 37C. Thereafter, the indicated primary antibodies were added to the wells and the percent inhibition by the competitor antibodies as compared to inhibition with l" ~ . sera or normal human sera was deter~uned.
The invention now being fully desclibed, it will be apparent to one of ordinaly skill in the art that various changes and ~ can be made without department from the spirit or scope of the invention.
SUBSTITUTE SHEET IRULE 26)

Claims (26)

-26-

1. A method of diagnosing chronic hepatitis B virus (HBV) infection in a subject comprising:
a. contacting a sample from a subject suspected of having HBV with an epitope that contains the amino acid sequence of a woodchuck hepatitis virus antigen wherein the epitope binds with antibody to woodchuck hep-atitis B core antigen; and b. detecting the presence of antibody to woodchuck hepatitis B core antigen in the sample.

2. The method of claim 1, wherein the sample is serum.

3. The method of claim 1, wherein the epitope is bound to a solid support.

4. The method of claim 1, wherein the detection includes the addition of second antibody that binds to the anti-woodchuck hepatitis B core antigen antibody.

5. The method of claim 4, wherein the second antibody is detectably labeled.

6. The method of claim 5, wherein the label is selected from the group consisting of enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, phosphorescent compounds, and bioluminescent compounds.

7. The method of claim 1, wherein the anti-woodchuck hepatitis B core antigen antibody is isotype IgG.

8. The method of claim 7, further comprising detecting the level of IgM anti-hepatitis B core antigen in the subject.

9. The method of claim 8, wherein the ratio of IgM anti-hepatitis B core antigen antibody to IgG anti-woodchuck hepatitis B core antigen antibody is determined.

10. The method of claim 1, wherein the chronic hepatitis is chronic acute hepatitis.

11. The method of claim 1, wherein the chronic hepatitis is chronic persistant hepatitis.

12. A method of diagnosing chronic hepatitis B virus (HBV) infection in a subject comprising:
a. contacting a sample from a subject suspected of having chronic HBV with a first reagent which binds with anti-HBe antibody, a second reagent which binds HBeAg/anti-HBe immune complexes (IC) and a third reagent which binds HBsAg/anti-HBs ICs; and b. detecting the presence of anti-HBe antibody, HBeAg/anti-HBe immune complexe (IC) and HBsAg/anti-HBs ICs in the sample.

13. The method of claim 12, wherein the sample is serum.

14. The method of claim 12, wherein the reagent is an antigen or an antibody.

15. The method of claim 12, wherein the reagent is bound to a solid support.

16. The method of claim 12, wherein the reagent is detectably labeled.

17. The method of claim 16, wherein the label is selected from the group consisting of enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, phosphorescent compounds, and bioluminescent compounds.

18. The method of claims 12, wherein the detecting is measured as a positive to negative (P/N) ratio.

19. The method of claims 18, wherein the P/N ratio is greater than 3.

20. A reagent kit for diagnosing chronic hepatitis B infection comprising carrier means being compartmentalized to receive in close confinement therein one or more containers comprising a first container containing woodchuck hepatitis B
core antigen epitope, and a second container containing an antibody that binds to a human antibody.

21. The kit of claim 20, wherein the antibody that binds to a human antibody is labeled.

22. The kit of claim 21, wherein the label is selected from the group consisting of enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemilumi-nescent compounds, phosphorescent compounds, and bioluminescent compounds.

23. A reagent kit for diagnosing chronic hepatitis B infection comprising carrier means being compartmentalized to receive in close confinement therein one or more containers comprising a first container containing HBc antigen, a second container containing an antibody that binds to HBeAg/anti-HBe IC and a third container containing an antibody that binds to HBsAg/anti-HBs IC.

24. The kit of claim 23, wherein the kit further contains a container with an antibody which binds to a human antibody.

25. The kit of claim 24, wherein the antibody that binds to a human antibody is labeled.

26. The kit of claim 25, wherein the label is selected from the group consisting of enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemilumi-nescent compounds, phosphorescent compounds, and bioluminescent compounds.