CA2223277A1 - Method for detection of antibody to hepatitis c virus second envelope glycoprotein - Google Patents

Abstract

A method for detecting antibody to HCV in a test sample. The method includes utilizing a recombinant protein that is the expression product of mammalian cells transformed by a heterologous expression vector comprising a DNA
sequence encoding an E2 truncated protein. Test kits which include this recombinant protein also are provided.

Description

wo 96/4ll96 PCT/US96/08536 MF 1 llOD FOR l~ETECTION OF A~TIBODY TO HEPATm.~ C V~RUS
S~COND ENVELOPE G~ l~COPl~OT13~IN
F~T T OF T~ T~V~l~TION
The present inver~ion relates genr. ~lly to a mrthr~l for ~
antibodies to hf~ ic C virus (EICV), and more particularly, relates to a m~.thr,~ for clet~ctin~ antibodies to an ~ lition~l marker for HCV infection, the second envelope glycoploL~iL, (E2) of HCV.

~CKG~OUND OF 1~ ~V13NTION
The intro~lctinn of first ~P..~ on hrp~titic C virus enzyme i... ~n~cc~ys (HCV 1.0 EIAs) in 1989 and second ge~ on HCV enzyme ;... ~.c.c~ys (HCV 2.0 EIAs) in 1992 has ~11,.. ,.~1ir~11y reduced ~he incidence of post-l~n--~rl~;ion HCV (PT-HCV) i~e~;Lio., in countries routinely screening donated blood using these tests tDonahue et al., N. PT~1. J. Med. 327:369-73 (1992); Klçinm~n et al., Tr~ncfusior~ 32: 805-13 (1992); Alter, in Vir~l atitis ~n(1 T~iver Disease 551-53 (Nishioka et al. eds., 1994)]. In the second Ye~ m assay, antibodies to HCV (anti-HCV) are clet~ctecl using recombinant pl~L~illS derived from the core, NS3 (viral protease) and NS4 (unknown fimt~ti~n) genes of the virus. Third gc;ll~ldlion screening assays (HCV 3.0 EIAs), which include an ~ litic n~l antigen from the NS5 region (viral polymerase and a second unknown function), are now available and are being used in Euro~e ~Lavanchy et al., J. Cltn Microbiol. 32 ~72-7s (1994)].

Additional unlicensed immlm-~blot assays such as an illllllllllodot assay or a strip immnn~blot assay (SIA) can be used for the det~ction of antibodies to individual HCV p.~,Lei~ s, including core, NS3, NS4 and NS5.
These assays are int~n~letl for use as an additional, more particular test for those human seru_ or plasma test samples found repe~t~ly reactive using a licensed anti-HCV screening assay, e.g., HCV 2.0 EIA. These assays are used as a means to CO~ reactivity in a HCV 2.0 Sclee~ g assay. According to the conventional hl~ ion of results for these assays, reactiviey to at least two HCV ~lol~ s cc~ s~?onding to antigens encoded by dilr~euL parts of the HCV
genome is iu~c~ led as positive. Reactivity to only a single HCV protein is d as in(~ r, Any donor demo~ antibodies specific for 2 or more HCV
S gene products should be regarded as having serocol,v~lL~d to HCV subse~ne-nt to inf~ction with this virus [Alter, 1994; Bl~L~ et al., Tl,...~rusion 33:634-38(1993); Sayers & Gretch, T.~ ru~ion 33:809-13 (1993)]. In ~r1(1ition~ bec~use of the high cl~ ci~y rate associated with HCV i.~re.;~io~- [Di Ri~cegli~ et al.,~epatolo~y 14:969-74 (1991); Alter et al., N. Fr~l. J. ~ed. 327:1899-905 10 (1992)], these donors should be considered potentially infectious. With respect to the inflrl~ r test samples, blood banks have a .~igniflr~ntcol~ elu with respect to how to handle indel~ ~i~Le cases and the added ~:A~e~ se of follow-up testing.
Leon et al., using multiple suppletn~ont~l HCV tests to evaluate 15 RIBA 2.0 intl~lr ...i.-~lt?/PCR negative samples, demo..~l.,.lr~l that true HCV
antibodies were present in 71.6% and 57.1% of core and NS3 in~lele~.--i-.i.l.;
spec;---e~ ;~e~;Lively [Vox S~ 66:245-46 (1994)]. Previous studies using PCR have inrlir~tlo(l as high as 75% of RIBA 2.0 inrlrle....i.~le sperimpn~
may be HCV RNA positive [Halfon et al., Journ~l of rnfectious Di.~
166:449 (1992); Buffet et al., J. Med. Virol. 43:259-61 (1994)]. A positive PCR result on inrlr~ .-.i..~le specimens is compelling evidence of HCV
)O~U1~. On the other hand, in-l~Lr. "~ samples which are PCR negative is not enough evidence to conclude false reactivity. While one previous study [Tobler et al., Tr~n~fusion 34: 130-34 (1994)] suggests that RIBA 2.0 2~ in-lel~ " . ,i "~l~ samples which are PCR negative are most likely to intli~-~t~ false reactivity for HCV, negative PCR results on in~ie~ samples need to be regarded as inconclusive due to factors such as low virus concentration [IJlrichet al., J. Clin. Jnvesti~ation 86:1609-14 (1990)], illLt.l..illr.ll viremia during HCV infection [Allain et al., J. Clin ~nve~ti~tio~ 88: 1672-79 (1991); Prince et al., J. ~nfPctious Diseases 167:1296-301 (1993); Peters et al., J. Med. Virol.
42:420-27 (1994)], and variability in PCR technique [Zaaijer et al., Lancet 341:722-24 (1993)].
S ~ C~ C, c~ ,c.~ion to HCV 3.0 selccl~ill~ assays w~la~lLS
careful scluLilly be~nse of the discordant results obtained bcLwccm HCV 2.0 and 3.0 version assays, particularly among specimens i~ tifi~-l with in-1Pte. . . ~ immllnc~blot reactivities, to ensure that all potentially infectious units ~ lcllLly ~l~tt~ctP~l by HCV 2.0 tests are also ~lP-tPctPd by the new HCV

3.0 assays. It is i~ ul~ll, Lhclc;roie, to further investigate and lm~ the true di~osiLion of HCV 2.0 positive spec-imPn~ co.~ antibody reactivity to only a single gene product.
Diffirlllti~s in the e,~lc~ion and pllrifi~tinn of the ~u~Livcc viral envelope ~,uLcins (E1, E2) have ~levt;llL~d ~let~ sealcll and possible incol~oldLion of these pLoLc;l~s as targets in blood screerung assays. The 5' end of the HCV genome encodes two ~uLaLive envelope ~roLel, s, El and E2. Both u~eills contain multiple glycosylation sites [T~kPuchi et al., J. Ge~ Virol.
71:3027-33 (1990)] and share some nucleotide and amino acid seqllen~e homologies with pe~livilus envelope ploLeills [Miller & Purcell, Proc. N~tl.
Acad. Sci. USA 87:2057-61 (1990); T~kPll~hi et al., supra, 1990; Choo et al., Proc. Natl. Acad. Sci. USA 88:2451-55 (1991)]. In addition, the E2 protein has been shown to contain a hy~el v~iable region with mutation ch~r~ctPri~ti~
- similar to that observed in the hypervariable V3 loop of gpl20 of the human immlln~deficiency virus [Kato et al., Molecnl~r ~n~lBiolo~ic~l Medic;n~
7:495-501 (1990); Houghton et al., Hepatolo~y 14:381-388 (1991); Weiner et al., Proc. N~tl. Aca. Sci. USA 89:3468-72 (1992)]. As envelope pl~L~ S, El and E2 are likely to be located partially or entirely at the surface of the virion particle. This association with the structural surface of the virus would seem to make these ~lolellls prime targets for humoral immlmP responses to infection with HCV. As evidence of this, the hypervariable region of HCV E2 appears to mutate in response to humoral i"",~l~,lts selective ul~s~ule [Tn~h~ncpe et al., W O96/41196 PCTrUS~6/Og~6 Proc. N~tl. Acad. Sci. USA 88:10292-96 (1991); Ogata et al., Proc. Natl.
Acad. Sci. US~ 88:3392-96 (1991); Lesniewski et al., J. Med. Virol.
40:150-56 (1993)].
However, prior to the present invention, some investig~t -r~
5 reported low reactivity between HCV E2 recombinant ~luL~i ls and the antibodies of individuals infected with HCV. Mita et al. [~iochemi~ n-l Riophysical p~ rch Co~ r~lions 183:925-30 (1992)] and Hsu et al.
atolo~y 17:763-71 (1993)] reported that antibodies to E2 were ~1~ottoctec~ in 17% and 10%, respectively, of patients with chronic type C hepatitis liver 10 disease. The co~ulluation and degree of glycosylation of the recombinant E2 protein may affect its ;Illlllll~ C~_Livi~y. Chien et al. reported that antibodies to E2 were clet~ctecl in 97% of chronic non-A, non-B h~?~LiLis (NANBH) paffents using a carboxyl-L.~",~ A E2 protein (amino acids [aa] 384-661) produced in CHO cells ll~n~et 342:933 (1993)].
Thus, there is a need for the development of additional assay reagents and methods to identify acute infection and viremia which may be present, but not ~;ullGllLly ~letPct~hle by comm~.rcially available SCl~;l,,,,~, and collr~ ol~ assays. These assay reagents and methods are needed in order to help ~ l, between those individuals with acute and pelX;~lelll, on-going 20 and/or chronic infection and those individuals whose HCV infections are likely to be resolved, and to define the prognostic course of NANB h~5.1;1i~ infection in order to develop ~.e\,~llLive or Lh~ uLic ~.Ll~Legies. The present invention provides a hc.etufol~ unrealized co"r;"~toly marker for ~lclr~ ",;";,-~ tlue HCV hlr~;Lion.
SUM~l~Y OF I~F TlWhNTIoN
The present invention provides an iul~oved method for cletectin~
the presence of antibodies to HCV antigen which may be present in a test sample colll~lisiulg cont~tin~. said sample wi~ HCV antigen and ~el~",~i";,~g 30 whether antibodies are bound to said HCV antigen, wh~l~iul the improvement co",~lises employing as said HCV antigen at least one recombinant HCV

CA 02223277 l997-l2-02 WO 96/41196 PCT~US96/08536 protein co",~ a recombinant polypeptide that is the ~ ssion product of ~ ~ ~;I " " ~ n cells Lld~r~ ed by a heterologous t:A~l~s~ion vector cu~ isillg a DNA sequence encoding an E2 Llu~cated protein, a DNA sequence encoding a rabbit heavy chain signal sequence and a DNA seqn~nre encoding an amino-5 t~rmin~l sequence of human pro-urokinase, wherein said HCV antigen DNA
sequence is located duw~ll~ to said other DNA s~q~lenr~.
The present invention further provides a test kit for dçt~cfing the ~ se~ce of antibodies to HCV antigen which may be present in a test sample, collll)l i~ a co~ el coll~ i"~ a recombinant polypeptide that is the 10 eA~l~ s~ion product of m~mm~ n cells lld~r~,lled by a heterologous ~A~l~ssion vector co",l" ;~ a DNA seqll~nre encoding an E2 L~
protein, a DNA sequence encoding a rabbit heavy chain signal sequence and a DNA sequence encoding an amino-t~rmin~l sequence of human pro-urokinase, wl~ ;ill said HCV antigen DNA seqll~n~ e is located dc w~Ll~ to said other DNA sequences.

l'~RlFh' DF.~C RIPI ION OF l~; DR~WINGS
Fig. 1 shows the immllnofluulescellL st~ining patte~ of HCV E2 antigen in transfected CHO cells using (A) rabbit anti-peptide serum (amino acids 509-551); and (B) HCV positive human serum.
Fig. 2 shows the results of a RIPA analysis of HCV E2 antigen produced in CHO cells. 35S-labeled E2 antigen was immlm~ eci~ led with:
pre-i."".. ,i-e rabbit sera (Lane 2), hypeli".,.. ~ serum from a rabbit i.. l.. ;,Pd with synthetic peptide aa 509-551 (Lane 3), normal human plasma (Lane 4), 25 and three dirrel~ HCV antibody positive human plasma (Lanes 5-7). T ~ne 1 contains radioactive molecular weight m~rk~r.c.
Fig. 3 is a SDS-PAGE gel of purified HCV E2 antigen produced in CHO cells. T ~ne 1: Molecular weight malh~l~. Lanes 2 and 3: Purified E2 antigen.

WO 96/41196 PCT~US96/08~36 nETA~n Dli ~Cl~rION OF T~ INV~NTIO~
The present invention provides an il~roved method for ~let~cting the ~lcsellce of antibodies to HCV antigen which may be present in a test sample C~ g cont~cting said sample with HCV antigen and tl~l~ "";";"g 5 whether antibodies are bound to said HCV antigen, wherein the hl~ VCllltllL
co...~ es employing as said HCV antigen at least one recombinant HCV
protein co. "~ g a recombinant polypeptide that is the c~ ion product of m~mm~ n cells Ll~srolllled by a heterologous c~rcssion vector co..~ a DNA seqllenre encoding an E2 llullcaLcd protein, a DNA sequence encoding a 10 rabbit heavv chain signal sequence and a DNA sequence encoding an aino-tPrmin~l seqnpnre of human pro-urokinase, wherein said HCV antigen DNA
sequence is located dowl~Lle~ll to said other DNA sequences.
Recombinant polypeptides produced as described herein in m~mm~ n e~lci,~ion ~y~L~ S provide ~,.I;g~ for ~ gnostic assays which 15 can be used to flP,tPrminP- the viremia of a patient based on a strong correlation between the presence of E2 antigen and patients found to be viremic using reverse l~ cl;l,Ldse polymerase chain reaction (RT-PCR) amplification. Ihe antigens also are useful as an early marker of seroconversion, and provide another means for ~l~lt;",.il~i"g true HCV exposure in in-lel~l",i..~l~ test 20 samples tested by commercially available col-ril "-~tory tests. The antigens also provide a means for resolving dis~ ~ll results between collllller~;ially ~! available second gene,dlion and third generation HCV scr~ g assays.
The present invention confers several te~hni~l advantages over the prior art. For example, the presence of antibodies to HCV E2 in s~ec."~t, s 25 already positive for HCV antibody provides additional, and therefore, more compelling evidence of true HCV infection. Furthermore, the ~l~se~-ce of antibodies to HCV E2, along with reactivity in a licensed HCV 2.0 screening assay as well as reactivity to the HCV core or NS3 protein in an immllnnblot assay, point to a co~ illL~l~letation that individuals with these serologic 30 profiles have had previous, or have ongoing, HCV infection.

.

W O 96/41196 PCTrU$96J08536 The recombinant polypeptides produced can be provided in the form of a kit with one or more co~ ; such as vials or bottles, with each cont~iner COll~ llg a separate reagent such as a recombinant polypeptide, packaged as test kits for the convenience of p~lrulll~hlg assays. Other aspects of the present invention include a recombinant polypeptide co",l., i~;..~ an HCVE2 epitope ~tt~eh~ to a solid phase.
The present invention provides assays which utilize the recombinant ~l~ leills produced as ~l~srrihecl herein in various formats, any ofwhich may employ a signal generating compound which g~ S a measurable 10 signal in the assay. All of the assays described generally detect antibody, and include cont~rtin~ a test sample with at least one HCV antigen provided herein to form at least one antigen-antibody complex and ~etecting the ~l~;sellce of the complex so formed. These assays are described in detail herein.
The term "test sample" refers to any culll~ollt;-lL of an individual's 15 body which can be a source of the antibodies of interest. These co~ uL~ are well known in the art. These test samples include biological samples which can be tested by the methods of the present invention described herein and include human and animal body fluids such as whole blood, serum, plasma, cel~los~i~l fluid, urine, lymph fluids, and various ext~rn~l section~ of the 20 r~ hatoly, i..~ l and g~~iluuli~y tracts, tears, saliva, milk, white blood cells, myelomas and the like, biological fluids such as cell culture ~u~".
f~ed tissue specimens and fixed cell specimens.
"Solid phases" ("solid supports") are known to those in the art but not critical and include the walls of wells of a reaction tray, test tubes, 25 poly~Lyl, .le beads, m~n~tic or non-m~gnPtic beads, nitrocellulose strips, membranes, microparticles such as latex particles, plastic tubes, glass or silicon chips and sheep red blood cells are all suitable examples and others. Suitable methods for immobilizing peptides on solid phases include ionic, hydrophobic, covalent interactions and the like. A "solid phase", as used herein, refers to 30 any material which is insoluble, or can be made insoluble by a subsequent reaction. The solid phase can be chosen for its intrincic ability to attract and CA 02223277 l997-l2-02 W O 96/41196 PCTrUS96/08~36 immobili e the capture reagent. ~llr .~I;vcly, the solid phase can retain an litinn~l receptor which has the ability to attract and immobilize the capture reagent. The aMition~l leceyLor can include a charged sllhst~nre that is oppositely charged with respect to the capture reagent itself or to a chal~ed 5 sllbst~nre conjugated to the capture reagent. As yet another ~ v~, the ~ ccyLor molecule can be any specific binding lllel.lbei which is ~tt~rh--~l to the solid phase and which has the ability to immobilize the capture reagent through a specific binding reaction. The rcccyL~l molecl-le enables the il~dircc~ binding of the capture reagent to a solid phase m~tçri~l before the y~lrollllauce of the10 assay or during the pclr~.. ,..~.~re of the assay.
It is col.~r~"l,lated and within the scope of the invention that the solid phase also can comprise any suitable porous m~t~ri~l with ~urrlcicuL
porosity to allow access by detection antibodies and a suitable surface affinity to bind ~ntipen~, Microporous structures are generally ~ler~lcd, but m~t~ri~l~
1~ with gel structure in the hydrated state may be used as well. Such useful solid SuyyOl~ include: natural polymeric carbohyd,dles and their ~y~ ;r~lly mollif~ cross-linked or substituted dclivdLivcs, such as agar, agarose, cross-linked alginic acid, substituted and cross-linked guar gums, cellulose esters, especially with nitric acid and carboxylic acids, mixed cellulose esters, and 20 cellulose ethers; natural polymers co.,~ nitrogen, such as ylOLcillS and d~ivdLivesl inrln~ing cross-linked or modified gelatins; natural hydrocarbon polymers, such as latex and rubber; synthetic polymers which may be yl~cd with suitably porous structures, such as vinyl polymers, inrln-ling polyethylene, polyyloyylene~ poly~Ly~ene, polyvinylchloride, polyvinylacetate and its partially 2~ hydrolyzed delivalives, polyacrylamides, polymethacrylates, copolymers and terpolymers of the above polycon~len~tçs, such as polyesters, polyamides, and other polymers, such as polyul~ anes or polyepoxides; porous il~ol~anic m~teri~l~ such as sulfates or carbonates of ~lk~lin~o. earth metals and m~ Psi...~, inrlllr1ing barium sulfate, c~lcillm sulfate, c~lcillm carbonate, Silic~t~s of alkali and ~lk~lin~ earth metals, ~lllmimlm and m~ s;.. ~; and ~ .;,.. or silicon oxides or hydl~es, such as clays, alllmn~, talc, kaolin, zeolite, silica gel, or W 096J41196 PCTrUS96/08536 glass (these m~teri~l~ may be used as filters with the above polymeric m~t~ri~l~); and ~Lul~,s or copolymers of the above classes, such as graft copolymers obtained by initi~li7in~ polymP.ri7~tion of synthetic polymers on a pre~Yi~tin~ natural polymer. All of these m~teri~ may be used in suitable 5 shapes, such as films, sheets, or plates, or they may be coated onto or bondedor ls....;..i1l~d to a~lupli~L~ inert carriers, such as paper, glass, plastic films, or fabrics.
The porous ~LIu~;Lul~ of nitrocellulose has exrellent absorption and adsorption ~ tiPs for a wide variety of reagents. Nylon also posse~ses similar characLeli~Lics and also is suitable. It is collL~ lated that such porous solid ~u~olL~ described herein above are preferably in the form of sheets of thic~Ps~ from about 0.01 to about 0.5 mm, preferably about 0.1 mm. The pore size may vary within wide limits, and is preferably from about 0.025 to about 15 mic~ s, especially from about 0.15 to about 15 microns. The snrf~res of such supports may be activated by chrmir~l processes which cause .! covalent linkage of the antigen to the support. The i~lcv~l~ible binding of the antigen is obtained, however, in general, by adsorption on the porous m~t~ri~l by poorly understood hydrophobic forces. Suitable solid ~iU~Ji)OlL:~ also are described in U.S. Patent Application Serial No. 227,272.
The "in-ljc~tor reagent" colll~lises a "signal gel~ldLillg compound"
(label) which gelleld~t;S a m~lr~ble signal detect~ble by e~trrn~l means conjugated to a specific binding m~mher for HCV. "Specific binding member"
as used herein means a member of a specific binding pair, that is, two dirre molecules where each of the molecules through chrmir~l or physical means specifically binds to the other molecule. An immlml~reactive specific binding member can be an antibody, an antigen, or an antibody/antigen complex that is capable of binding either to HCV as in a sandwich assay, to the capture reagent as in a competitive assay, or to the ancillary specific binding member as in an indirect assay. In addition to being an antibody member of a specific binding pair for HCV, the in~lir~tor reagent also can be a member of other specific binding pairs, including hapten-anti-hapten systems such as biotin or anti-biotin CA 02223277 l997-l2-02 W O 96/41196 PCT/U~ 3S36 and avidin or biotin, a carbohydrate or a lectin, a complementary nucleotide seqnen~e, an effector or a receptor molecule, an enzyme cofactor or an enzyme, an enzyme inhibitor or an enzyme, and the like.
The various "signal ~e"~ g compounds" (labels) contemplated 5 include chromogens, catalysts such as enzymes, Illmin~scent compounds such as fluc,lcsceLù and rhntl~mine, chemill"..;"Pscent compounds such as ~rri~linillm, ph~ "~ inillm and dioxetane compounds inr~ ing those described in co-~e--dLù~ U.S. Patent Application Serial No. 0_/921,979 collc~o~dL~ to EP
Publication No. 0 273,115, which enjoys common (.)WUe~ ) and which is incorporated herein by reference, rat1io~rtive element~, and direct visual labels.
Fx~mples of er~zymes include ~lk~line phosph~t~ce, horseradish peroxidase, B-galactosidase, and the like. The selection of a particular label is not critir~l, but it will be capable of producing a signal either by itself or in conjul,.;lion with one or more additional sub~ ees.
Other embo~liment~ which utilize various other solid phases also are co..~eLu~lated and are within the scope of this invention. For example, ion capture procedures for sepalalillg an immobilizable reaction complex with a negaLively charged polymer, described in co-~elldi lg U.S. Patent Application Serial No. 150,278 corresponding to EP Publication No. 0 326,100, and U.S.
Patent Application Serial No. 375,029 corresponding to EP Publication No. 0 406,473, both of which enjoy common owllcnsl~ and are incorporated herein by lcrc,~,..ce, can be employed according to the present invention to effect a fast solution-phase immllnochemi~l reaction. An immobilizable immlme complex is separated from the rest of the reaction llli~UlC by ionic ;..l~l~c~l;ons 25 between the negatively charged poly-anion/immnne complex and the previously treated, positively charged porous matrix and ~letecte~l by using various signalgener~tin~ systems previously described.
Also, the methods of the present invention can be adapted for use in systems which utilize microparticle technology including in ~uLoLuaLcd and 30 semi-auLuLuaLed systems wllclcLIl the solid phase cc,m~lises a uliclop~licle.Such systems include those described in pending U.S. Patent Application Nos.

W ~96/41196 PCTrUS96/08536 425,651 and 425,643, which correspond to published EP Publication Nos. 0 425,633 and 0 424,634, 1~S~e~ V~1Y~ both of which enjoy common OW11 and are inco~ Lcd herein by lcfcl~nce.
After plc~alillg the recombinant ploteins as rlescnhefl herein, ~ 5 these recombinant ~ eills can be used to develop unique assays as ~lescnbec~
by the present invention to detect the presence of anti-HCV in test samples.
For example, a test sample is cont~rte~l with a solid phase to which at least one recombinant HCV protein col~ ih~g E2 antigen is ~tt~rh-od ~he test sample and solid phase are ;,.~ for a time and under conditions sllffirient to form antigen-antibody complexes. Following ill.-~b~lio~, the antigen-antibody complexes are ~let~cte-l Tntlir~t 3r reagents may be used to facilitate detection, depending upon the assay system chosen.
In another assay format, a test sample is contacted with a solid phase to which at least one recombinant HCV protein CO~ illg E2 antigen produced as described herein is ~tt~rll~cl and also is contacted with a monoclonal or polyclonal antibody specific for the HCV protein(s), which preferably has been labeled with an in~lir~tor reagent. After incubation for a time and under contliti~n~ sufficient to form antigen-antibody complexes, the solid phase is sepdldted from the free phase, and the label is ~let~cte(l in either the solid or free phase as an in~ tion of the presence of anti-HCV.
Other assay formats lltili~ing the proLei ls of the present invention are collLc~ lated. These include cont~cting a test sample with a solid phase to which at least one recombinant HCV protein COlllpli~illg E2 antigen produced as described herein is ~tt~rh~tl inrub~ting the solid phase and test sample for a time and under conditions sufficient to form antigen-antibody complexes, and then co~t~-ting the solid phase with a labeled recombinant antigen to form antigen-antibody-antigen sandwiches. Assays such as this and others are described in U.S. Patent No. 5,254,458 which enjoys common ow~ sl~ and is incorporated herein by reference.
While the present invention discloses the ~lcrelc,~ce for the use of soiid phases, it is collLclll~lated that the proteins of the present invention can be W O 96/41196 PCTrUS96/08~36 12 utilized in non-solid phase assay systems. These assay systems are known to those skilled in the art, and are considered to be within the scope of the present invenhon.
The present invention will now be described by way of examples, 5 which are meant to illustrate, but not to limit, the spirit and scope of the invention.

M~TliRT~T ~ Al~ l~ETEIODS
F7 Clor~ir~ ~n(1 F~?ressio~
The viral source for the envelope gene was second passage HCV-H strainplasma [Ogata et al., supra, 1991~ from a rhi.,.~ e which e;~CllLj a Type la genoLy~e of HCV [Mishiro & Bradley, in Viral ~el)~titi~
~n~l T iver ni~ 283-85 (Nishioka et al. eds., 1994)]. The envelope gene cDNA was isolated by RT-PCR amplification. A L~ rdted E2 sequence was obtained using PCR amplification of the region coding for amino acids 388-664 of the large open reading frame of HCV. E2 complement~ry DNA (cDNA) was inserted into a plasmid vector dowl~tlc~.. of both a rabbit heavy chain signal sequence and a human pro-urokinase amino t~rmin~l seq~lenre to enh~nre signal proLease procç~ing, efficient secretion and final product stability in cell 20 culture fluids. The e~les~ion system is described more fully in co-pending U.S. patent application serial no. 08/_, [Atty. Docket No. 5763.US.01], filed CC~ ;ullcu~y hel~;wiL~ and which is incorporated herein by rc:re~ ce.
Briefly, plasmid 577 co"li,i.,;"g a 2.3 kb fragment of pBR322 including the b~t~ l gene beta~ t~m~e and the origin of DNA replication, a 1.8 kb 25 cassette directing expression of a neomycin rPci~t~nre gene under the control of herpes simplex virus-l (HSV-l) thymidine kinase promoter and poly-A addition signals, and a 1.9 kb cassette directing ex~l~,ssion of a mouse dihydrofolate re~ ct~e gene under the control of simian virus 40 (SV40) T-antigen promoter and 1, A I ~ÇI ;plion enh~nrer and poly-A addition signals (for selection and 30 amplification) was modified to include a 3.5 kb cassette directing expression of a tl~ lrA E2 gene under the control of SV40 T-antigen promoter and W096/41196 PCTrUS96/08~36 enh~nrP.r, h~ B virus surface antigen enh~nrer I and a fr~ment of HSV-1 genome col;~... g poly-A addition signals.
The 3.5 kb E2 e~ ssion cassette contained a duplex ~yllLlleLic oligonucleotide that had been digested with SpeI and XbaI inserted at the XbaI
5 cloning site of the c~ettP by sticky end li~ti~m The synthetic oligonucleotidesequence contained a sequence derived from a rabbit immllnoglobulin gam~a heavy chain signal peptide and other seq-lenres to create restrirtion sites for cloning ~ul~oses, inserted dow~.Lle~l of a promoter elemPnt and ~ C~ .l.lion start site. The E2 eA~les~ion cassette also contained the PCR-derived E2 10 fr~nent inserted as an XbaI fr~gmlont dow~ e~ of the rabbit sequence.
Tmm~ t~ly after the XbaI site, the sequence encoding the amino lr~ llAl sequence of human pro-urokinase--serine, ~p~r~gin~, gl~ - -ic acid and leucine (SNEL)--followed. The pro-urokinase sequence promoted signal pluL~ase p~uces~i..g, eîrl~;it;ll~ secretion and product stability in culture fluids.
Tmmrtli~t~ly following the SNEL seqllr-nre, the seqnenre encoding the amino acid sequence of the HCV ~uldLiv~ envelope gene from aa 338 to aa 664, a duplicate stop codon and a XbaI site for cloning purposes.
~hinese h~m~ter ovary (CHO) cells lacking dhfr (dhfr-) (Dxb-111) were transfected with the HCV E2 plasmid and stable cell lines were obtained after several rounds of meth~L~ Le selection. Uriacio, et al., Proc Acad. Sci. 77:4451~466 (1980). These cells are available from the ~merir~n Type Culture Collection (A.T.C.C.), 12301 Parklawn Drive, Rockville, MD
~! 20852, under ~ce~ion number CRL 9096. Cell cultures were grown on Ham's F12 custom _inus form~ tinn (without glycine, hypoxi1"~ "~ or thymidine) supplemrntr~l with methoL c~ate hydrate, 5000 nm, G418 at an actual concentration of 300 ug/ml, dialyzed 10% fetal calf serum and ~ S
buffer at a cullcellLldLion of 8 ml per 500 ml of media (for non-C02 inrn~tion) Ham's F12 Custom Minus Medium was overlayed onto just ~ collfluellL monolayers for 12-24 hours at 37~C in 5% C02. Then the growth mP~ m was removed and the cells were rinsed three times with phosphate buffered saline (PBS) (with calcium and m~ si~ ) available from Gibco-W O96/41196 PCT~US96/08536 BRL, to remove the ,~ g media/serum which might be present. Cells then were inr~lb~tP-l with VAS Custom Medium (VAS Custom formnl~tion with ..lil.P with HEPES without phenol red, available from JRH Bioscience, product number 52-08678P), for 1 hour at 37~C in 50% C02. As a final wash, 5 the VAS then was discarded. These procedures are ~let~ilPd in co-pending U.S.
Serial No. _ (Attorney docket number 5763.US.01), incorporated herein by ~crc~e~ce. Media co--l~ g secleLed E2 was collected off confluent cell monolayers, pooled and stored frozen until ~, ~- ;rir~l ion. The cells typicallywere 1~1 ~e~Lcd at six to seven day intervals.
An E2 antigen inhibition assay was used to screen the cells. A
value of 100% was given to a HCV positive standard which was diluted to achieve an al~sull,auce value of 1.0 in the assay. A standard curve was ltorl by spiking into the HCV positive standard known amounts of E2 antigen in the range of 0.4 ug/ml to 50 ug/ml. In the assay, 20 ul of the test sample (i.e., spiked standard or cell sup~. "~ "l) were inr~lb~tPd with the standard positive control for a~lv~ ly one hour at 40~ C and then inr~lb~tPd with an E2 peptide-coated bead (HCV ~yllLllcLiC peptide colll~ulisillg amino acids 509-551) for ~y~ ly one hour at 40~ C. The amount of E2 antigen produced was measured by adding a gamlna globulin specific goat anti-human-HRPO conjugate, inr~lb~ting, adding OPD substrate and ~ r~ the reaction with lN H2SO4. The unknown cell culture SUpt;~ l was read off the ~dald curve to ~PtPrmine E2 concentration. It was ~ f - ~ that 6-10 milligr~m~ of CHO-E2 were produced per liter of culture fluid using this method.
E~pression of E2 was ~letectecl by immnn~-lluolcsccllL ~ .;,.g using polyclonal rabbit sera from animals ;III.I~ Pd with an HCV sy,lLhetic peptide comprising amino acids 509-551 (anti-peptide 509-551 serum). Figure 1 shows the immnnofluolcsccllL staining pattern of HCV E2 antigen in Llal~rccLcd CHO cells using (A) rabbit anti-peptide serum (amino acids 509-551); and (B) HCV positive human serum.

CA 02223277 l997-l2-02 W096/41196 PCTrUS96fO8536 E2 expression also was tl~te~ctr-l by radio;,l.. (lpreci~iL~Liol-analysis (RIPA) of Iysed cell extracts from 35S-labeled CHO cell cultures.
Figure 2 shows the results of a RlPA analysis of HCV E2 antigen produced in CHO cells. Both rabbit and human ~nti~M specific for HCV E2 antigen 5 ' pleci~iL~Led a heterogeneous E2 protein.
F7 Purific~tion pnrifir~tion of E2 was achieved by first conc~ liu~ the cell SUpe- ~ i 50 fold followed by ion r~ch~n~e and lectin chluluaLography. The ion exc~ e ch~ togr~phy consisted of two columns, S-Sepharose and 10 DEAE-Sepharose. The hal v~L~ were clarified at 1500 x g for thirty Ll~illul~Sand the ~.~pe~ i were conce~ 1 to 50x with an Amicon stirred-cell co~-re~ and employing an Amicon YM10 membrane (available from Arnicon, Beverly, MA). The 50x concentrate was 0.2 u final filtered and then e~ nsively dialyzed against S-Sepharose lUL ~mg buffer coLu~LisiLIg 0.02M
15 sodium pho~ph~tr buffer (no salt), pH 6.5, conductivity approximately 2.0 mS.After dialysis, the supe. ,~ ..l was loaded onto an equilibrated 200 ml S-Sepharose col~ m at a flow rate of Sml per minute. The unbound flow was collected, coLIceL Ll~Led to original volurne and e~LeL~sively dialyzed against DEAE-Sepharose running buffer comprising 0.2M Tris buffer, 0. lM NaCl, pH
8.5, col~lu~LiviLy approxirnately 12 mS. After dialysis, the ~iu~le~ 11;11;1111 was loaded onto a 200 ml DEAE-Sepharose column at a flow rate of 5 rnl per minute. The unbound flow was collected, conne~tr~tr~l to original volume and e ~LeL~ively dialyzed against wheat germ ag~ l;..i.. (WGA)-Sepharose 6MB
lUlL~il g buffer COlu~liSiulg O.OlM sodiurn phosphate buffer, 0.13M NaCl, pH
7Ø After dialysis, the ~u~.,.lLa~L was loaded onto a 10 ml WGA-Sepharose 6MB column at a flow rate of 0.5 rnl per minute. The unbound flow was collected and recircnl~tto~l The column flow was reversed and the purified CHO-E2 antigen was eluted using 10 mM N,N'-diacetylchitobiose in WGA-'' Sepharose running buffer. Purified antigen was dialyzed against phosphate buffered saline and stored at -70~ C.

W O 96/41196 PCTrUS96/08536 Final purity was ~csesse-l using sodium dodecylsulfate poly~ yl;lll~ide gel electrophoresis (SDS-PAGE, a procedure well-known to those of oldi~y skill.. Figure 3 shows a SDS-PAGE gel of purified HCV E2 antigen produced in CHO cells. The purified E2 antigen ran as a heterogeneous band of a~pl.-x;,.,;1lely 62-72 kDa on SDS-PAGE. A similar heterogeneous band was co"li".,~l to be E2 by RIPA analysis (Figure 2). The final purity was e~,l;",;~lPcl to be greater than 90% using sc~nning de~i~ollletry of SDS-PAGE gels stained with Coomassie blue. During the harvest phase of growth, the CHO cells were grown in protein free media which greatly enll~nrecl the efficiency of pllrifir~tion of this glyc.,~lvk;in.
Prel;",;"~,y ~)L- ;"~entc with endoglycosidase H ~ e.~l (lem(J~ that the E2 protein appeared to be glycosylated as evi~l~-nrecl by the reduction in size from a 62-72 kDa heterogeneous band to a distinct 32 kDa protein band after tre~tment with endoglycosidase H.
Solid Ph~ce I"""~"oa~y~
Screening for HCV antibody was carried out using commercially available 2.0 and 3.0 HCV EIAs from Abbott Labol~tolies (North Chicago, ~! Illinois) and Ortho Diagnostics Inc. (Raritan, New Jersey). All testing was carried out according to the m~mlf~r*lrer's instructions. Supplernrnt~l, commercially available, imm-lnoblot assays (RIBA HCV 2.0, Chiron Corporation, Emeryville, California; MATRIX HCV 1.0 and MATRIX HCV
2.0, Abbott Labor~tories, North Chicago, Illinois) were used to establish the specific antibody reactivity patterns. RIBA HCV 2.0 and MATRIX HCV 1.0 assays were performed according to the m~mlf~rtllrer's instructions. MATRIX
HCV 2.0 is a second generation MATRIX immllnnblot assay which coll~hls an NS~ antigen in addition to core, NS3 and NS4. The assay procedure is the same as ~at for MATRIX HCV 1.0, as previously described [Vallari et al., L
Clin. Microbiol. 30: 552-56 (1992)].
Specimenc Chronic and acute NANBH specimens were obtained from multiple U.S. sites. Serially collected speci~ens from individuals WO 96~41196 PCTrU5~6;~ 6 serocollvclLiLIg to HCV antigens were obtained from commercial plasma vendors. Archived samples which were HCV RNA positive (N=495) were obtained from a large virology ler~lcl-ce laboratory in ~e U.S. without linkage to patients or donors. The RNA extraction and PCR amplification procedures - 5 have been described [Gretch et al., J. Clin. Microbiol. 30:2145~9 (1992)].
Additional HCV RNA positive specimens were collected from Japan (N=59) and The Neth~rl~nrls (N=33) [Zaaijer et al., J. Med. Virol. 44:395-97 (1994)].
Specimens (N=304) from blood donors at risk for HCV infection wi~h ALT
values greater than 100 IU/L were provided by Dr. Alfred Prince, New York Blood Center. Samples indeterminately reactive on MATRIX 1.0 for core (N=139) and NS3 (N=149) were obtained from the Abbott Virology R~relellce Lab~ uly, North Chicago, Illinois and lcplcs~llLed a mix of HCV
2.0 reactive blood donors and patients. A commercially available anti-HCV
panel colllplised of varying titer of anti-HCV (PHV203), well ch~rarteri7çd with regard to HCV serological markers, was obtained from Boston Binm~lic~, Incorporated (BBI), West Bridg~w~lel, ~c.c~l~h~ Ll~i. HCV 2.0 reactive plasma specimens were obtained from Nor~ ~",~ ir~ Biologicals I~col~ol~Led (NABI) of which only samples concordantly reactive in both HCV 2.0 EL~'s (Abbott and Or~o) were analyzed fur~er.
FXA~IP~,F~
Fx~ rle I
Anti-F~ Anti~pen F.T~
Purified HCV E2 antigen, plcpal~d as described above, was coated onto poly~Lylclle beads at a concentration of 1.0-2.0 ug1ml in a 0. lM
borate, 0.15M NaCl buffer, pH 9Ø The antigen coating buffer composition was adjusted with respect to pH and ionic ~Ll~L~Lh to provide opLi sensiLiviLy and specificity in the assay.
o Briefly, the assay procedure used was as follows. Specimens were diluted with specimen diluent and mixed. All specimens were tested at a 1:41 dilution in the assay. The specimen diluent co~ lised a phosphate and TRIS-WO 96/41196 PCT~U5~CI~ 6 EDTA buffered saline solution c~..li.i--i-.g bovine serum albumin, fetal bovine serum and goat serum with 0.002% Triton X-100~ deLelgenL. The HCV E2 antigen-coated beads were added, one to each well of the tray, and inr~ t~d at 37~ C for appro~cim~t~ly 60 ...i"~ s in the rotation mode. The unbound S m~t~ri~l~ were removed by washing the beads with water. Anti-E2 ~ g bound to the bead was ~et~ct~ by adding to each well co..l 1i..i..g a bead 200 ul of conjugate CO~ g goat anti-human IgG labeled with hor~er~ h peroxidase as the label [Dawson et al., J. Clin ~icrobiol. 29:1479-86 (1991)].
The beads were incubated at 37~ C for a~ laL~ly 30 l~i.."lrs in the rotation 10 mode. The unbound m~t~ri~l~ were removed by washing the beads with water.
Color development was obtained by adding o-Phenylen~ (OPD) solution co..l;1i..i..g hydrogen peroxide to the beads, and, after incubation for a~>pro~ima~ely 30 mimlt~c, a yellow-orange color developed in ~Lo~olLion to the amount of anti-E2 which was bound to the bead. The enzyme reaction was 15 stopped by adding 1 ml of lN H2SO4. The i,ll~iLy of the color was measured using a spectrophotometer at a wavelength of 492 mn.
Fx~ le 2 ~pecif1cit:y The specificity of the anti-E2 EIA described in Example 1 was 20 established by testing specimens from several populations of volunteer blood donors totaling 750 serum and plasma specimens. A cutoff value for the E2 antibody assay was established at a signal to negative control al)soll,~lce (S/N) ratio of 4Ø This cutoff lcpl~sellL~d a ...i,~ """ of six (range of from six toten) ~.L~dald deviations from the mean of the absolballce distribution for any of these populations.
Fx~n~ple 3 Corre1~tion betwee~ Anti-E2 FT~ and Prior Art HCV Assays One hundred fifty-nine patients diagnosed with chronic NANBH
were tested using the Abbott HCV 2.0 test and the anti-E2 EIA of Example 1.
Of those ~aLiell~, 147 (92.5%) patients were positive with HCV 2.0, while 141 (88.6%) patients also had antibody to E2. Overall, there was 96.2% agreement _ WO 9~41196 PCTrUS96108536 b~Lweell the HCV 2.0 and anti-E2 assays. A high correlation (94%) between HCV core and E2 antibodies also was observed in this population.
A similar high concor~ce was seen between the HCV 2.0 and anti-E2 assays in acute NANBH ~aLie~ . One hundred thirteen ~eci~Sells were 5 tested using the Abbott HCV 2.0 test and the anti-E2 EIA of Example 1.
Ninety-nine (87.6%) specilllells gave concordant results (51 positive and 48 negative), while 10 specimens reacted exclusively with HCV 2.0 and 4 speci~e~s were positive only in the E2 antibody assay. The overall reactive rates in acute patients for HCV 2.0 and anti-E2 EIA were 54% and 49%, 10 .~,specLively.
F~n~le 4 Seroconversion S~ >1es Serially collected specimens from five individual plasma donors s who seroco.lv~ d to multiple HCV antigens were tested using the anti-E2 EIA
15 of Example 1. In three of the five p::~ti~.ntc, anti-E2 was the first antibody ~etect~le during seroconversion. Anti-E2 eventually appeared in all five cases.
Table I. Seroconversion to HCV Proteins in Plasma Donors HCV Antibody Specificity Core NS3 NS4 NS5 E2 Donor No.(Days)- (Days)(Days)'(Days)'(Days)' Expressed as days to ~ ucu~ ion after the first ALT elevation (ALT=S0 IUiL or greater) "O" Indicates ;~elucuL~ iu~ to that ;narke~ occurred by the ti~ne the ALT value reached 50 IU/L
NS: No sclucc,~ ,iu.~ detected WO 96/41196PCT~US96/08536 The results of Table I show that antibodies to E2 as ~t~ct~o~l by the anti-E2 EIA are a good index of HCV exposure by demon~ that all five individuals who seroco~ Led to other HCV proteins also seroco~ . led to E2. E2 antibodies a~e~ed as the first serological marker of HCV infection in 5 three of the five cases in-lie~tin~ that E2 antibodies are produced early after HCV infection in some individuals.
Fx~n~le 5 ~A Positive Specim~n~
In order to establish how frequently E2 antibodies appear during 10 active HCV inf~ction, 587 HCV RNA positive plasma specimens, i~l~ontifi~-l inthe section "Spec;...~ " above, were tested using the anti-E2 EIA of ~.Y~mrle 1 as well using the MATRIX HCV 2.0 assay to test for other individual HCV
antibodies. As shown in Table II, 571 (97.3 %) of these RNA positive specimens were shown to contain antibodies to E2 (Table II), in~ ing 56 (94.9%) of the 59 ~yee~ ells collected in Japan. All E2 ~osiLive samples c~ ed other HCV antibodies as ~ettocte~ by MATRIX but no single antibody occurred with greater frequency than E2 antibody in this population.

l~able II. Correlation of HCV E2 Reactivity with Pl~,sence of HCV RNA
No. Specimens No. E2 Antibody %
Panel Testes No. PCR Posidve Posidve Total 587 587 571 97.3 E2 antibodies were found in 97.3% of these patients demo~L~ g that the presence of E2 antibody and HCV RNA, as ~l~tPcteA by PCR, are very closely and positively correlated. Since 94.9% of 59 HCV
patients from Japan (where Genotype lb predo,~ ec) were reactive for E2 antibodies, it appears that there must be conserved E2 epitopes among Type la and Type lb viruses. The close correlation between HCV RNA and E2 antibody suggests that the presence of E2 antibody alone is not sufficient for W ~ 96141196 PCTrUS96108S36 virus cl,o~r~n~e and calls into question the role these antibodies may play in virus neutr~ tinn Fx~n~?le 6 HCV Populatio~
S Several HCV populations, identifi~(l in the section "S~ec; . . .~
above, were tested in the anti-E2 EIA of Example 1. Among the cohort of New York Blood Center blood donors with ALT values greater than 100 IU/L, 48 (15.8%) were positive for E2 antibodies. Samples reactive with E2 antigen were v~lirled to tl~le~ "~ whether ~e samples reacted with additional HCV
serological .. ;.,k~l~. Forty six (95.8%) of these 48 donors also were reactivein the Abbott HCV 2.0 EIA and were confirmt--cl positive (except for one sample which tested core antigen-reactive only) in the MATRIX HCV 2.0 assay. Thus, the data int1i~te that a po~itive E2 antibody test has a high positive predictive value for HCV exposure.
Among the specimens ,L~".l~Led as in~ te. .~ by MATRIX
HCV 1.0, 59 (42.4%) of 139 core reactive only specimens were found to contain E2 antibodies and 23 (15.4%) of 149 NS3 reactive only ~eci~
were found to contain E2 antibodies. The data show that the combined high specificity and high predictive value of the anti-E2 EIA of Fx~mple 1 is useful in ev~ln~tin~ in(1rle, Illii~Alt-, samples.
F~m~?le 7 S~mI7les with l:)iscoldallL Results between HCV 2.0 ~n~l HCV 3.0 ~ssays BBI Panel PHV203 specimen members were evaluated with the Abbott HCV 3.0 EIA and anti-E2 EIA of Example 1. Testing results from the cc,l~ ;ially available assays Abbott HCV 2.0 IA, Or~o HCV 2.0 ~A, Ortho HCV 3.0 EIA, MATRIX HCV 1.0 (Abbott) and RIBA HCV 2.0 (Chiron) were provided by BBI. Two panel members reported by BBI as HCV
negative were also E2 antibody negative.
Eighteen ~78.3 %) of 23 HCV 2.0 EIA positive (by both Abbott and Ortho assays) specimens tested anti-E2 positive. Among the 23 HCV 2.0 concordantly positive ~ec"llells, six samples (26.1%) tested negative in ~e Ortho HCV 3.0 EIA but reIn~in~l reactive in the Abbott HCV 3.0 EIA. Table m sets forth the serological profile for each of these samples. As shown in Table III, three (50%) of the six specimens (panel members -01, -02 and -10) contained antibodies to multiple HCV pL~L~ s, of which two samples (panel W O 96141196 PCT~US96/08536 members -02 and -10) had antibodies to E2. The two anti-E2 positive samples also were reactive to core in both the RIBA and MATRIX assays.
Thus, the data show that two (33.3%) of six BBI panel memherS~
which were concordantly positive in both m~mlf~ rer's HCV 2.0 EIAs but negative in the Ortho 3.0 EIA, col-la~ed both core and E2 antibodies.
The anti-E2 EIA of Example 1 was used to help evaluate NABI
plasma donor specimen~ with discordant reactivity between lir.to.n~e(1 HCV 2.0 EIA tests and HCV 3.0 EIA tests ~ lllly sold only outside the United States.
In order to remove any pre-selection bias, only those donor ~ec ~l~S
~ (N=104) repeatedly reactive by both the Abbott and Ortho 2.0 EIAs were tested using HCV 3.0 blood SC;lc;elf~g tests from each of these cu,.,l.,."i~s.
Thirteen (12.5%) of 104 specimens, r~le~e~ 12 unique donors, were non l~a~;Live in either one or both HCV 3.0 EIAs. A complete analysis of the HCV antibody p~ "~ present in these 13 specimens was done using RIBA 2.0, MATRIX 2.0 and the anti-E2 EIA of Example 1. Table IV
sets forth the serological profile for each ~ecilllell. Specimen numbers 5 and 8 re~lesellL sequential donations from an individual donor.

Table m. Serological Profile for HCV 2.0/3.0 EIA Discor~
BBI Panel Members HCV Scr~nin~ ET~'s ~ 1 T~ctinc~
Abbot~2.0QrthQ~Q Abhott 3.0 Or~o 3~0 ,~ntih~ c Detected E2 Panel Member S/CO S/CO S/CO S/CO RIBA 2.0 MATR~ 1~0 S/N
PHV203-01 1.4 1.9 1~6 0.6 Core, NS4 Core, NS3, 2~4 PHV203-02 1.7 2.5 2.7 0.8 Core Core 13 PHV203-03 2.2 1.9 2.6 0.3 NR NS4 1.6 PHV203-10 1.3 2.3 2.7 0.8 Core Core. NS3 21.

PHV203-24 1.4 1.6 6.0 0.2 Core Core 3.2 PHV203-25 2.3 >4.4 2.9 0.2 NS4 NS4 1.6 S/CO: Sample to cutoff value, considered positive if = 1.0 or greater S/N: Sample to negative control absorbance ratio, considered posirive if = 4.00 or gn~ater NR: N ~ ~.

W O 96/41196 PCTAUS96i~36 Table IV. Serological Profiles for Thirteen HCV 2.0 Positive/HCV 3.0 Negative Plasma Donors Screer~ing Assay ReactivitY
HCV 2.0 HCV 3.0 HCV 3.0 Su~ 1 T~
Spel~im~ n Abbott/ AWottOrtho Antihodies Detected E2 EIA
Number Ordlo S/CO S/CO RIBA 2.0 MATR~ 2.0 S/N
+/+ 2.90 0.70 CORE CORE 5.92 2 +/+ 1.59 0.31 CORE CORE 2.80 3 +/+ 1.96 0.32 CORE CORE 2.40 4 +/+ 1.99 0.79 CORE CORE, NS4Y- 10.12 +/+ 2.72 0.52 CORE CORE 4.92 6 +/+ 2.24 0.68 CORE CORE, NS32.40 7 +/+ 3.55 0.08 CORE NS4Y- 0.80 8 +/+ 3.01 0.47 CORE CORE 5.52 9 +/+ 1.97 0.96 CORE CORE, NS50.60 +/+ 2.09 0.04 NS4 NS4Y- 1.00 11 +/+ 1.51 0.15 NS4 NR 1.20 12 +/+ 0.65 0.05 NS4 NS4, NS51.40 13 +/+ 0.27 0.0S NS4 NS4Y' 1.20 S/CO: Sample to cutoffvalue, co~ Cd positive if = 1.00 or greater 25 ? * Denotes .~,a,livily to the c100 (NS4) antigen w~ ,;.scd in yeast. Reactivity to both the yeast and E.coli c100 andgens rnust occur in order to be co~;d.,l~,d reactive to NS4 on MATRIX 2.0 NR: NO. cacli~.
S/N: Sample to negative control al)s~l,a lce ratio, co~id~ ,d positive if = 4.00 or greater As shown in Table IV, specimen numbers 5 and 8 which lc;~ selll r seq~lenti~l donations from an individual donor are core and E2 antibody ~osiliv~
demo~lldling the reproducibility and specificity of these assays. Four specimenswhich reacted to core antigen on both RIBA 2.0 and MATRIX 2.0 also contained antibody to E2. These four spechllens were reactive in the Abbott HCV 3.0 assay W O 96/41196 PCT~US96/aF'~6 (S/CO values 1.99-3.01) but were neg~live in the Ortho HCV 3.0 assay (S/CO
values 0.47-0.79).
Three other specimens had antibodies to two distinct HCV plo~eil,s other than E2 on MATRIX (core/NS3, core/NS5 and NS4/NS5). Of these S ~ec;,..~ , the two core reactive spechlle~. were positive rn the Abbott HCV 3.0 EIA (S/CO values 2.24 and 1.97) and negative or borderline negative in the Ortho3.0 EIA (S/CO values 0.68 and 0.96). The other specirnen which collL~.. ed antibodies to NS4 and NS5 was negative in both the Abbott and Ortho HCV 3.0 EIA.
The data show that four (30.7%) of 13 samples which were concordantly ~o~ in both m~nllf~r~lrer's HCV 2.0 EIAs but negative in the Ortho 3.0 EIA, contained antibodies to both core and E2.

Claims (17)

WHAT IS CLAIMED IS:

1. In a method for detecting the presence of antibodies to HCV
antigen which may be present in a test sample comprising contacting said sample with HCV antigen and determining whether antibodies are bound to said HCV
antigen, wherein the improvement comprised employing as said HCV antigen at least one recombinant HCV protein comprising a recombinant polypeptide that is the expression product of mammalian cells transformed by a heterologous expression vector comprising a DNA sequence encoding an E2 truncated protein, a DNA
sequence encoding a rabbit heavy chain signal sequence and a DNA sequence encoding an amino-terminal sequence of human pro-urokinase, wherein said HCV
antigen DNA sequence is located downstream to said other DNA sequences.

2. The method of claim 1, wherein said DNA sequence encoding an E2 truncated protein comprises amino acids 388-664 of the large open reading frame of HCV.

3. The method of claim 1, wherein said HCV antigen comprises an E2 truncated protein and at least one other HCV antigen selected from the group consisting of core, NS3, NS4 and NS5.

4. The method of claim 1, wherein said contacting step comprises incubating said test sample with a solid phase to which at least one recombinant HCV
protein comprising E2 antigen is attached for a time and under conditions sufficient to form antigen-antibody complexes.

5. The method of claim 4, wherein said determining step comprises contacting the solid phase with a member of a specific binding pair.

6. The method of claim 5, wherein said specific binding member is an antibody.

7. The method of claim 6, wherein said antibody is anti-human IgG.

8. The method of claim 5, wherein said specific binding member is a recombinant antigen.

9. The method of claim 5, wherein said specific binding member is conjugated to a signal generating compound.

10. The method of claim 9, wherein said signal generating compound is selected from the group consisting of chromogens, enzymes, luminescent compounds, chemiluminescent compounds, radioactive elements and direct visual labels.

11. The method of claim 1, wherein said contacting step comprises incubating said test sample with a solid phase to which at least one recombinant HCV
protein comprising E2 antigen is attached and an antibody specific for said at least one recombinant protein for a time and under conditions sufficient to form antigen-antibody complexes.

12. The method of claim 11, wherein said determining step comprises contacting the solid phase or free phase with a member of a specific binding pair.

13. The method of claim 12, wherein the specific binding member is an antibody.

14. The method of claim 13, wherein the specific binding member is anti-human IgG.

15. The method of claim 12, wherein said specific binding member is conjugated to a signal generating compound.

16. The method of claim 15, wherein said signal generating compound is selected from the group consisting of chromogens, enzymes, luminescent compounds, chemiluminescent compounds, radioactive elements and direct visual labels.

17. A test kit for detecting the presence of antibodies to HCV.
antigen which may be present in a test sample comprising a container containing a recombinant polypeptide that is the expression product of mammalian cells transformed by a heterologous expression vector comprising a DNA sequence encoding an E2 truncated protein, a DNA sequence encoding a rabbit heavy chain signal sequence and a DNA sequence encoding an amino-terminal sequence of human pro-urokinase, wherein said HCV antigen DNA sequence is located downstream to said other DNA sequences.