CA2179609A1

CA2179609A1 - Monoclonal antibodies against hev orf-2 and methods for using same

Info

Publication number: CA2179609A1
Application number: CA002179609A
Authority: CA
Inventors: Deborah A. Paul; Mark F. Knigge
Original assignee: Individual
Current assignee: Abbott Laboratories
Priority date: 1993-12-22
Filing date: 1994-12-16
Publication date: 1995-06-29
Also published as: EP0736087A1; WO1995017501A1; AU1437695A; JPH09507025A

Abstract

Monoclonal antibodies are described which specifically bind to Hepatitis E Virus (HEV), and more particularly to HEV orf-2 antigen. Also provided are hybridoma cell lines which secrete these monoclonal antibodies, methods for using these monoclonal antibodies, and assay kits which contain these monoclonal antibodies.

Description

.
21 7~6G~
wo 95117501 Pcr/uss4/14sos METHODS FOR USING SAME
1~ A A k pr~ n ,i of rh ~ ll This invention relates generally to antibodies which specificaily bind to Hepatitis E Virus (HEV), and more particularly, relates to a novel hybridoma cell line which secretes ,.~ antibodies to HEV orf-2 antigen, and methods for using these mr--AIrlr-~ql antibodies.
HEV, variously referred to as .. . ' epidemic or entericaily transmitted non-A, non-B hepatitis (ET-NANBH), has a globai ~iictrih~ n and has been noted as the cause of major endemic outbreaks of viral hepatitis in developing countries. D. W. Bradley et al., Br. ~r~i B--ll 46:442-461 (1990).
Sporadic cases of ET-NANBH, as well as imported travel exposure, have been reported in developed countries. S. J. Skidmore et al., Lancet 337:1541 (1991).
15 Although the fecal-oral route of i IJl ' limited person-to-person routes of exposure have been suggested in some ~ .i.l. .,.;nl.~; Al studies.
O. Velasquez et al., J. Am~r. Mr~i Accnr 363:3281-3285 ( 1990). This disease has been ~1 ' as having a high mortality rate of alu~l~ "/ 20% in pregnant women infected during their third trimester of pregnancy. See D. W.
20 Bradley etal., suvra.
Molecular cloning of the putative agent of HEV has been hampered by the lack of a tissue culture system for virus ~lu~ ,atiull. However, the use of available animal models and a newly developed non-specific A . ~l~l; r;. A: ;, .. .
procedure have allowed the i~l. .l;I ;. -: ;. ." of a unique cDNA clone (identifled as 25 "ET 1.1") obtained from bile of cyrlvlllulg~.. macaques infected with a Burmese strain of HEV. A. G. ~ - ;. I et al., Vovr. Vir~lc~l 1:73-80 (1986), D. W.
Bradley et al., Proc. NAri A~ i Sci. U,~ 84:6277-6Z81 (1987) and G. W.
Reyes et al., Science 247:1335-1339 (1990). Successful ~ of the viral originofthiscloneledtothei~l~ ..:;r;. -~;.,.. of similarsequencesirlhumanfecal 30 specimens collected from ET-NANBH epidemics m Somalia, Tashkent, Borneo, Pakistan and Mexico. See G. R. Reyes et al., supra. cDNA libraries also have been prepared from human stool samples obtained during an ET-NANBH
outbreak in Mexico. G. R. Reyes et al., G. - l ~. 26:142-147 (1991). IIlllllUllv~,lC~ ;llg of these cDNA libraries led to the ' ~ of two 35 cDNA clones which encode epitopes specific for HEV. P. O. Yarbough et al, J.
Yir~L 65:5790-5797 (1991). The isolation and sequencing of a set of U,~ J ,,g cDNA clones led to the recognition that this fomm of hepatitis is caused by a novel WO95/17sol 2 1 7 9 6 0 9 2 ~ PcT/Uss4/1is0s virus unlike any of the other ~ ~ul~ly ~ ;, d agents of viral hepatitis. A.
W. Tam et al., Virolo~ 185:12-131 (1991).
Varions regions of the HEV genome have been cloned and expressed in E
~li as fusion proteins with glutathione-S-transferase (GST). ~ç~, for example, S.
J. Skidmore et al., ~ . Four of these 1~ antigens, two derived from a Burmese (B) strain of HEV and two derived from a Mexican (M) strain of HEV, have been shown to contain antigenic sites recognized by antibodies from individuals previously exposed to HEV. ~ , P. O. Yarbough et al., ~_. The two antigens from the Mexican strain, named M3-2 and M4-2, correspond to amino acid sequences at the carboxy-terminus of the second open reading frame (ORF-2) and the third open reading frame (orf-2), I~ Li~"ly. The two antigens from the Burmese strain, B 3-2 and B 4-2, correspond to amino acid sequences at the carboxy-terminus of orf-2 amd orf-2, ~ ly. The M 3-2 and B 3-2 , ' antigens both comprise 42 amino acids from the carboxy ternunus of ORF-2. The degree of amino acid homology between these sequences of 42 amino acids is 905%. I~l. The M 4-2 and B 4-2 .-- ~."l,~ amtigens each comprise 33 amino æids from the carboxy terminus of orf-2; the degree of homology between these two sequences of 33 amino acids is 73.5%. L.
Tests developed for detection of HEV must contain reagents which are useful for ~' ,, the specific presence of the virus in a test sample. The need therefore exists for reagents, such as ~ antibodies, capable of reacting only with HEV. Additionally, the ability to produce pure, specific ., .. ,. " ~ .; ri..
antibodies is clearly of great importance for accurate i, l. . ,l i ri. i. ,. "
. 1. -- ,.. ;, ~ ;-.1 " and purification of the HEV antigen.
While methods are available to confirm the presence of screening assay results for agents such as Hl~l, these techniques are not yet available for confirming the presence of HEV. Methods such as culturing HEV in vitro and a viral-based Western blot test are not available. While the detection of HEV nucleic acid may be done by performing PCR, this technique is tedious and expensive, requires special equipment such as a ill~,.lll~,l~,., and turn-around time is up to 24 hours. ' ' .,.. u~ y (~M) has been used to confirm the presence of amb-HEV anbbody, but the use of EM is cost prohibitive as a routine y tool.
It therefore would be ddva~ )u~ to provide a " " ."...-1. ." ~1 anbbody which can be used in accurate, rapid and cost effecbve methods for screening forHEV antigens or HEV antibodies in a test sample.

WO 95/17501 PCT/US94/1450!i ..rnm~ly of th(- rnvrnti~n The present invention provides a highly specific and novel antibody or fragment thereof that can be employed for the detection of HEV orf-2amtigen. The " " ""~ antibody specifically binds to the HEV orf-2 protein.
5 The hybridoma which secretes the ' ' antibody is identified as:
Hybridoma cell line HICI 19 (A.T.C.C. deposit No. HBI 1521, secreting " ~ amtibody (HlCl l9). The specificity of this ' -' antibody enables the a lv ~ ;. ", of HEV-orf-2 antigen, which itlrntifir~fi~n can be useful in viral dil~tl~ n studies as well as in the diagnosis and o evaluationofHEV infections.
T_e ' -' antibody of the present invention can be used in an y for the detection of HEV antigen or antibody. Many such y formats are known in the art and can be modified by adding a known amount of a ' ' antibody which specifically binds to HEV orf-2 1 5 antigen.
A ~al Li-,ul~uly preferred assay format is a u.~ ,LLi ~ assay for ~' ~ the presence and amount of HEV antibody which may be present in a test sample. Tbe assay involves contacting a test sample suspected of containingHEV antibodies with a solid phase coated with HEV antigens and an indicator 20 reagent comprising a signal gene}ating compound and a, . ,~ antibody which ~;rl~,.l y binds to HEV orf-2 amtigen, for a time and under conditions sufficient to form Oll~ L/a '-' ~/ complexes of the test sample and solid phase andlor indicator reagent and solid phase; and ~' 3 the presence of HEV
antibody present in the test sample by detecting the reduction in binding of the25 indicator reagent to the solid phase as compared to the signal generated from a negative test sample to indicate the presence of HEV antibody in the test sampleAssay kits which contain the ' ' antibody of the present invention are also described.
30 Brief Drc~rmtif~n of the Drawinpc Figure I is a schematic ., . of the plasmid used for production of orf-2 CKS/HEV SG-2 antigen from plasmids pJ0201 and pGEX-HEV-0RF2-SG-3.
35 DPf~ A Dl~c~rmfit~n of th~ Tnv~nfil~n The l"" .~ antibody of the present invention can be employed in various assay systems to determine the presence, if any, of HEV antigens or HEV

2 i 7 9 6 0 9 4 PCT/IIS94114505 antibodies in a test sample. Fragments of the mnnor~ l antibody provided also may be used.
The ,,,. ,..,~,1.,,, l antibody of the present invention may be screened for as follows. R~ ' or synthetic HEV orf-2 antigen is used on a solid phase 5 (preferably, polystyrene beads). For example, the CMP-KDO synthetase (CKS) HEV l~ ' orf-2 (SG-3), obtained as described in Example l, below is coated on the solid phase in the unpuriFled (extracted and solubilized) form.
Alternately, an ;..~ .y (EIA) which uses the synthetic peptide spB3-~
(obtained as described in copending application U.S. Serial Number 08/089,877 o Flled July 9, 1993) on the solid phase is utilized. Detection of non-specific binding is ~ ; h 1 by coating the solid phase with CKS alone or with a non-HEV
CKS l~ ' protein. Test samples (mouse serum, tissue culture rn~nt or mouse ascites fluid) are serially diluted in a specimen diluent and a portion is incubated with each solid phase for 1-2 hours at 40C. Beads are thenwashed with buffer and bound antibody is detected using horseradish peroxidase (HRPO)-labeled second antibody. Beads are incubated with conjugate for a sufficient time at 40C, and washed as before. An appropriate substrate solution is added in the dark at room ~ "l'- .~al~lr for 30 minutes. Sulfuric acid is added to stop the reaction, and the amount of color generated is determined by measuring the absorbance of the substrate at 492 nm within 2 hours of sulfuric acid addition.
Details of this method are described in the example section, below For example, in a preferred assay for~nat, the ' -' antibody of the invention can be employed as a competitive probe for the detection of antibodies to HEV antigen. For example, HEV antigens coated on a solid phase, are contacted with a test sample suspected of containing antibody to HEV, and incubated with an indicator reagent comprising a signal generating compound which generates a measurable signal attached to the ~ nnrlor~ antibody of the invention for a timeand under conditions sufficient to fomm i~ 1. ' ly complexes of the test sample to the solid phase or the indicator reagent to the solid phase. The reduction in binding of the """,-~ antibody of the invention to the solid phase, as evidenced by a reduction in the generated signal, can be ~ measured.
A measurable reduction in the signal compared to the signal generated from a confirmed negative HEV test sample would indicate the presence of anti-HEV
antibody in the test sample.
In an alternative assay method,for detection of HEV antigens, a polyclonal or ....~ l anti-HEV antibody or a fragment thereof, which has been coated on a solid phase, is contacted with a test sample which may contain HEV antigens, to form a mixture. This mixture is incubated for a time and under conditions .. . . . . . .

WO 95117501 5 2 ~ 7 9 6 0 9 PCI/US94/14505 sufficient to form ~ i6~ complexes. Then, an indicator reagent comprising a, . ,~ " .~ 1.... 1 or a polyclonal antibody or a fragment thereof, which specifically binds to HEV antigen, to which a signal generating compound which generates a measurable signal has been attached, is contacted with the 5 ~.:;6_.-/~.~il,ody complexes to fomm a second mixture. This second mixture then is incubated for a time and under conditions sufficient to form ~llibody~ i6~ icatul reagent complexes. The presence of HEV antigen present in the test sample and captured on the solid phase, if any, is determined by detecting the measurable signal generated by the signal generating compound. The0 amount of HEV antigen present in the test sample is l--u~,u- liullal to the signal generated.
Alternatively, a polyclonal or m~ r~ l anti-HEV antibody or fragment thereof which is bound to a solid support, the test sample and an indicator reagent comprising a .. ,.. ~. 1.." .1 or polyclonal antibody or fragments thereof, which 15 specifically binds to E~V antigen to which a signal generating compound whichgenerates a measurable signal is attached, are contacted ~i~ l 'S, to form a mixture. This mixture is incubated for a time and under conditions sufficient toform _.A.iI.u~,' ,, 'indicator reagent complexes. The presence, if any, of HEV
antigen present in the test sample-and captured on the solid phase is determined by 20 detecting the measurable signal generated by the signal generating compound. The amount of HEV antigen present in the test sample is ~lu,uulLiullal to the signalgenerated. In this or the assay format described above, the, . ,....~ antibody of the invention can be employed either as the capture phase or as part of the indicator reagent.
In yet another detection method, the ' ' antibody of the present invention can be employed in the detection of E~V antigen in fixed tissue sections, as well as fixed cells by ' ' ' analysis, by standard methods well-known to those skilled in the art.
In addition, the . . " .~ antibody can be bound to matrices similar to CNBr-activated sepharose and used for the affinity I ~ of specific HEV
antigens from cell cultures, or biological tissues such as blood and liver.
The ' ' antibody of the invention also can be used for the generation of chimeric antibodies for therapeutic use, or other similar "~
The ' ' antibody or fragrnent thereof can be provided individually 35 to detect EIEV antigen. C~ 1.: ;"r: of the "", " 1, ", ,1 antibody (and fragments thereof) of the present invention provided herein also may be used in 1 l ,. . ,1,~
with other "" " ,~ antibodies that have differing ~ for E~V as '""'1'""` '~ in a mixture or "coclctail" of HEV antibodies, each having different wo 95117501 2 1 7 9 6 0 9 6 PCTIUS94/14505 binding ~ Thus, this cocktail can include the -' I antibody of the invention directed to orf-2 protein from the H:EV genome, along with different antibodies directed to other HEV regions, such as the orf-3 HEV
proteinorotherbindingsitesonorf-2HEVprotein. Thiscocktailof,...~ ~cl~
s antibodies would then be used in place of the single ' ' antibody as described in the assay formats herein.
The polyclonal antibody or fragment thereof which can be used in the assay formats should specifically bind to HEV antigen. The polyclonal antibody used preferably is of ' origin and includes but is not limited to human, o goat, rabbit or sheep anti-HEV polyclonal antibody. The polyclonal antibodies used in the assays can be used either alone or as a cocktail of polyclonal antibodies. Since the cocktails used in the assay formats are comprised of either .",.".~1.~-- ~1 antibodies or polyclonal antibodies having different ED3V specificity, they would be useful for diagnosis, evaluation and prognosis of HEV infection, as well as for studying HEV protein J;rrclcllt~ iu~l and specificity.
Test samples which can be tested by the methods of the present invention described herein include human and animal body fluids such as whole blood, serum, plasma, C.,ll;lJlU:.~J;lldl fluid, urine, biological fluids such as cell culture fixed tissue specimens and fixed cell specimens.
The "solid phase" is not critical and can be selected by one skilled in the art. Thus, latex particles, u~ s, magnetic or non ~ ' beads, ...... ,1., ~.. 1. ~ plastic tubes, walls of microtiter wells, glass or silicon chips and tanned sheep red blood cells are all suitable examples. Suitable methods for ~ ~li7in~ peptides on solid phases include ionic, l~yJIu~llul~ covalent 25 - and the like. A "solid phase", as used herein, refers to any material which is insoluble, or can be made insoluble by a subsequent reaction. The solidphase can be chosen for its intrinsic ability to attract and immobili_e the capture reagent. Altematively, the solid phase can retain an additional receptor which has the ability to attract and immobili_e the capture reagent. The additional receptor 30 can include a charged substance that is oppositely charged with respect to the capture reagent itself or to a charged substance conjugated to the capturc reagent.
As yet another alternative, the receptor molecule can be any specific binding member which is; . , ~ I upon (attached to) the solid phase and which has the ability to immobili_e the capture reagent through a specific binding reaction. The 3 5 receptor molecule enables the indirect binding of the capture reagent to a solid phase material before the ~.r~ of the assay or during the p.,lru~ cc of the assay. The solid phase thus can be a plastic, derivati_ed plastic, magnetic or non-magnetic metal, glass or silicon surface of a test tube, microtiter well, sheet, 2 l 7q60q bead, u~O~ L~.le, chip, and other . ~. known to those of ordinary skill in the art.
It is ~ .,r ~l and within the scope of the invention that the solid phase also cam comprise any suitable porous material with sufficient porosity to allow5 access by detection antibodies and a suitable surface affinity to bind antigens.
~Lulul~uluu~ structures are generally preferred, but materials with gel structure in the hydrated state may be used as well. Such useful solid supports include:
natural polymeric ~ubul~y~' ' and their synthetically modified, cross-linked or substituted derivatives, such as agar, agarose, cross-linked alginic acid, o substituted and cross-linked guar gums, cellulose esters, especially with nitric acid and carboxylic acids, mixed cellulose esters, and cellulose ethers; natural polymers containing nitrogen, such as proteins and derivatives, including cross-linked ormodified gelatins; natural llydlUUalllUII polymers, such as latex and rubber;
synthetic polymers which may be prepared with suitably porous structures, such 15 as vinyl polymers, including pol~ '.yl~ uly~)lu~)~lull~., poly~lylc.l~
poly v ylullloride, puly V;lly; and its partially hydrolyzed derivatives, poly~lyl~ulli~i" ~ùlyll~~ ,ly copolymers and t~l~)UIyll~l~ of the above pc ly~ ' such as polyesters, polyamides, and other polymers, such as pOlyl Clllall.,~ or pul~ù,.i-L,i" porous inorganic materials such as sulfates or20 carbonates of alkaline earth metals and n~ ci..~, including barium sulfate, calcium sulfate, calcium carbonate, silicates of alkali and alkaline earth metals, aluminum and .. c" ~ . and aluminum or silicon oxides or hydrates, such as clays, alumina, talc, kaolin, zeolite, silica gel, or glass (these materials may be used as filters with the above polymeric materials); and mixtures or copolymers of the above classes, such as graft copolymers obtained by initializing luuly~ aLu of synthetic polymers on a pre-existing natural polymer. All of these materials may be used in suitable shapes, such as films, sheets, or plates, or they may becoated onto or bonded or laminated to appropriate inert carriers, such as paper,glass, plastic films, or fabrics.
The porous structure of ll;t~uu.,llulu~ci has excellent absorption and adsorption qualities for a wide variety of reagents including antibodies. Nylon also possesses similar l - ~ r ;~ and also is suitable.
It is . ~ ' I that such porous solid supports described l.~,.~,i.l~.'oc.~., are preferably in the form of sheets of thickness from about 0.01 to 0.5 mm, preferably about 0.1 mm. The pore size may vary within wide limits, and is preferably from about 0.025 to 15 microns, especially from about 0.15 to 15 microns.

WO 9~J17501 2 1 7 9 6 0 9 ~ PCT/US94114505 To change or enharlce the intrinsic charge of the so~id phase. a charged substance can be coated directly to the material or onto ~ u~ i-,lc, which then are retained by a solid phase support material. Altematively, ~ ,lu~ Lil,l~,S can serve as the solid phase, by being retained in a columm or being suspended in the mixture of soluble reagents and test sample, or the particles themselves can be retained and;, -,. ~ by a solid phase support material. By "retained and ' ' '" is meant that the particles on or in the support material are not capable of substantial movement to positions elsewhere within the support material. The particles can be selected by one skilled in the art from any suitable type of particulate material and include those composed of pbly~lylclll~, p~lylll~.Li~yl~ly- pbly~nu~y~cll~"latex,polyt i " c~ yl~ c, polyacrylonitrile, pûly~,alL , or similar materials. The size of the particles is not critical, although it is preferred that the average diameter of the particles be smaller than the average pore size of the support material being used. Thus, . ~ ' which utilize various other solid phases also are , ' ' and are within the scope of this invention. For example, ion capture procedures for an irrrnt~hili7~hlr reaction complex with a negatively charged polymer, described in co-pending U. S. Patent Application Serial No. 150,278 rorrP ' to EP Publication No. 0326100, and U. S. Patent Application Serial No. 375,029 (EP Publication No. 0406473), can be employed according to the present invention to effect a fast solution-phase - ' ' reætion. An ;".. 1.;1;,~1l1~ immune complex is separated from the rest of the reætion mixture by ionic interactions between the negatively charged poly ' complex and the previously treated, positively charged porous matrix and detected by using ~5 various signal generating systems previously described, including those described m, ' ' signal " ~ as described in co-pending U.S. Patent Application Serial No. 921,979 Cbl~ ' g~ to EPO Publication No. 0 273,115.
Also, the methods of the present invention can be ædapted for use in systems which utilize - ic ul~.uLi~,lc technology includjng automated and semi-automated systems wherein the solid phase comprises a -~-ulJalLi-,lC. Such systems include those described in pending U. S. Patent Application 425,651 and U. S. Patent No. 5,089,424, which correspond to published EPO a~ Liu Nos. EP O 425 633 and EP 0 424 634, ."~ ,Li~.,ly, and U.S. Patent No.
5,006,309.
The mdicator reagent comprises a signal generating compound (label) which is capable of generating a measurable signal detectable by external means conjugated (attæhed) to a specific binding member for HEV. "Specific binding member" as used herein means a member of a specific binding pair. That is, two .

woss/17sol 9 21 7960q PCT/US94114505 different molecules where one of the molecules through chemical or physical means specifically binds to the second molecule. In addition to being an antibody member of a specific binding pair for HEV, the indicator reagent also can be a member of any specific binding pair, including either hapten-anti-hapten systems5 such as biotin or anti-biotin, avidin or biotin, a 10lbullyl' or a lectin, a . 1 y nucleotide sequence, an effector or a receptor molecule, an enzyme cofactor and an enzyme, an enzyme inhibitor or an enzyme, and the like. An UlCU,~ specific binding member can be an antibody, an antigen, or an ulllibvvy/ull~ll complex that is capable of binding either to HEV as in a sandwich o assay, to the capture reagent as in a competitive assay, or to the ancillary specific binding member as in an indirect assay.
The various signal generating compounds (labels) c~n~ . ' ' include UIIIUIII()~ catalysts such as enzymes, ' compounds such as fluorescein and rhodamine, r h~mi!~ cu...l, ', radioætive elements, 15 and direct visual labels. Examples of enzymes include alkaline ~
hr rsP.r2irlich peroxidase, bet~ g~ , and the like. The selection of a particular label is not critical, but it will be capable of producing a signal either by itself or in: ; with one or more additional substances.
Other ..,.1,o.li..,.. t~ which utilize various other solid phases also are 20 . ' , ' ' and are within the scope of this invention. For example, ion capture proccdures for imml)hil g an ~ reaction complex with a negatively charged polymer, described in co-pending U. S. Patent Application Serial No.
150,278 cullco~ullvill~ to EP publication 03261û0, and U. S. Patent Application Serial No. 375,029 (EP publication no. 0406473) both of which enjoy common 25 ownership and are i~lCul~ ' ' herein by reference, can be employed according to the present invention to effect a fast solution-phase ' ' reaction. An ;",..",1,;1;,_1 .lr immune complex is separated from the rest of the reaction mixture by ionic ~ betwcen the negatively charged poly ' complex and the previously treated, positively charged porous matrix and detected by using 3 0 various signal generating systems previously described, including those described in ' ' signal , as described in co-pending U.S. Patent Application Serial No. 921,979 ~ to ~PO Publication No.
O 273,115, which enjoys common ownership and which is ;IICUI~U~ ' J herein by reference.
Also, the methods of the present invention can be adapted for use in systems which utilize l.u,,lul,uli icl~ technology including in automated and semi-automated systems wherein the solid phase comprises a IIU~IUUUIL;CI~ Such systems include those described in pending U. S. Patent Arrli~iionc 425,651 and wog5/17sol 2 1 7 ~ 6 0 9 ~ PcTNssal4sos 425,643, which correspond to published EPO ~ Nos. EP 0 425 633 and EP 0 424 634, I-,DY~L~IY, which are i..cu.~, ' herein by reference.
The use of scanning probe l..;.,.U~U~Y (SPM) for , ;, alsû is a technology to which the mnn~lnnql antibûdies of the present invention are easily5 adaptable. In scanning probe microscopy, in particular in atomic force Illh,lU~UI~y, the capture phase, for example, at least one of the ,. .., ~
antibûdies of the invention, is adhered to a solid phase and a scanning probe microscope is utilized to detect ~i,, '~It L,o.ly complexes which may be presenton the surface of the solid phase. The use of scanning tunneling Illi~,lU~.~y o eliminates the need for labels which normally must be utilized in many ~ systems to detect ~lLis.,~ iL~udy cûmplexes~ Such a system is described in pending U. S. patent application Serial No. 662,147, which enjoys common ownership and is ~,, ' herein by reference.
The use of SPM to monitor specific binding reactions can occur in many 15 ways. In one .. . ,l ,n,l; I . .. :, one member of a specific binding partner (analyte specific substance which is the mn-nrlnnol antibody of the invention) is attached to a surface suitable for scanning. The attachment of the analyte specific substance may be by adsorption to a test piece which comprises a solid phase of a plastic or metal surface, following methods known to those of ordinary skill in the art. Or, 20 covalent attachment of a specific binding partner (analyte specific substance) to a test piece which test piece comprises a solid phase of derivatized plastic, metal, silicon, or glass may be utilized. Covalent attachment methods are known to those skilled in the art and include a variety of means to irreversibly link specific binding par~ners to the test piece. If the test piece is silicon or glass, the surface must be 25 activated prior to attaching the specific binding par~ner. Activated silane compounds such as triethoxy ar~ino propyl silane (available from Sigma Chemical Co., St. Louis, MO), triethoxy vinyl silane (Aldrich Chemical Co., Milwaukee, WI), and (3-mercapto-propyl)-trimethoxy silane (Sigma Chemical Co., St. Louis, MO) can be used to introduce reactive groups such as arrino-, vinyl, and thiol, 30 ~ .,ly. Such ætivated surfæes can be used to link the binding partner directly (in the cases of amino or thiol) or the ætivated surfæe can be further reacted with linkers such as ;,' ' ' ' yl~ bis (~u1~,ill;lllhlyl) suberate, SPPD 9 succinimidyl 3-[2-1uyliJy' ' ' ~] propionate), SMCC ( Iyl~[N-' yl] ~Y~ ' ' l-carboxylate), SL~ U~ idyl [4-iodoæetyl]
35 cu, L ),andSMPB( ~14-[1- ' ' ,' yl]butyrate)to separate the binding partner from the surfæe. The vinyl group can be oxidized toprovide a means for covalent attachment. It ,Iso can be used as an anchor for the pUlylll".;~iu,. of various polymers such a3 poly ærylic acid, which can provide ~ WO 95/l~501 11 2 1 7 9 6 0 9 PcTlUS94/laOS
multiple attachment points for specific bindjng partners. The amino surface can be reacted with oxidized dextrans of various molecular weights to provide hydrophilic linkers of different size and capæity. Examples of oxidizable dextrans include Dextran T ~10 (molecular weight 40,000 daltons), Dextran T-l 10 (molecular weight 110,000 daltons), Dextran T-500 (molecular weight 500,000 daltons), Dextran T-2M (molecular weight 2,000,000 daltons) (all of which are available from Pharmacia, Piscataway, NJ), or Ficoll (molecular weight 70,000 daltons (available from Sigma Chemical Co., St. Louis, MO). Also, p~ ulyl~
intr~rrir~nc may be used to immobilize a specific binding partner on a surface of a o test piece by using techniques and chemistries described by pending U. S. Patent ~rrlir~ionc Serial No. 150,278, filed January 29, 1988, and Serial No. 375,029, filed July 7, 1989, each of which enjoys common ownership and each of which is ull ' herein by reference. The preferred method of attachment is by covalent means. Following attachment of a specific binding member, the surface may be further treated with materials such as serum, proteins, or other blockingagents to minimize non-specific binding. The surface also may be scanned either at the site of ' ~ or point of use to verify its suitability for assay purposes. The scanning process is not anticipated to alter the specific binding properties of the test piece.
~o While the present invention discloses the preference for the use of solid phases, it j5, . l l ~ that the - ' ' antibody of the present invention can be 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 invention.
The "- " ,-~ antibody of the invention can be used as a positive control rn an assay which is designed to detect the presence of HEV antibody. In an assay which detected the presence of HEV antibody in a test sample, HEV
amtigens would be used as a capture phase. These HEV antigens could be prepared by various means from viral Iysates, synthetic peptides of various >~ regions of the HEV genome, and/or ' proteins produced by using either synthetic or native antigens or epitopes of antigens. It also is. l ' ' that these types of HEV antigens could be employed in a variety of assay fommats including those described herein as either the capture phase or detection phase. The use of the - ' ' antibody of the invention would 3 5 ensure that the reagents provided to detect HEV antibody were performing adequately by being used in place of a test serum in the p.,lrullll~ .,e of the assay, according to procedures known to those of ordinary skill in the art.

wo 95~l750l 2 ~ 7 9 6 0 9 12 PCIIIJS94/145û5 ~
It is ~ that the reagent employed for the assay can be provided in the form of a kit with one or more containers such as vials or bottles, with each container containing a separate reagent such as a l l antibody, or a cocktail of ".~".n~ l antibodies, employed in the assay. These kits also could 5 contain vials or containers of other reagents needed for performing the assay, such as washing, processing and indicator reagents.
The following examples ~' the advantages and utility of the HlCll9",.~,~n.1.",~1antibodyoftheinventionbydescribingmethodsforthe , epitope mapping and clinical utility of the 10 """,,.~ lantibodies. Themethodsusedfor 1 1antibody dLV~
follow procedures known in the art and detailed in Kohler and Milstein, Nature 256:494 (1975), and reviewed in J.G.R. Hurrel, ed., Mnnn~lnnAl HvbrirlnmA
Antihnflipc TP~ hniQ ~P~ An~1 Applications. CRC Press, Inc., Boco Raton, FL
(1982).
To practice the invention, a ., ' DNA clone was constructed to contain the gene encoding the full length orf-2 of HEV as a fusion protein with CKS. This antigen (designated SG-3 protein) expressed by this clone was used to immunize a mouse from which an immune splenocyte was fused to a SP2/0-Ag 14 myeloma cell to produce a hybridoma cell line that secretes a mnnn- lnn ~1 antibody 20 of ~ . lin (Ig) class Gl (IgGI) reactive with HEV orf-2. The resultant O. l ~ was produced in mouse ascites fluid and was purified by affinity Llll~ o 1 .Y
The exarnples set forth below are meant to illustrate, but not to limit, the spirit amd scope of the invention.
Example I
t'~nPrAtinn ~S Cll - ". ,. . i, .~ inn of ~yhri~1nm~ ('rll T .inP
A. Production of .. ' HEV Protein.
Plasmid pGEX-HEV-0RF2-SG-3 (Figure 1) was obtained from Genelabs, Inc., Redwood City, California and encodes the carboxyl termins 327 amino acids of orf-2 antigen of HEV as a glutathione S-transferase (GST) fusion in the pGEX expression system. The gene encoding orf-2, designated as the SG-3 protein, (see P.O. Yarbough, et al., supra) was cloned and expressed as a chimeric fusion protein with CMP-KDO synthetase (CKS) in p~asmid pJ0201 in E. coli according to methods known in the art. The CKS/HEV-SG-3 protein was expressed in E. coli strain XLI-Blue at greater than 20% of total cell protein after induction with isopropyl b-D ~ 1r (IPTG). The clones were propagated in 10 liter fermentors, yielding 170-260 grams of wet cell paste per rr.",~. ,~ l;,~,, 2 ~ 79609 B. CKS-HEV Protein Snl,.1,;1;, 1;""
The ~. coli cells expressing the SG-3 protein were Iysed at pH 10 in the presence of various protease inhibitors. The CKS fusion proEein was insoluble and remained in the pellet aftem,~L iruodLiu~l. Pellets were washed with variousdetergents to remove non-specific proteins. Following snll~hili7~inn in Tris, pH8.5 containing 0.5% SDS, the CKS fusion protein was found to represent 50 to 60% of the total protein as evaluated by SDS-PAGE and gel ~II ~;Lull.~ily. The solubilized protein was further purified by Sephacryl S-300HR column .,11~ .y . Frætions were analyzed by SDS-PAGE, pooled and evaluated for purity by gel ~ y. Final purified protein was greater than 90% pure.
C T of Mice The regimen (4 mice) consisted of an initial; 1 .. .. .
witb additional at six and nine weeks. For the primary 10 ,ug CKS-HEV orf-2 SG-3 extracted, solubilized and purified Ir. ~""1.: -~ protein, prepared as described above, was emulsified in complete Freund's adjuvant. Four mice (Balb/c) were inoculated i,.~ lly with tbis emulsion. At three weeks post- mouse serum was screened for enzyme .y (EIA) ~,~Livi~y as described below. The serum anti-HEV titer tested against beads coated witb BV SG-3 was 2 x 104 in specimen diluent containing E. coli and CKS Iysate and 103 against beads coated with CKS alone. The mouse serum antibody titer against beads coated with HEV
orf-2 synthetic peptide spB3-2, prepared as described in copending U.S. patent application Ser. No. 07/089,877 filed July 9, 1993 was 2 x 102.
Four weeks after the first three mice were boosted , with 10 llg of the ~ protein in incomplete Freund's adjuvant. At week nine, post anti-BV titers from boosted mouse No. 4 against beads coated with protein HEV SG-3 described above were 1.5 x 106 irl specimen diluent with no additives to block out unwanted reactivity, 2 x105 in specimen diluent with additives and 5.0 x 103 against spB3-2. This mouse was then boosted .~,..u .IJ (via the tail vein) with 30 llg of the 7 protein and used for fusion 3 days later.
D. r~ of the H~l,.idu"ld~
Normal mouse ~ ,..0~ were prepared by aseptically removing the spleen from a non-immunized mouse and crushing it using the plunger of a syringe through a screen that fits into a 60 x 15 mm Petri dish containing a small amount of Dulbecco's' Minimum Essential (DME) medium. The splenocyte so]ution was washed with DME medium, and the red blood cells were iysed by adding I ml of 0.83% NH4CI in 10mM Tris to the cell pellet for I minute. The cells were again washed and I~U~ ,lldl~Xl in 40 mls of DME medium containing 20% Fetal Bovine Serum (FBS).
On the day of the fusion, the immunized mouse was sacrificed, and the spleen was removed and p}ocessed as described herein for the preparation of normal ~ul~,n;: uy t~ except that the splenocytes were , ~ 1 in 10 mls DME
medium and counted. Splenocytes were fused in a 1:1 ratio with the SP2/0 o myeloma cell line using the described " .~. l; r,~ ~ il " . of conventional methods (Kohler and Milstein, supra). Cells were plated at IX106 ,pll,llot~t~" per well of a 24 multi-well tissue culture tray in 1.5 ml DM~ medium containing 20% FBS, and thymidine (HAT). After 24 hours. 200,000 normal ~pl~,n;,~yt.,, were added per 24 multi well (0.5 ml). HAT medium was added on day 5 and replaced on day 7.
Two of twenty-nine irlitial hybrids from the fusion were positive for antibodies to HEV. Hybrid #1 (H1) had an optical density (O.D). of 1.155 when tested at a 1:10 dilution against SG-3 antigen- coated beads and was negative against CKS-only coated beads, and HEV spB3-2 beads. Hl was then cloned at one cell per well by limiting dilution tt-chnique as described in Godirlg, M~ "~orl. . ~1 Antihnr~ l Prartir~ c 2nd ed, Academic Press, New York [1986).
E. r~ Of Clones By Limiting Dilution The viable cells in the antibody positive wells were counted, and for each 96 multiwell tissue culture tray, an aliquot containing 100 cells was added to 20 mls of DME media containing 20% Fetal Bovine Serum (FBS) and Sx] o6 normal mouse ~I.,ll~yt~,~. An eight channel pipetman adjusted to 0.2 ml was used to plant the cell suspension. The trays were incubated at 37C in a humidified 5%
C2 incubator and refed on days 5 and 7 or as needed because of ~,~alJul~liu~l.
When the wells containing growth were 30-50% confluent (usually between 10-21 days), those wells containing only I colony were sampled and tested for antibodyactivity. Several positive wells from each hybrid were selected and the cells were expanded. A clone designated HICI 19 resulted which had reactivity against HEV
orf-2(SG-3).

21 7960q F Production and r, ~ of the HICI 19 Mrnrrlr~ ~1 Antibody Clones selected for furLher evaluation were scaled up in tissue culture T-flasks and 106 cells were injected into the peritoneal cavity of pre-pristaned BALB/c mice (Charles River B ' ' Services, Inc., Wilmington, MA) (see s Hurrell, s~pra). The resulting ascites fluid was harvested 7-10 days after injection" r V I and frozen. The antibody was affinity purified on a Protein A Sepharose CL4B (Pharmacia-LKB Riot~rhnnl~j~ C Piscataway, NJ). Bound . 1 antibody was eluted from the columrl at a pH of 5.5, indicating its subtype as IgGI.

G. Screeningand~ l;.. of HlC119~ Antibody To screen and ~ - r~ the antibody, enzyme - y, (ElA's) were utilized which contained different antigens on the solid phase (~ C
beads): the CKS-HEV ~c~" ' orf-2 (SG-3) (prepared as described in Example 1) was coated on the solid phase in either the unpurified (extracted andsolubilized) form (ca. 50-60% pure HEV protein) or a purified protein (>90%
pure), another EIA used the synthetic peptide spB3-2 on the solid phase, and to detect non-specific binding the solid phase was coated with CKS alone or a non-HEV CKS IC~,~ ' ' ' protein.
For all assays, mouse serum, tissue culture c,.r.-~ ,r,~.lt mouse ascites fluid samples or ' ' antibody (MAb) HICI 19 obtained above were serially diluted 5 to 10-fold in specimen diluent containing phosphate-buffered-saline/Tris/EDTA, pH 7.8 with 20% goat serum, 10% fetal calf serum, 1% bovine serum albumin, 0.2% Triton-X 100, 0.1% sodium azide, with or without 1% E.
coli Iysate and 0.01% CKS added, then 200 1ll was incubated with each solid phase for 1-2 hours at 40DC. Beads were then washed 3 times with 5 ml distilled water. Bound antibody was detected using 200 1ll HRPO-labeled second antibody (Goat anti-mouse IgG H+L) from Kirkegaard and Perry Labs) at 0.3 llg/mL in conjugate diluent containing Tris-buffered-saline with 10% goat serum, 10% fetalcalf serum and 0.15% Triton-X 100. Beads were incubated with conjugate for I
hour at 40C, and washed as before. O-PI~ J (OPD) substrate was freshly prepared by adding I OPD tablet (Abbott) per 5 mls of OPD Diluent (Abbott), and 300 ~1 of OPD substrate solution was then added to each washed bead and incubated in the dark at room ~.ll~l~L,., for 30 minutes. One ml of sulfuric acid added to stop the reaction, and the amount of color generated was determined by measuring the absorbance of the substrate at 492 nm with 2 hours of sulfuric acid addition. Results given in Table I below indicate that the antibody was specific for the orf-2 region of HEV.

Wo 95117501 21 7 q 6 0 q 16 PCTIUS94/14505 ;. ." of T~ l C119 Mnnnrlrn~l Ailtihn ly Absorbance at 492 nm ~?~nmh~ HEV R., . ,.. }. - - - .l CKS
Dllutlon (-1) SG-3-coated bead coated bead I x 103 >2 0.017 10 x 103 1.762 ND
100 x 103 0.756 ND
I x lo6 0.120 ND
(~nm,IPti~ive ~cc~v for Antihn~y to HFV:
Solid phase was coated with HEV orf-2 (SG-3) or a rnmhinorinn of proteins coding for the orf-2 and orf-3 regions. The solid phases were pre-incubated with a 1:2 dilution of HEV negative human plasma (negative contro~) orhuman plasma known to contain HEV Ab (sample) in specimen diluent overnight at room ~ . 'l" . .t. . . ~ Beads were then washed and " " . ~, i, ,. ~l antibody (MAb) of the invention against HEV orf-2, diluted to give an O.D. of 1.000 - 2.000 when no inhibitor is present, was added to the appropriate beads and incubated for I
hour at 40-C. The beads were then washed and HRPO-goat anti-mouse IgG (H +
L) was added aW ubcubated for 2 hours at 40 C. The beads were washed and label detection was performed using OPD substrate as described above.
Tllc~/le~i~,dly, a positive HEV Ab containing sample should reduce the signd 50%. In the example provided the HEV Ab positive sample showed 20.0%
inhibition of MAb HICI 19 when the SG-3 bead was used, and 54.2 % inhibition when the HEV orf-2/orf-3 Combo bead wæ used. The results are given in Table n below.

wo 95/17501 2 1 7 9 6 0 9 PcTlUsg4/lao5 ~able II
~'om~Pti~iye Acc~v for Antiho ly to ~F.V ~rf-2 HEV BEAD
~mhin ~r Antigen Rtc~ ' Antigens from orf-2 from orf-2 and orf-3 O.D. % ~hibition O.D. % Inhibition Pre-incubation with~:
Specimen Diluent Only 1.370 --- 2.197 . ----Negative Humam Plasma 1.448 --- -1.955 ---Human anti-HEV + 1.159 20.0 0.895 54.2 Plasma 5 Example 3 't~mrrrifive A cc ?y for orf-2 An~i,~n to ~FV
Solid phase is coated with HEV orf-2 (SG-3) protein. Ml antibody (MAb) HICI 19, at a: previously determined to give an O.D. of 1.000 - 2.000 in this assay format, is pre-incubated with sample at a time and i l _ long enough to allow binding of HEV amtigen in the sample to MAb (30-120 minutes, at room i r ' C to 40C). The solid phase is then added, and the mixture and bead are incubated at a time and tClll~ long enough to aDow any remaining unbound MAb to bind to the solid phase (i , range:rooml , to40C,timerange: I -16hours). Binding i 5 of MAb to the solid phase is then detected either using a labeled second antibody (e.g. h~rg~r51~1ich peroxidase (HRPO)-labelled Goat anti-mouse IgG) wit~h incubation of 1-2 hours at 40C, or MAb itself could be labeled, thus making theassay potentially ~- r.~ in one step. Label detection is then performed (i.e.
if using HRPO labeled Ab, add OPD substrate, incubate 30 minutes, add sulfuric ~o acid to stop the reaction, and read at 492 nm). A positive, HEV antigen containmg sample will inhibit the MAb from binding to the solid phase amd reduce the signal at least 50%.

Claims

WHAT IS CLAIMED IS:

1. A monoclonal antibody or fragment thereof which specifically binds to HEV orf-2 encoding protein wherein said monoclonal antibody is secreted by hybridoma cell line A.T.C.C. deposit No. HB 11521.

2. A monoclonal antibody or fragment thereof secreted by A.T.C.C.
deposit No. HB 11521.

3. A hybridoma cell line which secretes a monoclonal antibody or fragment thereof which specifically binds to HEV orf-2-encoding protein having the identifying characteristics of hybridoma cell line A.T.C.C. deposit No.
HB11521.

4. A hybridoma cell line A.T.C.C. deposit No. HB11521.

5. In an immunoassay for HEV antigen or antibody, the improvement comprising the step of adding a known amount of a monoclonal antibody or fragment thereof which specifically binds to HEV orf-2 antigen.

6. A competitive assay method for determining the presence of HEV
antibody which may be present in a test sample, comprising:
a. contacting a test sample suspected of containing HEV
antibodies with a solid phase coated with at least one HEV antigen and an indicator reagent comprising a signal generating compound which generates a measurable signal and a monoclonal antibody which specifically binds to HEV orf-2 antigen, for a time and under conditions sufficient to form antigen/antibody complexes ofthe test sample and solid phase and/or indicator reagent and solid phase; and b. determining the presence of HEV antibody present in the test sample by detecting the reduction in binding of the indicator reagent to the solid phase as compared to the signal generated from a negative test sample to indicate the presence of HEV antibody in the test sample.

7. The method of claim 6 wherein said monoclonal antibody is secreted by the hybridoma cell line A.T.C.C deposit No. HB 11521.

8. The method of claim 6 wherein the signal generating compound is selected from the group consisting of a luminescent compound, a chemiluminescent compound, an enzyme and a radioactive element.

9. An assay kit for determining the presence of HEV in a test sample comprising a container containing at least one monoclonal antibody or fragment thereof which specifically binds to HEV orf-2 region.

10. The assay kit of claim 12 wherein said monoclonal antibody has the binding specificity of the monoclonal antibody secreted by the cell line A.T.C.C.
deposit No. HB11521.