CA2191430A1 - Detection of different hiv genotypes utilizing a synthetic peptide-modified immunoassay - Google Patents

Abstract

An assay to simultaneously detect the presence of one or more HIV subtypes of HIV-1 antibody and/or HIV-2 antibody in a test sample is disclosed. The analytes are captured on the same or different solid phases and the presence of the analytes is determined by detecting a signal generated by using a cocktail of synthetic and recombinant antigen-containing indicator reagents. Preferred indicator reagents include a synthetic 19 amino acid cyclic peptide homologous to an immunodominant region of HIV-1 gp 41 and a synthetic 19 amino acid cyclic peptide homologous to an immunodominant region of HIV-2 gp 36.

Description

~WO9!i/33206 2 1 9 1 430 IIU~,", 1'71 DETECTION OF 1)l~ T ~TlV CT~,~OTYPF.~ UTIT.T~;T~G A SYNT~,TIC
PT1',PTIn~. MODT~T~,n TlVrMUNO.~ 5AY
This slrrlir~Ti~m i8 a rnntim1~ti~.n-in-part of U.S.Serial No. 08/204,703 5 filed March 2, 1994 which is a c~ntinll~tirln in part of ~rFlir~ti-ln of U.S.Ser.
07/866,380, filed April 9, 1992, whic_ is a ~ n-in-part arFlir~t~n of U.S.Ser. No.07/787,710 filed November 4, 1991, which is a ~
lir~t;~ln of U.S.Ser. ~o. 07/361,733 filed June 2, 1989 (~h~n~ nP~l), which is a ~ .I.;....~I - .-in-part ArrlirAtir~n of U.S.Ser. No. 07/320,882 filed March 9, 1989 1 0 (ah~nfl~mP-l), which is a ~ ; . .. _l nn ~rFlir~tirln of U.S.Ser. No. 07/020,282 filed February 27, 1987 (~h~n~ npd) which is a ~ - .-in-part ~rFlir~tir~n of U.S.Ser. No. 06/811,240 filed December 20, 1985 (~Ih~n~nPA), all of which enjoy common ownership and are incvll,u.ale.l herein in reference.
15 Field of thP Invontion This invention relates to human immllnr~PfiriPnry virus (HIV) and imm-ln~-~cS~ys. In particular, this invention relates to an immun~qR:~y and reagents for the gim-llt~nPouc detection of more than one HIV ~ ol~,ues.
More particularly, this invention relates to an imml~n"~Cc~y for detecting HIV-

2 0 1 antigens and/or HIV-1 ~nt ho~liPs and HIV-2 ~ntiho~liPR in a test sample.
Prmln~ of tho Inven~ T~
Acquired imnmlnrldPfiriPnry syndrome (AIDS) is a disorder of the immune system ~cRori~ted with U,U~JOllUI- blAic infections and/or nPorlAcnn~
2 5 which has reached epidemic proportions in the United States as well as in Europe and in central Africa. The Pp: lPmi~lngirsll data suggest that AIDS is caused by at least two types of human immllnn~lPfiriPnry viruses, collec-t*ely APRi~n~ted as HIV. HIV type 1 (HIV-1) has been isolated from patients with AIDS and AIDS-related complex (ARC), and from healthy persons at high risk

3 0 for AIDS. See, for example, F. Barre-Sinoussi et al., ~n~ 220:868-87191983); M. Popovic et al., ~ 224:497-500 (1984); and R. C. Gallo et al., ~
224:500-503 (1984). HIV-1 is tr~ncmitfocl by sexual contact, exposure to blood and certain blood products, or from an infected mother to her fetus or child. P.Piot et al., ~iç~ 239:573-579 (1988). The prevalence of HIV-l ~nbho~liPR in 3 5 AIDS and ARC patients and persons at risk is high, and the virus can be WO 95/33206 ~ r~,l"J.,,~ 1'''1 2191~30 isolated from nearly 90% of all 6.,.u~uOa;Liv~ individuals. See, for example, M.G. S~rn~rlh~ran et al., ~ 224:506-508 (1984); and D. Gallo et al., . 25:1291-1294 (1987).
In 1986 a second human immlln/~rlPfi~Pnry -virus, HIV-2, was isolated S from patients with AIDS in west Africa. F. ClaYel et al., ~ 233:343-346 (1986). HIV-2 infections al60 have been identified in individuals from several countries outside of west Africa. See, for example, A. G. Saimot et al., I~
i:688 (1987); M.A. Rey et al., I.an~ i:388-389 (1986); A. Werner et al., Lanceti:868-869 (1987); G. Brucker et al., AIDS 2:141(1988); and K Marquart et al., ~ 2:141 (1988). Although at the present time HIV-2 appears to be endemic only in west Africa, it appears likely that, based on the PYrPripnrp with HIV-1,HIV-2 will spread to other parts of the world.
HIV-2 virus is similar to HIV-l virus in its morphology, cell tropism, Lioll with the CD4 cellular receptor, i~a~ u,uc.lI ic effect on CD4 cell6, overall genomic structure and its ability to cause AmS. F. Clavel, A S
1:135-140 (1987). However, HIV-Z differs from HIV-1 in several respects. See F.
Clavel, Ibid and R.A. Weiss et al., ~ 2:95-100 (1988).
Recently, new HIV subtypes have emerged. In 1990, DeLeys et ~1.
identified a new HIV isolate ~P~i~"~l~d subtype ANT 70. J. Virol. 64: 1207-1216 2 0 (1990). Further reports of HIV-1 related viruses in wild 1.; ~ n~ in Cameroon and Zaire were published by Peters et c~l. AIDS 3: 625-630 (1989) What is currently thought as a possible HIV-1 subtype with prevalence in the Cameroon area of Central Africa has been lP~iFn~tpd as subtype 0. This subtype, which has been problematic in currently available assays for HIV
2 5 detection is currently clL~ L~ d as having a,u~ul u~hll~lely 65% homology to HIV-1(- .~ - c, sequence, alU,ULU~illLaL~Iy 55% homolgy to HIV-2 c- . -~
sequence, and ~,uluLu~hllàlely 85% homology to ANT 70 sequence.
S~ ,, r~l tests indicate that HIV-I and HIV-2 and their emerging subtypes share multiple common epitopes in their core antigens, although 3 0 their envelope ~l~.,u~u~uL~ s are much less cross-react*e. F. Clavel, ~gp~a.
This limited cross-reactivity of the envelope antigens may explain the failure of most currently-available serological assays for HIV-1 to react with certain serâfrom individuals with antibody to HIV-2 and with emerging HIV subtypes. F.

W095133206 P~ '01?~

--~3 --Denis et al., J. (~lin Mirro. 26:1000-1004 (1988). A ~u~ l~ally available assay fûr HIV-VHIV-2 antibûdy, recently available frûm Abbott La'uvl~Lu.;c6, Abbott Park, IL 60064, A~Dci~n~t~d as the Abbott HIVAB@) HIV-l/HIV-2 (rDNA) EIA, - uses recAmhinnnt antigens C:UII ~I~.. ~Ain~ to the twû viral proteins, HIV-1 envelope and HIV-2 envelope. The use of these l~.. l.;.. ~.. l antigens allows for the improved detection of anti-HIV-1 and/or anti-HIV-2 ~---.~i~;..;..~ test samples, while ...;..;...~ non-specific reactions largely due to cross reactions with whole virus or viral lysate. The use of at least one l~
HIV protein to detect HIV antibody in a test sample with the use of labeled 1~.. 1.;.. 1 HIV antigens is described in the parent patent ArrAlir5lt;r~nR
previously ihl~ull ul~L~d herein by reference.
Tl.,..."l.A r~ yD have been previougly described to detect the presence of nntihoAiPc to the HIV virus in human sera used an enzyme linked ;.... A~... I,tl~l assay (ELISA) method employing as the antigen reagent 15 ill_Li~Led whole virus obtained from a cell line capable of virus replication .
~l.Re~...... ~.. l ;.. ~A~ yD for HIV describe the use therein of polypeptide 8e~ DC obtained by rernmhinS-nt DNA mDt,'~ "y. See Cabradilla, et al., Bio/T~l -' Oy, 4: 128-133 (1986). However, such previously described yD lack 6t:~D;Li~/iLy and D,u~rl~;Ly which could per_it test 20 samples, such as blood products C---.~A;..;..g virus to escape detection and thereby potentially result in the infection of those patients receiving, for example blood products. The lack of specificity (i.e., false positives) in such ;I,,,,,l~lIA.. yD i8 often due to nnncrerific binding of imml1nnglnbl-1inc to cellular protein in the viral lysates, or in the case of recnr~hin~nt antigens, lack 2 5 of D~r~Ly may be caused by shared epitopes with viruses unrelated to AIDS.
For e~ample, Gallaher, Cell, 50: 327-328 (1987) has reported that a region of HIV-1 gp41 shares an antigenic region with the I~D~ Luly syncytial virus and with the measles virus F1 oly.,u,ul ~Lt:il.. Thus, even highly purified 1~ ~..~.; ..~.~l HIV polypeptides could potentially be rDRrnnCihlP for false 3 0 positives. In either case, such false positives could result in the mic~ingnAcic of AIDS.
Based on the nllr1Dot;de analysis of the viral genome the HIV genomic RNA encodes (h~ at the 5' end):

W0 95133206 2 1 9 1 4 3 0 r~ 21 ~
(i) a ~ gene PYtPnflin~ between nllrl~ot~ c 310 to 1869 and encoding for the internal structure core or mlrlDor rcifl proteins including p24, the most antigenic core protein;
(ii) a ~QI gene extending between ~ t~ 1,629 to 4,673 and S encoding for the enzyme, reverse (~ ce; and (iii) an env gene ~ between nIlrlP~ c 5,781 to 8,369 and encoding for the envelope ~ u~ult ill including gp41, the most antigenic envelope protein. Ratner et al., ~aha~ 313:277-284 (1985).
One of the challenges faced by today's medical r~ y is the 10 protection of blood products from ~ n by HrV, which has been found in blood products (as well as other human body fluids), and which reportedly has been ~ d in the blood supply. Several assays are available to date, including the assay described in U.S. Patent No. 4,520,113 to Gallo et al.
Also, other assays which can detect HIV arltigen or HIV antibody are 15 known. Such assays include the anti-HIV-1~IIV-2 assay described hereinabove and disclosed in the ULC ~;UUDIy ,~r~.C..~e~ patent ,~ .1;rAI.....
ul~ulc.l~d herein by reference, as well as those taught by U.S. Patent No.

4,748,110 to D. Paul, U.S. Patent No. 4,983,529 to J. Stewart et al., and U.S.
Patent Arrlir .~;~m Serial No. 07/204,798., all of which enJoy common UW~ lDIliy and are illc~l~ul~L~d herein by reference. However, all known federally-approved assays for detection of HIV antigen analyte or HIV antibody analy~e are only capable of A~ r~ly detecting either HIV antigen analyte or HIV antibody analyte in a test sample. No known Culll~ lly available, federally-approved assay is available for detection of both HIV antigen analyte 2 5 andlor HIV antibody analyte in a single assay using a test sample.
The detection of more than one analyte in a test sample usually involves the separate detection of each analyte in a separate assay. Such detection methods have been preferred since they allow for stringent quality ~6DUlallCe dPt~rmin~tiAnc to be pelrul,.led for each analyte to be tested.
3 0 Advances in medicine have brought a rerognitiAn of new markers for many diseases and clinical crn~liti~nAc, along with the demand for clinical tests for these markers. Laboratories are faced with the problem of providing ,r.L ~lg amounts of tests in a timely manner while ~ to keep costs ~ W095/33206 - 21 91 430 r~
, -~5 -down. For example, the testing ~6~lui~t~Lu~Ls of blood banks have increased dramatically due to the addition of Human T-T,PllkPmiA Virus Type 1 (HTLV-l), HIV and Hepatitis C Virus (HCV) to the panel of agent6 tested in these lal u.d~,u.;~ on donor blood for the presence of or exposure to these agents.
One possible solution to reducing the laboratory workload brought about as a result of testing . ~u ui. ~u~x, especially in blood banks, is to find ways to combine assays. However, .. l.;.. ;.. ~ assays v~ithout ~UlulJIUUPil~ulg their individual p~.r~l Uol~ standards is difficult and more il~ull~llily, the problems involved in mAnllfA~t~lrin~ and quality control, can be 10 insu~Luuu~ ble.
Assays to cim~ .xly detect more than one analyte in a test sample would be adv~ u .c since the time involved in detecting more than one analyte in the test sample would diminish cnnr;~lPr~hly, and the cost of each assay would be lowered since less technical ti~e, reagents, and P.l..;l. l ..
15 would be required to perform such an assay.
For example, U.S. Patent No. 4,315,907 to Fridlender et al. teaches a h~k,~u~t:ueuu~ specific binding assay system wherein cPpArAti~n of bound-species from a free-species form of the labeled reagent occurs.
U.S. Patent No. 4,378,344 and EP 027008 to Zahradnik et al. teach a solid 2 0 phase device for d,~ l LUilUllg the presence of each analyte ..l..; ~ a receptacle and an insert wherein the presence of each analyte is ~ ...;-.Pd by the claimed assay method.
Great Britain Patent No. 2188418 teaches an assay tray assembly having reaction wells each with openings in the top surface from which a ~UI~; '- iS
2 5 extended and wherein the inner surface of each well sidewall and the outer surface of each projection may be; ~ d X;,--~ ,Po~ly for detecting two or more specific ~ c present in a specimen which has been uu~ludu~d into the reaction wells.
EPA No. 0 351248 to applicant ~)EXX Corporation discloses a 3 0 ximl1ltAnPollc immllnnAccAy for feline viruses or HIV in which an antigen andlor antibody member of a single binding pair are flPt~rtql,l~ Also, U. S.
Patent No. 5,039,604 to Papsidero teache6 an immlln.lACxAy which cimllltAnPollcly detects two HTLV or HIV ~ntiholliPx by adding two different W095/33206 2 1 9 1 430 Y~ o~

antigens and then a single labeled antibody w_ich i6 reactive with both antigens. In addition, U.S. Patent No. 4,870,003 to Kortright et al. discloses asolid phase immlln~cR~y for detection of an antigen and/or antibody of a single binding pair utilizing an antigen "spike" of il~ iVCLi~ arltigen.
Also, the detection of one or more analytes using two or more solid phases is the subject matter of co-pending U. S. Patent Alu,ulic~ iu~ Serial No.574,821, which enJoys common ownership and is i~,ul~uL~ d herein by reference.
Factors which have been identified for the sllr~PRcfill development of 0 Rimlllf~nPOllR assays are that the two assays to be p~, rl,. --Pd cimllllnll~ol~l~ly must have the same sample volumes, identical inrllh~fi~n times and identicsl cut-off ~ fi.~nR Such a Rimlllt InP~-lc assay also should be capable of being separately quality controlled for each analyte, both at the ...~-. r~ . and at t_e laboratory using the assay, to ensure the ~ D~tivily~ specificity and 1 5 reproducibility of the;, . . . ", ., ..~n: ~ ~y~
It tberefore would be adv,...l~G~u~ to provide an assay wherein the presence of more than one HIV subtype analyte, i.e., ~ 1 and/or HIV-2 1~nflhorliPR or antigens could be Rimlll~ ly detected, yet each separate analyte to be detected could be illLYid~r.lly quality controlled. Such an assay 2 0 would be an h~ u~ llt over ot_er known assays since the ~cimlllt~nP~.lc tlPi~l",;llnl.;....R of the presence of either HIV antigen analyte and/or HIV
antibody analyte would be p~ ru ....~d in one well, sPr~r~t;~n of solid phase "~ would not be required if more t_an one solid phase was utilized, and the assay could be quality Culltll 11_' for individual analytes which were to 25 be detected in the Rimlllf~nPollR assay.
Fulll~ uùl.3, it would be Elv...~G~euu~ to provide a l;r.G.. -l:G assay employing synthetic HIV peptide antigens having unique and highly conserved epitopes of the HIV virus. Such synthetic HIV antigens are beneficial because of the relative ease and lower cost with which they can be prepared, and more 3 0 hllluul L~Uy, because of the reduced risk of obtaining false positives due to impurities or presence of shared epitopes with viral proteins not related to AIDS. Most ilU,UUI i~ll~ly, synthetic peptide HIV antigens would provide increased s~ ,iviiy in detecting emerging HIV subtypes. Thus it would be ~ W095133206 21 91 430 r~l,u~

tldvn~ ul~A to proYide a ~u~dl~;ally available assay employing synthetic HIV peptide antigens to rAnA;~P~ltly and reliably detect HIV genotypes, such tlS subtype O AAnhhrJriiP~ in test samples. The lack of such an as6ay presents amajor challenge in worldwide detection of HIV detectiûn in blood and other 5biological samples.
Sllmm~ry nf thP InvPnt;An According to the present invention, an ;.. ,.. n^AA~y for detecting one or more Hn ~t,..uLy,ues is provided c~ , ;A.,.e the steps of rnnt~rt;n~ a test 10 sample with ~ 7.;~ t antigens (h~l~hl~, "capture reagents") of (a) HIV-2 gp36 env, (b) HIV-1 gp41 enY, and (c) HIV-l p24 gag-immnhili7pd on one or more sûlid materials to form a first mixture. The first mixture is ;....~ d for a time and under rnnriit;~nA sufficient to form HIV env antibûdy/ l~ .. l.. ~.. i antigen rnmpl~YPA and Hn ~ae antib~dy/.~ -- .hin~lnt antigen, -..~ and5 the resulting rc~mrl^yQA are contacted with (a) an improved indicator reagent a synthetic antigenic site-directed cyclic HIV-2 env peptide labeled with a signal gPnpr~tin~ c ..l -~ i in the presence ûr absence of a AIII HIV-2 env peptide labeled with a sgnal ~ li--g ~.................. I.u.. ri (b) an improved indicator reagent r~ g a synthetic antigenic site-directed 2 0 cyclic HIV-1 env antigen labeled with a signal generating -- ..I~v ~ in the presence or absence of a ,. ~..."l.;"~.,t HIV-1 env antigen labeled with a signal r.~il.g c~.llp~ul.d, and (c) a ~ r.;~ t HIV gaL antigen labeled with a signal ~ g ~ u~ r~ to form a second mixture. The second mixture is inrllh~tPd for a time and under ~ A sufficient to form HIVantibody/
2 5 capture reagent/indicator reagent; , 1 - The presence of A ~ C~ AA to one or more HIV ~ u~y,u~s, particularly, HIV-1 env ~ -o.l;Ps, HIV-2 ~ iP~A, HIV subtype O ~ntihorii~ps~ and/or HIV ~ ~nhhoriiPA in the test sample is riptDrminpd by detecting the total signal generated by the, , l Improved sensitivity of the assay of the present invention was found 3 0 when at least one indicator reagent ~ ;r n~ a sy-nthetic cyclic site-directed HIV ~ peptide having an i~ UllUlt;~.~,l.iVe specificity cht~ ib~ic of an immllnnd.. ;.. n.ll region of gp36 of HIV-2 or gp41 of HIV-l. In particular, the present inYention .,,,~I.C,~?rily and bul~ul~bill~;ly found that the inclusion of a .

WO95/33206 2 19 1430 r~ ol~7l HIV-2 gp36 synthetic peptide in the assay of the present invention oig,nifirsntly reduced the Ltlu~uG~ of HIV-2 false positives as compared to HIV
;".. ,.,.ns1 . ~ D , 1 1illg only 1~ ~.. l.;.~,.. f antigen in the indicator reagent.
Further, the present invention ~ -lly and sullu.;Dhlf,ly found that the

5 inclusion of a HIV-1 gp41 synthetic peptide in the assay of the present invention increased the ~_.IDi iVi y of the assay to HIV subtype 0 in test samples as compared to HIV immllnAsAesys; ~ 1 ~hlg only recnnnhins~nt antigen in the indicator reagent.
Test kits for pt:~Ul L dllg the assays of the present invention also are l O provided.
DPt~ilP~f DPcf~rintion nf t.hP Invention A CR9 v Form Atg Tthe detection of HIV antigen analyte and/or HIV-1 and HIV-2 antibody l 5 analy es in a test sample of the present inYention can be p r. .. F d according to various h~.l.lG~;GIleuuD and hG . c,~ ùuS assay formats known in the art where the various reagent additions as described above can be pG.~u.G~F d FiimllltAnPû~lcly or APqllPntislly. The assay of the present invention is bly in an immllnA,sccsy format, although the present invention is not 2 0 limited to i_munoreactive assays. For example, any assay utilizing specific binding members can be ~_. f.,.... ~1 A "specific binding member," as used herein, is a member of a specific binding pair. That is, two different mnlP~llPRwhere one of the mnlPclllPg through chemical or physical means ArerifirAlly binds to the second molecule. Therefore, in addition to antigen and antibody 2 5 specific binding pairs of common; ~ - - nAAA~ ~ D~ other specific binding pairs can include biotin and avidin, carbohydrates and lectins, ~ , 1 ~
nllrlPot~i~p S~1U- ~ ~, effector and receptor mnl~ 1PC, cofactors and enzymes, ~nzyme inhibitors and enzymes, and the like. Fu~ L...~G, specific binding pairs can include members that are analogs of the original specific binding 3 0 member, for example, an analyte-analog. Immunu.c~ivG specific binding members include antigens, antigen fragments; Antihor.iPc and antibody fragments, both mnnnrlnn~l and polyclonal; and rnmrlpyps thereof, including those formed by recnmhinAnt DNA methods.

~ W09S/33206 2 1 9 1 430 _9 "Analyte," as used herein, is the s l ,- ~ ~ to be detected which may be present in the test sample The analyte can be any ~ 17~ for which there exists a naturally occurring specific binding member (such as, an antibody, and more Q~p~Prifir~lly an HIV antibody), or for which a specific binding 5 member can be prepared Thus, an arlalyte is a sl~hQ~onrp that can bind to one or more specific binding members in an assay. "Analyte" also includes any antigenic s- -l ~ r, haptens, ~ ~ ihG l;Pr~, and rnmhin~t;~nQ~ thereof As a member of a specific binding pair, the analyte can be detected by means of naturally occurring specific binding partners (pairs) such as the use of0 intrinsic factor protein in the capture and/or indicator reagents for the of vitamin B12~ or the use of a lectin in t_e capture andlor indicator reagents for the ~ - ",;"~1:..., of a l~bG~yd~ rc. The analyte can include a protein, a peptide, an amino acid, a hormone, a steroid, a vit~min, a drug i-.~lu~lu-g those ~ d~ for ll-~ .~cu~ic purposes as well as those 15 ~ cd for illicit purposes, a 1,~ ; , a virus, and mPt~hnlitPc of or slntihoAiPc to any of the above sllhr~snrPc The test sample can be a m~mm~ n biological fluid such as whole blood or whole blood ~ including rèd blood ce~ls, white blood cells including ly.~ i,e or ly.ll,uhO~ I c subset preparations, platelets, serum and 20 plasma; ascites; saliva; stool6; cc.cb.v,,u;-~l fluid; urine; sputum; trachael aspirates and other rnna~;tllPntC of the body which may contain or be su6pected Of ~ lln;ll;l g the analyte(s) of interest The test sample also can be a culturefluid ~u,uc~aL~L~, or a s lgpPnr;~ln of cultured cells Mamm~lc whose body fluids can be assayed for HIV antigen analyte or HIV antibody analyte 2 5 &~ L..g to the present invention include humans and primates, as well as other ~ la who are sllcpected of c ~ these analytes of interest It also is ~ ",~ . d that non-biological fluid samples can be utilized The methods of the present invention are advn ~ u ~ly used in solid phase hcic.u~c..eous binding assays which include both sandwich and 3 0 c.,.--~ ., assay methods Hetdrogeneous binding assay t~rhniquPa involve the use of a solid phase material to which a member of the binding reaction becomes bound Prior to detecting the label which indicates the presence or amount of analyte in the test sample, the immrhili7Pd reaction rnmrArlPnt is W0 95/33206 2 1 9 1 4 3 0 r~

separated from excess sample and assay reagents by removing the solid phase from the reaction mixture.
In a solid phase sandwich assay, a capture reagent as defined below typically involves a capture binding mernber which has been bound to a solid 5 phase material. For e~ample, the specific binding member can be an immnhili7.Pd antibody which will bind to an antigen-analyte in the test sample, or the specific binding member can be an immnhili7Pd antigen which will bind to an antibody-analyte in the test sample. The capture reagent is contacted to atest sample, s~lRperted of c .~ ;..; .~ the analyte, and to an indicator reagent o ~ e a second specific binding member that has been labeled; for e~ample, a labeled anti-analyt~ artibody or labeled antigen. The reagents can be mixed Riml11t~nP~ lRly or added seqllpntislly~ either singly or in rnnnhin~tinn A binding reaction results in the fnTm~t;~n of a capture reagent~analyte/indicator reagent complex. The assay can also comprise t,he 15 step of S~u~ g the resultant complex from the excess reagents and test sample. The complex retained on the solid phase material is detected by e the solid phase for the indicator reagent. E analyte is present in the sample, then label will be present on the solid phase material. The amount of label which becomes ~ with the solid phase is directly ~ ,UUI liUllal to 2 0 the amount of analyte in the sample.
The assays of the present invention can be carried out using any of the sandwich assay formats, including the forward, reverse and Rim11lt~nP~lR
..P~ Typically, a forward assay involves the contact of the test sample to the capture reagent followed by an; . .~ period which is in turn followed 2 5 by the addition of the indicator reagent. A reverse assay involves the addition of the indicator reagent to the test sample followed by the addition of the capturereagent after an inrl1h~tinn period. A Rim111t~nPouR assay involves a single ; "- 1 .,. 1.;. . step as the capture reagent and indicator reagent are both contacted to the test sample at the same time.
3 0 Cu~lJt liLive assays can also be carried out using the antigens of the present invention. In a solid phase cu..-,u~ilivt: assay, the capture reagent again typically involves a capture binding member which has been affixed to a solid phase material and which is c~ntacted with both test sample and an ~ wo9sJ33~06 21 91 4 30 P~ - at~2l indicator reagent. The indicator reagent, however, can be formed from an analyte or analyte-analog which has been ~ d with a label. A binding reaction occur6 and results in the r....~ . of rnmpl^YPC of (1) imml ~hili7ad capture .~4_.~ ulalyte complex and (2) ;.. -~ d capture 5 reagentlindicator reagent complex. AlLelu~lliv~,ly, the immr~ 7Pd specific binding member can be an analyte or analyte-analog with which the test sample analyte competes for binding to the indicator reagent. In the ~u uut ~i~iv~ assay, the amount of label which becomes Ir 1 or ~ d with the solid phase is inversely related to the amount of analyte in the sample. Thus, a 10 positive test sample will generate a decrease in signal.
In these binding assays, the presence or amount of the analyte in the test sample is usually rl~ ..;,.Pd by detecting the presence or amoumt of the label which has become ~ d with the solid phase, although free or unbound indicator reagent may also be detected. In the ~,U~ Ut ~ assay, the more 1 5 analyte present in the test sample the lower the amoumt of label present on the solid phase. In the sandwich assay, the more analyte pre6ent in the sample the greater the amount of label present on the solid phase.
TndirQtnr RP,oFPnt.~
2 0 The indicator reagents of the present invention comprise a specific binding member of each analyte -..j~ d to a signel generating ~
(label). Each indicator reagent produces a ~' ~1 signal at a level relative to the amoumt of the analyte in the test sample. In a preferred ~,..I.G.l.,....1 each indicator reagent, while ~nmrricinE a specific binding member of a different 2 5 analyte, is ~v.lju~;a~ed to the same signal k~ e ~ I u- ll~. which is capable of ~ illg a flPtP~ol-l^ signal. In general, the indicator reagent is detected or measured after it is captured by the capture reagent. In the presentinvention, the total signal ~ d by the indicator reagent(s) indicates the presence of one or more of the analytes in the test sample. It is ~.... 1~...l.l 1.~.l 3 0 that different signal ~ i..g rrmrolln~lC can be utilized in the practice of the present invention. Thus, for example, different lluoldsc~lli cnmrollntl~ could be utilized as the signal ~tllo.~l~illg compounds, one for each indicator reagent, and detection could be rl~ ~....;..Pd by reading at different wavelengths. Or, a W095/33206 ~ r~ u,.,~..'ll1~1 2~9~430 ,2 short-lived rhPmil~nninPærpnt ~,v."~ such as an acridinium or rh~....,.l.l.. ;--~illm compound and a long-lived rhP~nilllminPcr~Pnt..- -l~v ~
such as a dioxetane can be utilized to generate 6ignals at diL;ferent times for different analytes. Method6 which detail the use of two or more 5 rhPmilllminPgc~nt ~..."~ .."~ which are capable of ~LL~laLil~g signals at different times are the subject matter of co-pending patent ~rrlir~;nn U.S.
Serial No. 636,038, which enjoys common v...~ iAu and is iLI~v.yulaL.,d herein by reference. Acridinium and rhPn~nthrirlinillm cuLlL~,~lds are described in co-pending U. S. patent Arrlir~ti~ln Serial No. 07/271,763 filed June 10 23, 1989, which enjoyæ common ownership and is ill~vl~JvLakd herein by reference.
In addition to being either an antigen or an antibody member of a specific binding pair, the specific binding member of the indicator reagent can be a member of any 6pecific binding pair, including either biûtin or avidin, a 1 5 carbohydrate or a lectin, a cn~n~ A ' y nucleotide sequence, an effector or a receptor molecule, an enzy-me cofactor or an enzyme, an enzy~ne inhibitor or an enzyme, and the like. An ilL l~Lullvlea~Li~e 6pecific binding member can be an antibody, an antigen, or an antibvuly/ ILLi~;t ll complex that is capable of binding either to the analyte as in a sandwich assay, to the capture reagent as 2 0 in a cuL..~LiLi~ assay, or to the ancillary specific binding member as in anindirect assay. If an antibody is u6ed, it can be a mnnr,rlnn~l antibody, a polyclonal antibody, an antibody fr~EmPntl a rPcnmhin~nt. antibody, a mixture thereof, or a mixture of an antibody and other specific binding members. The details of the preparation of such ~ntiho-liPe amd their suitability for use a6 2 5 specific binding members are well known to those in the art.
In one ~...hG.l;...~..l. the HIV antigen of the inGlicator reagent is a synthetically- or recnmhin~nt.ly-produced antigen capable of binding to an j~"""".n~ l region of the core and env proteins of HIV-I and~or the ~nY
region of HIV-2. In a prefered PmhoAimPnt. the antigen used in the HIV-2 3 0 indicator reagent is a synthetically produced cyclic peptide cu~ -l;..E to an imm7lnn~lnmin~nt. region of gp36 of HIV-2 and is ch~LIa~LtL;~5dd by its ability to hluLLuLlùl~z~,L with ~ntihotliPg induced by HIV-2. The synthetic peptide is frombetween about 1û amino acids to about 5û amino acids in leng~ and includes ~ wossl332~6 21 91 430 r~ UAI~7~
- 1.3 -the amino acid residue sequence CycAla-ph~ArgGAm-val~s(-cAFRQvc-) in which the two cysteines have been oxidized to their disulfide cyclic form. Preferred synthetic peptides include, but are not limited to:
(a)Lys-Asp~n-AlaGLA.-LAd~AsnSer-Tr~GAIy-7 A ~ A~"GLA~-val-cys-l~s-Thr~(pEprmEI);
(b ) Arg-Val-~r-Ala-De-GAlu-Lys-Tyr-LA~Lys-A~Gln-Ala-Gln-Ler~AA.ASer-Trp GAly-C~,, Al- Fl~Arg-Gln-Val-Cy8-Hi9-Thr;OE~lDEll);and (c) VA~-thr-Ala-Ile-Glu-Lyfi-Tyr-Leu Glu-Asp-Gln-Ala-Arg-Leu-Asn-Ser-Trp-Gly Cys-Ala-Phe-Arg-Gln-Val-Cys (PEP'lA-~E m).
In another prefered ~mho~li.omt~nt) the antigen used in the HIV-l indicator reagent i6 a synthetically produced cyclic peptide ~ AP'....~ to an immllnr~l.,...i,..."l. region of gp41 and is characterized by its ability to immunoreact with ~ntiho~ A induced by HIV-l and other HIV subtypes. The synthetic peptide is from between about 10 amino acids to about 50 amino acids 2 0 in length and includes the amino acid residue sequence -CysSer-GAIy-Ly~ Le~ne~
CSGKLIC-) wherein the cysteine residues are oxidized to their disulfide cyclic form. Preferred HIV-l synthetic peptides include, but are not limited to:
2 5 a~]n~ln-LAcu-Leu~-l~Gly~-6~M~Ly~Leu~fAThr wherein~s Arg-Ile LAcu-A]a-Val~lu-Arg-Ty-lA-LAcu-Ly6-Asi. orArg-Ile-Lcu-Ala-Val-Glu-Arg-T~ir-Leu~n-Asn;
,,is Glyor Ser;
~ is ne or Leu;
3 0 ~ is Ser or Lys;
is Ile or Val; and Af is Thr or Tyr.
Most preferred HIV-l peptides include but are not limited to:
- (a)Arg-~e-Leu-Ala-Val-GAIu-Arg-Tyr-Le~Lys~AspGln{~ln-LAd~Le.}Cdy-ne,TrpGly-CysSer-GAIy-Lys-Leu-ne~Thr-Thr,(PEPTn)E IV);
4 0 (b)Lys-lle-LeDtAla-Val-GAIu-Arg-Tyr-LA~Lys-A~G~n~n-LAd~LAd}~y-ll~Tr~Gly-

6 , ~ .'C I 121 Cy~&r-Cly-LyE La~D~Cy~Thr-Thr(PEPT~lE V);
(c)Ly~La~Ala-Val Glu AIg-Tyr-LEu-Lys As~G~n{~ln-L~#La~Gly-D~T~ly-Cys Ser-G~y-Ly~La~D~Cy~Thr-Thr(PEPTmE VI);
(d~ Ly~Ala-Val-Gl~A~-Tyr-L~Ly~A~Gln4~1n-Lsu L~y-D~TrpC31y-CyE&r-Gly-Ly~LE~DecYEThr-Thr~DEv~
(e) Ly~Val-Ca~-Tyr-La~Ly~ AapGln Gln-Leu La~ly-D~TrpGly-C~Ser-(~y-Ly~L~DE-Cy~Thr-Thr(PEPl~lDE Vl:~;
1 5 - - --- ~
(f)Lyn (~Arg-Tyr-LE~Ly~AspGin Gln-Le~La~Gly-D~T~(~y-(~&r~y-Ly~LE~D~(~Thr-Thr, I ( PEPI'IDE
n~
2 0 (g) LyE-A~-Tyr-L~Ly~ Asp,G~n-G~n-Leu La~Gly-D~T~Gly-Cys~r Gly-Ly~Leu De{~Thr-Thr;
OE~DE X~
~) Ly~Tyr-Leu~Ly~A~;pGln~n-La~LE~Cay-ll~T~G~y-Cy~Ser{~ly-LyE-L~D~}Thr-Thr, 1 ~PEPlIDEX~
OLy~L~}Ly~Asp~n Gin-La~L~Gly-D~T~Gly~br Giy-LyE LE~DE-CyE-Thr-Thr;and (j)Ly~Ly~ A~n{~n-LE~L~y-D~T~G~y-(~ Gly-LyE-L~D~Cy~Thr-Thr.
3 0 1 1 (PEPl'IDE ~m) It should be lln~1pret~od that a peptide of the present invention need not be identical to the specific amino acid ~P~ , presented above 60 long as the peptide contains the sequence CAFRQVC or CSGKLIC or homologs in their 3 5 oxidized form thereof and are able to bind with ~ntiho~iPc induced by genotypes of HIV. Moreover, the synthetic peptide of the present invention can be subject to various changes, such as insertions and deletions, and 5llhctihlt;~nC of one amino acid for another, either cu~ ,l Vd~ , or n----- ~ . Y~ in nature.
The peptides of this invention are prepared using a variety ûf known 4 0 methods. Conventional solid phase synthesis is most lul~f~l_bly emplûyed as is described in B. Erickson, et aZ. Solid-phase peptide synthesis, The Pro~Pinc VolII, 3rd ed. Af J1-1Pm;C Press, New York, New York (1976) and E. Atherton, et al.Solid Phase PeDtide Svnthesis . A Practical Al7~r~rh IRL Press, Oxford, (198g). IIowever, other well known methods of peptide synthesis may also be ~ W095133206 2 1 9 1 430 P~ 21 used. The resin support is any suitable resin conventionally employed in the art for solid phase preparation of peptides, preferably p-methylbG~ lu,.y~lcoholpoly~Ly.G..e and p-methylbenzydrlamine resin. Following the coupling of the first protected amino acid to the resin support, the amino ~luL~,Lillg group is 5 removed by standard methods conventionally employed in the art of solid phase peptide synthesis. After removal of the amino protecting group, remaining a-amino protected and, if nc~E__-y, side chain protected amino acids are coupled, seq Pnt;slly, in the desired order to obtsin the product. If required,cyclization between two cysteines is ~ (1 by diluting the crude peptide into an oxidizing Gllvi.u.. el~L that promotes disulfide bond fnrrnAt;~n The cyclized peptide is then purified and Iyophilized for storage.
Cyclic peptides of this invention (Peptides I- XIII) are prepared by the direct oxidative iUll~ iUll of protected or ull~.Lu~.Gd SH-groups to a disulfide bond following techniques generally known in the art of peptide synthesis. The 15 preferred method involves the direct oxidation of free SH-groups with p~ .. Lll;.~cu ide. Such cyclic peptides are believed to assume more rigid .r....~l:on with may favor binding to HIV Antiho~liPg The selection of an appropriate coupling reagent follows ~ ~ h lighPd art.
For instance, suitable coupling reagents are N,N'-diisopropylc&ll,~ lihllide or 2 0 N,N'-L~,IûllGAylcarboiimide (DCC) either alone or preferably in the presenceof 1-hYd~U~YbG11~U; - '~ Another useful coupling ,U1U~GdU~G makes use of performed by.. ,~l ;. ~1 anhydrides of protected amino acids.
The label of the indicator reagent is capable of ~.lG-~.Li-lg a measurable signal ~ L1_ by e~ternal means. The labels ~ d include, but are 2 5 not intended to be limited to CI~1UU1O~G11~ catalysts such as enzymes for e=ple, horseradish pero~idase, alkaline rl~ ce~ and B-~P1A(~ lh~;
minPgCPnt cnmroln~c such ag fluoregcein and .l,n~lh.,...,~;
rh~smil11min~5cPnt. compounds such as acridinium compounds, rhPnAnthridinium compounds and dioxetane compounds; radioactive 3 0 elements; and direct visual labels. The selection of a particular label is not critical, but it will be capable of producing a signal either by itself or in conjunction with one or more A~l~liti~mAl sl1hr~snrPg A variety of different woss/33206 21 91 430 r~ ,.,J.~

indicator reagents can be formed by varying either the label or the specific binding member.
Preferably Peptides I - XIII of the instant invention are labeled with horseradi6h p~-u~ida3~ (HRP0) using conventional Schiff base chemistry as described by Nakane, P., e~ ~1., J. ~T;~ .. Cvtochem. 22: 1084-1091(1974).
Preferably, aldehyde groups on HRP0 are ~HII ~ by oxidation of the C611VU.~V~ H residues with sodium ~ ;V~ These reactive aldehyde groups are allowed to interact with amino groups on the peptide, which are preferably at the amino terminus or the the e-amino group of Iysine. The 1 0 Schiff s base that forms is s11hseqll~ntly stabilized upon reduction with sodium borohydride and the resulting conjugate is stored until use.
C~llre R~ nt~
The capture reagents of the present Invention comprise a specific 15 binding member for each of the analytes of interest which are attached to at least one solid phase and which are lml~h~l~(l Although the capture reagent is specific for the analyte as in a sandwich assay, it can be specific for indicator reagent or analyte in a cv...,ut ~iiivt, assay, or for an ancillary specific bindir~g member, which itself is specific for the analyte, as in an indirect assay. The 2 0 capture reagent can be directly or indirectly bound to a solid phase material before the p~. I'o~ of the assay or during the p ~ re of the assay, thereby enabling the separation of immnhili7Pd rnmrl - from the test sample. This ~ l can be achieved, for e~ample, by coating the specific binding member onto the solid phases by absorption or covalent couplirlg.
2 ~ ~oating methods, and other known means of .~ ..t, are known to those in the art.
The specific binding member of the capture reagent can be any molecule capable of sperifi~lly binding with another molecule. The specific binding member of the capture reagent can be an immunoreact*e ~ v~ such as 3 0 an antibody, antigen, or antibody/antigen comple~. If an antibody is used, it can be a mnnr,rlnn511 antibody, a polyclonal antibody, an antibody fragment, a ~ecnmhin~nt antibody, a mixture thereof, or a mi~ture of an antibody and other specific binding members.
.

W095~33~06 2 1 9 1 4 30 ~ u~ .~4~7r The "solid phade" is not critical and can be selected by one skilled in the art. Thus, latex particles, microparticles, magnetic or non-magnetic beadd and mi~.uy~lL;dc~, membranes, plastic tubes, walls of wells of reaction trays, glass or silicon chips and tar~ned sheep red blood cells are all suitable 1 - ~ Suitable methods for immnhili7 nE~ capture reagents on solid phases include iorlic, hydrophobic, covalent interactions, and the like. In one exampleof the present invention, 60-well pol.~dLyl~le reaction trays and V4 inch puly~Lyl~..e beads are utilized, while in another example, a 96-well reaction tray is the only solid phase utilized. It is r~nntPmrl qtpd that all solid phases be I O present during the u,~ m of signal, thus .-1;...;..,.l;..~ the need to separate dolid phases for detection of signal.
A "solid phase", as used herein, refers to any material which is insoluble, or can be made insoluble by a ~"h6~ ..l reaction. The solid phase can be chosen for its intrinsic ability to attract and i.. ~l-;l~ P the capture15 reagent. All~l~lhLi~,ly, the solid phase can retain an Qrlflit;nnsll receptor which has the ability to attract and immnhili7P the capture reagent. The ~-litl~ln~l receptor can include a charged ....1.~ that is o~.~.ur,;L~ly charged with respect to the capture reagent itself or to a charged 511h~qnrP ~ d to the capture reagent. Ad yet another alternative, the receptor molecule can be any 2 0 specific binding member which is immnh-ili7pd upon (attached to) the solid phase and which has the ability to immnhili7P the capture reagent through a specific binding reaction. The receptor molecule enables the indirect binding ofthe capture reagent to a solid phase material before the p~lrUllU~e of the assay or during the p rv. ..u.~. e of the assay. The solid phase thus can be a 2 5 plastic, d~ Li,.ed plastic, magnetic or non-magnetic metal, glass or siliconsurface of a test tube, microtiter well, sheet, bead, ~lu~ualLidc, cbip, and other configurations known to tho6e of ordinary skill in the art.
It is rnntpmrlslt~d and within the scope of the invention that the solid phase also can comprise any suitable poroud material with sufficient porosity 3 0 to allow access by detection qntiho~liP~ and a suitable surface affinity to bind antigens. Mic.u~vlu.ls ~LIur,~ul~s are generally preferred, but materials with gel structure in the hydrated state may be used as well. Such useful solid supports include but are not intended to be limited to natural polymeric W0 95/33206 ., . r~ 5 carbohydrates and their 6ynth~tirJllly modified, cross-linked or suh~;tlltPd derivatives, such as agar, agarose, cross-linked alginic acid, s~lh~;b-tPd and cross-linked guar gums, cellulose esters, especially with nitric acid and ~6lbu~ylic acids, mixed cellulose esters, and cellulose ethers; natural polymers5 rAntslinine nitrogen, such as proteins and dc liYc~ 3~ including cross-lirlkedor modified gelatins; natural llydlu~lb~l~ polymers, such as latex and rubber;
synthetic polymers which may be prepared with suitably porous Dl.l U~l.UI t~
such as vinyl polymers, including pol.~,llylt.,a, pûly,u~u,uyl~lle, pûly~yl.,..e, pûlyvill~l~Lloride, polyvilly'a ' ' and its partially Ly~lul.~ d~;v. lAi~
10 pol~ l~des, polymethacrylates, ~U,UUIylll(~.D and l,~I,UUIy.' _.D of the above polycnnA~n~t~, such as polyesters, polyamides, and other polymers, such as polyurethanes or polyepoxides; porous inorganic materials such as sulfates or I,ullal~ of alkaline earth metals and .. ~ ;.. , including barium sulfate, calcium sulfate, calcium carbonate, silicates of alkali and alkaline 15 earth metals, aluminum and l~h~ l; and ~ minllm 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 cu,uoly~ .c, obtainedby init;oli7in~ poly.... ~ n of synthetic polymers on a pre^existing natural 2 0 polymer. All of these materials may be used in suitable shapes, such as films, sheets, or plates, or they may be coated onto or bonded or l~min~tPd to ul;~ie inert carriers, such as paper, glass, plastic films, or fabrics.
The porous structure of nitrocellulose has e~cellent absorption and adsorption qualities for a wide variety of reagents including .. n~l~.. hl 2 5 ~ntih~iP~ Nylon also possesses sirnilar ~ and also is suitable.
It is rnntsmrlstpd that such porous solid supports described hereinabove are ~I~.c~.ably in the form of sheets of thickness from about 0.01 to 0.6 mm, preferably about 0.1 mm. The pore size may vary ~-vithin wide limits, and is r~..bly from about 0.025 to 15 microns, especially from about 0.15 to 15 3 0 microns. The surfaces of such supports may be activated by chemical processes which cause covalent linkage of the antigen or antibody to the support. The irreversible binding of the antigen r,r an~ibody is obtained, WO9S/33206 2191430 ~ a~
however, in general, by adsorption on the porous material by poorly uulde l ~,I,uod hydrophobic forces.
Preferred solid phase m~tPri~lr~ for flow-through assay devices include filter paper such as a porous fiberglass material or other fiber matrix 5 m~tPri~l~ The thickness of such material is not critical and will be a matter of choice, largely based upon the 1,l V,U~I L6s of the sample or analyte being assayed, such as the fluidity of the test sample.
To change or enhance the intrinsic charge of the solid phase, a charged s. .l .~ e can be coated directly to the material or onto LU CI V,U~I Licles which 10 then are retained by a solid phase support material. Alternatively, LUL~L Op&l L~lc~ can serve as the solid phase, by being retained in a column or being, u~ d in the mixture of soluble reagents and test sample, or the particles themselves can be retained and immAhili7Pd by a solid phase support material. By "retained and immAhili7Prl" is meant that the particles on or in 15 the support material are not capable of b~ 1 LU()Vt:lU~ , 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 polystyrene, polyLut~hyla~ laL~ pvly~.u,uJltlle, latex, polytetrafluulvc,~llyltue, poly~ lu.liLI;le, poly~l~ulla~, or similar materials.2 0 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, Pml.~.l;.. I.~ which utilize various other solid phases also are ~nntPmrlqted and are within the scope of this invention. For example, ion capture ~uu~iduu~s for immAhili7in~ an immAhili7~hlP reaction 2 5 complex with a negatively charged polymer, described in co-pending U. S.
Patent A~ : . Serial No. 150,278 ~,UIL~ ,7, tA EP Pul,li~,lliull No.
0326100, and U. S. Patent ArrlirP~iAA Serial No. 375,029 (EP Publication No.
0406473), which enjoy common ownership and are ill~vl~ul~ted herein by reference, can be employed according to the present invention to e;fect a fast 3 0 solution-phase immllnArhPmirsll reaction. An immAhili7~hlP immune complex is separated from the rest of the reaction mixture by ionic interactionsbetween the negatively charged poly-anionlimmune complex and the previously treated, pVDi~iVt~ly charged porous matrix and detected by using W095133206 . 21 91 430 r~ 5~ 2l ~

various signal ~ g systems previously d~Pcrrihe~ including those described in rhPm~ minPscPnt signal ~ as described in co-pending U.S. Patent ArFlirAti~n Serial No. 921,979 ~,UII' ~ to EPO
pllhlir~t;~.n No. 0 273,115, which enjoys common uw.~..,LI. and which is 5 incu.~ul~.~t,d herein by reference.
A180, the methods of the present invention can be adapted for use in systems which utilize ~ L u~. Licle technology including ~ and serni-~..I,- ....lPd systems wherein t-h-e solid phase ~ul..~ es a mi~lu~ . Such systems include those described in pending U. S. Patent ArrlirAtinn 425,651 1 0 and U. S. Patent No. 5,089,424, which cul l c b~ulld to published EPO ArFlirAti/~n~
Nos. EP 0 425 633 and EP 0 424 634, l~a,u~ .ivt ly, and U.S. Patent No. 6,006,309 all of which er~Joy common ownership and are il.~l,uul~.~ed herein by reference. Such systems also include U.S. Patent ArFlir~ti~n Serial No.
07/859,218 filed March 27, 1992, which enjoys co_mon uwll~ l i,u and is 15 ill.,ul~ul~d herein by reference.
~ n the practice of one ~.(.ho~ of the present invention, a test sample CI.crprtpd of . -.~ -;..;..g any of the HIV antigen analyte or HIV antibody anslytes of interest is cimllltAnPollcly contacted with a solid phase to which afirst specific binding member of a first analyte is attached, and a solid phase to 2 0 which a first specific binding member of a second analyte has been attached,thereby forming a miYture. The specific binding members serve as capture reagents to bind the analyte(s) to the solid phases. If the specific binding member is an illll..ul.ul~actant, it can be an antibody, antigen, or complex thereof, specific for each analyte of interest. If the specific binding member is 2 5 an antibody, it can be a .. ,n~l.. Al or pol~,lu.. Al antibody, an antibody fragment, a recnmhin~nt antibody, as well as a mixture thereof, or a rniYture ofan antibody and other specific binding members. This miYture is inrllhAted for a time and under csnAition~ sufficient for a bindnng reaction to occur and which inrllhA~ion results in the fnrmAt;~n of capture reagent/first analyte 3 0 cnmpl^YPc of the first analyte if it is present in the test sample, andlor the f~rmAtinn of capture reagent/second analyte ~nmrl - of the second analyte if it is present in the test sample.

~ W0 95/33206 2 1 9 1 4 3 0 P~ ,."
Then, an indicator reagent for each analyte i8 contacted with the rl; The indicator reagent for the first analyte c~ a specific binding member of the first analyte of interest which has been labeled with a signal ~;G~ illg -nmrol-n~ The indicator reagent for the second analyte 5 ~ P~ a specific binding member of the second analyte of interest which has been labeled with the same signal generating ~ -u- ..rl as the indicator reagent for the first analyte, thereby forming a second mixture. This, second mi cture is inr77h~tPd for a time and under ....,.1;1:... ,~ sufficient to form capture .~a~e..llL.~ analyte/i..di.,~lo. reagent cAnnrl-YP,s and/or capture 10 .ea~G..lJ~c v..d analyte/indicator reagent cnnnrlPyp~ The presence of either analyte is .l~ Pd by detecting the signal generated in rnnn~ct;~n with the ~nnnrlPYP~ formed on the solid phase as an in~lir~t.inn of the presence of one or more analytes in the test sample. If the indicator employs an enzyme as the signal ~rllr.,-l;ll~ nnnnro ln(l (label), then the signal can be detected visually or 15 --~&,,.,-ed ~,u~_~- u~ ;rAlly. Or, the label can be detected by the measurement of fluorescence, chPmilllmin~rpn~p~ radioactive energy emission, etc., ~PpPn~in~ on the label used to generate the signal.
The capture reagents can be attached to the same solid phase, or can be attached to different solid phases. It is . - .~ AI~d that all capture reagents2 0 could be attached to the same solid phase, or that each capture reagent could be attached to a separate solid phase, or that cnnnhin~tinn of capture reagents could be attached to separate solid phases. For example, if . i~.u~v~lid~ were the solid phase of choice, then separate llli~,LU,U~Il Licles could have at least one capture reagent(s) attached to it. A mixture of microparticles (solid phases) 2 5 could be used to capture the various analytes which may be present in the test sample by using the mixture of ~i~. u~u~- ~icles. It is ~ d that different ratios of capture reagents attached to solid phases could be utilized in such an assay, to optimi~e analyte(s) detection.
In the ~ .ho-li....~ described hereinabove, it is preferred tbat the specific 3 0 binding member used as a capture reagent for the HIV-1 antibody analyte be HIV-l gp41 antigen, and that the specific binding member used as the capture reagent for the HIV-1 antigen analyte be anti-HIV-1 gp24 antibody. It is most preferred that the HIV-I gp41 used as capture reagent be a r~Pcnmhinslntly WO95/33Z06 21 91 430 r ~ o~
-2a prepared antigen (protein). Also, it is preferred that the specific binding member for the antibody analyte indicator reagent is a HIV-l gp41 recrlnnhin~nt protein or synthetic peptide described above, labeled with an enzyme, and that the specific binding member for the antigen analyte indicator 5 reagent is anti-HIV p24 antibody, labeled with an enzyme. It is most preferredthis HIV-l p41 antigen be synthetically produced, and that the enzyme be horseradi6h peroYidase (HRPO). Synthetic peptide XIII de6cribed above cnnjl~E~tsd to HRPO is a prefered indicator reagent.
In another PnnhotlimPn~ of the present invention, a te6t sample suspected 10 of c~m~sinin~ any of the analytes of interest is ~im~ ly contacted with a first solid phase to which a first specific binding member of a first analyte and a first specific binding member of a second analyte have been attached, an indicator reagent for the first analyte ~ E a specific binding member for the first analyte labeled with a signal generating ~u...l.u~ and an indicator 15 reagent for the second analyte ~ a specific binding member for the second analyte labeled with a signal ~;c~ g c ~ to form a mixture.
The specific binding members serve as capture reagents to bind the analyte(s) to the solid phases. If the specific binding member is an illllllu~lu~cd~l~ll, it can be an antibody, antigen, or complex thereof, specific for each analyte of 2 0 interest. If the specific binding member is an antibody, it can be a ",.~",~lr..~l or polyclonal antibody, an antibody fragment, a rec~nmhin~nt antibody, as well as a mixture thereof, or a mixture of an antibody and other specific binding members. The indicator reagents comprise specific binding members of the first and second analytes of interest which have been labeled with a signal 2 5 ~CI.~ illg crnnrolln-l This mixture is incubated for a time arld under rnn~itinn~ sufficient for a binding reaction to occur and which inrllh~t;nn results in the form~t.iûn of capture reagent/first analyte/indicator reagent c~ P~ of the first analyte and/or capture reagent/second analyte/indicator reagent c ,,..I,l - P~ of the second analyte, if either or both the first or second 3 0 analyte are present in the test sample. The presence of either analyte is determined by detecting the signal generated in rfSnn~c~inn with the cr~nr1P~Ps formed on either or both solid phases as an inr~irs-t.i~n of the presence of thefirst analyte and/or the second analyte in the test sample. If the indicator WO 9~il33706 I~~ 1171 employs an enz~zme as the signal generating cAmrolln(1 (label), then the signal can be detected visually or ~,zaDu.,zd D~o~,l.lu~h ~ trically. Or, the label canbe detected by the IlleaD~I z~uent of nUv.~.,c~...,e, rhPr.A,il~"";"...~_......
.ALoa.,Li~ energy emission, etC~"l~l, ...1;"~ on the label used. Al60, it is 5 ~ ' zd that the assay can include the use of a hapten-anti-hapten system, in which case the indicator reagent c~m further comprise a hapten such as biotin. The use of a biotinlanti-biotin system for assays is the subjectmatter of cop-pending U.S. Patent Application Serial No. 687,785 which ~,ullrzD~V~db to published European Patent ArrlirAt;~-n No. 0160900 (pubhshed 10 November 13, 1985), which enjoys common Vll~lDIli~J and is i--~u-~v,~lLzd herein by reference.
In the ~r.Aho~hr~A~Ant described h~ a'vv.~, it is preferred that the specific binding member used as a capture reagent for the HIV-1 antibody analyte be HIV-1 gp41 antigen, and that the specific binding member used as the capture reagent for the HIV-1 antigen analyte be anti-HIV-1 p24 antibody. It is most preferred that the HIV-1 gp41 used be a 1~ 1~l~AI~I1Y prepared antigen (protein). Also, it is preferred that the specific binding member for the antibody analyte indicator reagent is HIV-1 p41 antigen, labeled v~ith an enzyme. It is most preferred this HIV-1 p41 antigen be recAmhinant.ly or synthetically 2 0 produced, and that the enzyme be hul Dr~l~.viDIl perAxidase (HRPO). It is most prefered that this HIV-1 gp41 antigen be ~nthPti~llly produced. Solid phases preferred include a magnetic or non-magnetic bead, a well of a reaction tray, and lui~u~.l-iicles, either alone or in any cAnnhin~tiAn Positive and negative controls can be included in the assay of the present 2 5 invention to ensure reliable results. A blank solid phase(s), to which no capture reagent has been attached, can be utilized as the negative reagent control. Positive controls can include a positive control for each analyte whichcontrol is tested separately, and a combined positive control wherein the presence of all analytes to be detected in the asgay are ~l~t~rminA~
3 0 As previously stated, it is preferred that l~l~.~l~ly-prepared antigens be used as capture reagents on the solid phase and that synthetic HIV peptides cvllju~dLed to exnzme be used as indicator reagents in the assay.
However, it is tA be understood that the present invention is not limited to the VVO 9.;/33206 2 1 9 1 ~ 3 0 rrmhinAt;~n of ~.. ~- .. ,.. l proteins reagents and synthetic peptide reagent6 as de6cribed above, but that other rrmhinAt;~nc are r~ . For example, one or more or all of the capture reagent proteins can be ~y~lL~ ,dlly produced as can the protein of the labeled reagents. R~..,.1-.;..A.~I. HIV-1 and5 HIV-2 may labeled with ezyme may also be used in romhinAt;~m with sy-nthctic peptides as indicator reagents. Moreover, viral Iysates or isolates of the specific analyte can be employed, provided that a labeled HIV-2 synthetic peptide reagent of the present invention is employed.
Expression of Hn gp41 or parts of HIV gp41 have .l~.. ,- l ~l~d the 10 utility of rer!nnhinAnt DNA (rDNA) derived HIV erlvelope 9t~ in tli~gnr~L;c assays. Wood et al., Crltl S~rin~ ~Arbor Sy~w. ~ on R~A
l'llmor V;rLlcPc Cold Spring Harbor, New York, May 22-26 (1985); Chang et al., RirLo~ -ev 3:905-909 (1985); Crowl et al., Cell 41:979-986 (1985); ÇAhrp~illA etal., Bi~ -l^ev 3:128-133 tl986). While it is general knowledge that viral 15 proteins e~pressed in l~i or other U~ 5..~8 have potential utility in tli~g,nr.6L;~ assays, development of irnmllnrARCAys using these reagents, which also will have the specificity and s~~ y equal to or greater than the native viral protein6 derived from the cell culture has been a difficult task. Further,the ~ of Hn gag proteins in ~QIi have indicated that the HIV ga~
2 0 proteins produced by rDNA L~Lllolo~y could have potential fliAg,n~ value.
Wood et al., Cold Svrin~ ~Arbor S~.u~ on RNA Tllmnr virllcpc Cold Spring Harbor, New York, May 22-26 (1985); Dowbenko et al., PNA~ U.~A
82:7748-7752 (1985); Ghrayeb et al., DNA 5:93099 (1986); Stei~ner et al., ~Iroloev 150:283-290 (1986).
25 The present invention utilizes l.~.. l.;.. ~.ltly-produced HIV envelope proteins as assay reagents. The cloning of the Hn genome and t .IJles&;ul~ of Hn envelope and core protein in E. coli, the pllrifirsltirn and ~,..
of gp41 and p24, and various assay formats which utilize these 1~ "
proteins are described in U,S. Patent Ar~lirAt;~n Serial No. 07/020,282 filed 3 0 February 27, 1987 and previously incorporated herein by reference, from which this present invention claims priority. Briefly, HIV-infected HT-9 cells were harvested and totl cellular DNA was isolated an subjected to digestion. The DNA segments encoding for the core protein and for the envelope ~

WO 9~/33206 1 ~

were further 6~lh~Alnnpd into bscterial t~-,Ul~;UII vector6 u6ing well-known rernmhin~Ant ~^rhn^l~,y U.S.Patent Arrlir. t~An Serial No. 07/020,282 al60 tesches that, in the detection ûf HIV-1 antibody, the u6e of 1.,- l.;.. .l antigens as the capture reagent and the indicatAr reagent allows for the detection of anti-HIV-1 AntihoriP~ ûf different ;.. ~.,ijlnh.. lin classes. These i"""""A~ nb-llin clag6e6 include IgG, IgA, IgE and IgM. The detection of anti-HIV-l IgG, IgM and IgA u6ing the Abbott HIVAB~9 HIV-I/HIV-2 (rDNA) EIA
a66ay ha6 been de6cribed in an ab6tract by J. L. Gallarda et al., fith ,~nn~
Forllnn on ~ms. He~Aatihc ~n~ Ot.hPr Bloûd-Bûrne DiRPACPc Atlanta, Georgia, 10 March 29-April 1,1992.
It i6 ~n- ~ ,ul ~ Pd and within the 6cope of the present invention that ."h;..A..~ antigen6 produced in h~L~ ^ 60urce6 or synthetic HIV
peptides can be utilized in the assay and will cAntrihute an even greater les6ening of fal6e po6itive re6ult6. For exa~nple, if an l~,~i prepared 1. ~.. l.. _.. i. antigen 6uch a6 gp41 i6 u6ed a6 the capture reagent, then a ..... l.. _.. ~. antigen gp41 produced in any 6uitable 6ûurce different than ~li, 6uch a6 in a 6uitable yea6t hû6t or otber suitable ho6t 6uch as ~
mp~n~ ,. can be uged. The use of h.,~.~ ,u6 60urce6 of antigen6 in assays, including I~Cv .~h;~A~t antigens, is the subject matter of co-pending 2 0 U.S. Patent A~ v ~ Serial No. 07/701,626, which enjoys common ~ ,L,u and is iL..~ uL _L~ d herein by reference.
Further, although the present invention preferably utilize6 re~^AnAhinAntly produced antigen6, it i6 well within the 6cope of the invention to utilize 6ynthetic protein6 in6tead of l.~ ''''1';llAIII1Y produced antigen6. Al'hu6, 2 5 variûus 6ynth^t;^Ally prepared HIV peptide6, of varying length, a6 de6cribed above, can be u6ed.
ALhe present invention alsû utilize6 A .. l ;l~û~l;Pc which crerifirAlly bind toHIV antigen analytes. In a preferred ~ .ho~ anti-HIV p24 antibody is u6ed. In a mo6t preferred PmhorimPnt a mixture of ... .A l~ ;l V l PC, 3 0 both 6pecific for HIV p24 antigen, is u6ed. In thi6 mixt~ire, one ",,,.,nrl.."~l antibody whic_ 6pPrifirAAlly bind6 to an epitope on HIV-1 p24 to which epitope human anti-HIV-1 p24 IgG dûes nût ~vLu~ Lil,i~cly bind i6 u6ed with another mnnnrlAnAl antibody which cperifirAlly binds to a different epitope of HIV-1 p24 WO95/33206 ~ '011~1 219~430 to which different epitope human anti-HIV-l p24 IgG does ~,ulu~liL~ly bind.
Further, the ~"--~nrl~ l antibody which dûes not ~;u. .~u~f,i i~,ly bind human anti-HIV-l p24 IgG also cperifir~lly binds to HIV-2 p24 antigen. These ..,rrl~ nt;horliPs and their uge in HIV antigen assays are the subject S matter of co-pending U. S. Patent Arrli^ ~;~ n Serial No. 07/204,798, whichenjûys commûn uwl.t,l~L,u and is hl~ul uu.~.f d herein by reference. These ~"~.,,,~rl~ nt;horfiPc are ~ Pd as 31-42-19 and 31-90-25. HyL~;du~ d cell line 31-42-19 ~u~ulu~llg mr~nnrll~n~l antibody 31-42-19 was deposited at the AmPrir~n Type Culture Cûllection, 12301 Parklawn Drive, Rockville, 1 0 Maryland, 20852 on May 26, 1988 and has been accorded ATCC Deposit Nû. HB
9726. Hyl~;du ua cell line 31-90-25 ,u~udu~ ...nrl~...Al amtibody 31-90-25 was deposited at the AmPrir~n Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, 20852 on May 26, 1988 and has been accorded ATCC
Deposit No. HB 9725. The use of these .. . ~ .ncl - ,~l - - I ;I -o-l;~ as antibody 15 ~ in Hn antigen assays also has been described in U.S. Patent No.

7,204,79B, which enjoys common uw~ l iu and is il~ ,uu~a.~d herein by reference.
It is cr~ntrmrl ~ted and within the scope of the invention that the detection of HIV-2 antigen is possible with the assay of the invention. In this assay 2 0 format, HIV-2 p41 would be attached to a solid support as the HIV-2 antigen capture reagent, in addition to the previously-described HIV-l p41 antigen capture reagent and HIV-l antibody capture reagents. The solid support can be the same solid support to which all other capture reagents are attached, it can be the same solid support to which Hn-l ~ antigens have been 2 5 attached, or it can be attached to a solid phase to which no other capture reagent (except fûr Hn-2 p41) has been attached. The assay p~u~idu~ would be the same as described hereinabove for the various ~ "ho ~ ; of the invention. The HIV-2 antibody analyte indicator reagent wûuld comprise ....l.;"~.~f. and/or synthetically-prepared HIV-2 gp36 antigen attached to a 3 0 rfPtPCt~hl^ label. In one PmhorfimPnt., the rPcr~mhin~ntly prepared HIV-2 p41 is utilized. The sequence for the Hn-2 virus (including p41 antigen) is described in EP 0 347,365, published December 20, 1989 to Diagen Corp, which is incul,uu.d.~d herein by reference. A most preferred HIV-2 rer~mhin~nt W0 95~332()6 , r~" :

antigen encodes the first 104 amino acids of the HIV-2 p41 antigen. The resulting plasmid ~lecienAted as pJC104 e2presses the HIV-2 ~nY protein as a fusion with CKS protein. This plasmid encodes a . ~ ~v~ . protein ~v .~ ~;.. ;.~ the first 239 amino acids of the CKS protein, 13 amino acids from the pTB210N multiple restriction site linker, 104 amino acids from the HIV-2 env protein (amino acids 506-609), and an a~itlrnAl 15 amino acids from the pTB210N multiple restriction site linker, following the methods disclosed by Bolling and MAn~Prki, "CKS Method of Protein Synthesis," U.S. Patent ArrlicAti~n Serial No. 167,067, filed March 11, 1988, which enjoys common ownership and is incu.l,u.c.~ed herein by reference. In another prefered ,,,~\ho.l;, ~ sy-nthetic HIV-2 peptides of varying lengths, as described above, d to enzyme are used as indicator reagents. Most p~E~ h peptide I cOllju~;uled to HRPO is the indicator reagent.
The present invention will now be described by way of PY~mrlPc~ which are meant to illllPtrAtP, but not to limit, the spirit and scope of the invention.
li:~AlVI PLT~.~
E~ample 1 2 0 Cr,~tinF Plv~ e Ucin~ Two Solid PhACPC
This ~ulu~,~lule utilized 1/4 inch polystyrene beads (available from Abbott Laboratories, Abbott Park, IL 60064) and a 60-well PUIYDLYIe1IC reaction tray (available from Abbott T.AhorAt~riP~, Abbott Park, IL 60064). Two different anti-HIV-1 p24 """,rrl ",Al Antiho~iPC were coated on the beads, as follows. The 25 beads were coated at a ~u.l~GLc.~ion of 8 ~glml (u~lu~kly 1.6 ,ug/ml/bead) in a 0.25 M sodium citrate buffer (pH 7.2) for twû hours at 45C. The beads thenwere washed in the 0.25 M sodium citrate buffer (pH 7.2), and then they were reacted with a detergent solution ~U..I ~;..;..e 0.1% Triton X-100~9 (pulyu~ ylene ether, available from Sigma Chemical Co., St. Louis, MO) for 3 0 one hour at 45C. The beads neYt were blocked with 1% bovine serum albumin (BSA) in 0.25 M sodium citrate buffer (pH 7.2) for 30 minutes at 45C, and then u~ ûD~,d with 2% sucrose, 1% rh.-srhAtP glass for 15 minutes at 15-30C in 0.25 M sodium citrate buffer and allowed to dry. The two nnrnrrlr~nAl n,ll;h~.l;P

W0951~3206 2 1 9 1 4 3 0 P~.l. s ~4'71 used are rlPRienAt~pd as 31-42-19 and 31-90-25. They are the subject matter of apatent ArrlirAti~n U.S. Patent Application Serial No. 07/204,798 that describes their d~ u~ l and uses, previously ill~ u~,vu~_led herein by reference Their use also has been described in U.S. Patent No. 7,204,798, which erJoys S common ownership and is il~ vl~vv~ld herein by reference. H~l,.;du~ cell line 31-42-19 ,vl~UWIlg .. ~.~1.. ,A1 antibody 31-42-19 was deposited at the AnnPrirAn Type Culture Collection, 12301 Park awn Drive, Roc_ville, Maryland, 20852 on May 26, 1988 and has been accorded ATCC Deposit No. HB
9726. ~ u.\.a cell line 31-90-25 ~Ivvu~llg l,.-. lrrlr~ ~Al antibody 31-90-25 was ~eposited at the Amprir~An Type Culture Collection, 12301 Parlawn Drive, Roc_ville, Maryland, 20852 on May 26, 1988 and has been accorded ATCC
Deposit No. HB 9725.
Next, the wells of the 60-well reactiûn tray were coated with HIV antigen, as followæ. The l~ ~ .. 1.;, A ~ l l protein HIV-l p41 ~ protein ~1F ;'~ a6 15 pTB319 was added to each well at a ~UIl~ c..I,l-lLon of 1 llg/ml in 0.1 M 3-[cyclohexylamino]-1-~,.v~ lfsnir acid (CAPS buffer, pH 11), and inrllhAtetl for two hour6 at 40C. The wells then were washed twice with 400 111 of rhn~l.hAIP buffered saline (PBS, pH 7.5), reacted with 0.1% Tween-20~l9 for one hour at 40C, and then blocked with 3% BSA in PBS for one hour at 40C. The 2 0 wells nelct were ov O. ~ùcllod with 5% sucrose in PBS for 20 minutes at room t~ O and allowed to dry.
The pTB319 plasmid Aulu~u~g l~ ~--~;I~AIII, protein pTB319 is the subject matter of a patent Arplir~t;~n to Bolling and M~nrlPcki~ "CKS Method of Protein Synthesis," U.S.Patent ArrlirAt;~.n Serial No. 167,067, filed March 11, 2 5 1988, ~u~,v;uu~ly il~,u~u~_lod herein by reference. pTB319 was produced by inserting a synthPt;~Ally-produced DNA fragment which encoded the carbo~y terminal 42 amino acids of HIV-l pl20 into the plasmid pTB315, as described in Bolling and M~n'lPrki, D2~-3 0 Exarnple 2 Siml-lt~n~ g Acc~v for HlV slnt~en slnr1 F~ nt.ihrdv The two solid phases prepared as described in E~ample 1 were used in an assay for detection of HIV antigen and/or HIV antibody in a test sample, as W0 95133206 1 ~

follow6. An HIV-1 S_.U.,Ull~O.D;~tll panOI, which ~n~<~;nPd 65 sOrum samplOs derived from rline HIV-1 infected individuals u ld~.~ uulg ~_.UI~UII~.D;Oll, wasusOd in thô assay. Each sOrum samplO was diluted in a separate well of the 60-well tray prOviously prepared in EAample 1 by adding 150 111 of thô serum S aar~ple to 50 111 of specimen diluent, which contained 2% Tween 20~
(P~ UA~ U1Yl~ t1~t;~ available from Sigma Chemical Co., St. Louis, MO).
Then, a bOad ~tl~i.; tUDIy coated with anti-HIV p24 ~n~;hoAiP~c as describOd im E~amplO 1 was placed in each well .- .~:..;.~ a serum sample. The wells of each tray were ;.. - ~ d for 60 minutes at 40C under r~tntim~ c rotation.
I O Following i .. l .~ l ;.. , each well of the 60-well reaction tray was washed with 15 rnl of deiorlized water (dH20) in the Abbott Parallel Plu~o~ .-g Center~M (PPC, available from Abbott Labu..~Lu.;es, Abbott Park, IL). 200 111 of an HIV p24 antibody probe reagent (rabbit polyclonal F[ab']2 anti-HIV-l [active Ill~,~otLolli/:
anti-p24 antibody at a ~UIl~oll~ .Lu.. of 2 to 6 ~Lg/r~l] in an antibody diluentl 5 (2.25% BSA, 7.5% calf serum, 7.5% goat serum, 25% humarl recalcified plasma, 0.1% sodium azide) was added to each wOll/bead and then the resulting mixture was inr-lhAtPd for 60 minutes at 40C without rotation. Each bead/well in the reaction tray was washed with 15 ml of dH20. Then, 200 ~1 of conjugate diluent (0.18% Tris, 1.19 % Tris-HCl, 0.38% NaCl, 9.0% calf serum, 2 0 0.9% goat serum, 10.0% human calcified plasma, 4.5% Triton X-100~), 0.013%
~..1,...,;l .. sulfate, 0.009% U ilueluD~d) which contained a mixture of rPc~-nnhinAnt HIV-l p41 antigên labelled with IlulD~ iDll p.,.UAilaSe (pTB319 coupled to HRPO), and HRPO-labelled goat anti-rabbit IgG antibûdy were added to each bead/well of the reaction tray and allowed to incubate for 60 minutes at2 5 40C without rotation. Each bead/well of the 60-well reaction tray was washed with 15 ml of dH20 as ~ul~iouDly APcrriheA Then, 300 Ill of o-phenylPnPAiAminp-2Hcl (OPD) was added to each well/head and then was in~lhAted for 30 minutes at room ~ .o in the dark. The reaction then was stopped by adding 300 ~ of a stopping reagent (1 N H2SO4) to each 3 0 welVbead. The reaction was read using the Abbott PPC which measured the optical density of the reaction at 492 nm using a 630 nm reference. The cutoff value was r~ -liRhPd as 0.1 OD + mean OD of the negative control. Thus, W095133206 21 91 430 r~ o~

serum samples were considered reactive (posit*e) if the sample to cutoff value was greater than 1.
All 65 serum samples from the 9 individuals described h~ ~u~ were tested following this ~L~ luLe. The results obt~ined then were compared to 5 the results obtained for the same serum sample when using an HIV antigen assay (HIVAG~19, available from Abbott T.ol.. l.. ;rr, Abbott Park, IL) and an HIV antibody assay tHuman Tmn -ln~ Virus Types 1 and 2: E.~i.
and B....rc~ . ;"... recomhinont antigen, Abbott HIVAB~9 HIV-l/EIIV-2 (rDNA) EIA; available from Abbott Lal,~ o.;~s, Abbott Park, IL) following o ..~.... r,.. ~ S directions as provided in each product insert. The data are reported in Table 1, wherein "OD" refers to the optical density reading, "S/CO"
means r~omp~ ll.-off value, "Result." refers to the illLtl,~ LtiUII of the test,"HIV-1/2 Ab HIV-l Ag Comb" .1P~ P~ the assay of the invention, "HIV-1/2 Ab" ~^ignot~ the HIVAB~8) HIV-l/HIV-2 (rDNA) EIA assay and "HIV-l Ag"
15 ~Pr~ieno~qr~ the Abbott HIVAG~) assay.

~ W~95133206 21 91 430 r ~

~mplelD ~V-~/2Ab ~V-Il2Ab HIV-lAg ~IIV-lAgCamb S!CO Result S/CO R~ult YCO Rl#;ult SVO 121 1 L3~ + Q33 - 110 +
2 L92 + 2i55 + L80 +
3 516 + 632 + 20 +
4 L79 + 3.65 + Q~0 -SV0031 6 0~3 - Q13 - Q40 6 Q87 - 0.13 - Q30 7 Q8~ - Q13 - Q,35

8 Q78 - 0.~5 - Q~5 1 5 9 Q95 - 0.17 - 040 Il Q62 - Q10 - Q,35 L2 ~40 + 9C4 + 1~2) +
13 3.40 + 11~09 + 8.0 +
2 0 14 3.40 + 3.0D + ~D.50 +
16 3.40 + ~24 + fi40 +
16 L87 + 6.25 + 3,~ +
17 L52 + 7.3~ + Q80 18 L~8 + 7.24 + LO +
SV0151 19 Qg7 - 0.40 - L70 +
221 + 149 + 150~ +
21 265 + 26'i + 7.9~ +
2~ 254 + 7.30 + 20 +
3 0 23 267 + 6.51 + L80 +
24 L89 + 3.81 + 0.40 SVOO91 25 2.40 + 0.24 - 18.80 +
a6 3.40 + 0.44 - 71.60 +
3 5 ~7 3.40 + 2.81 + 5.20 +
28 3.40 + 2.75 + 1.90 +
29 2.71 + 2.43 + 0.66 30 2.23 + 2.26 + 0.66 31 1.64 + 3.72 + 0.49 4 0 32 1.35 + 10.57 + 0.41 33 nt~
S~Olll 34 3.40 + 0.12 - 32.80 +
35 3.40 + 0.46 - 42.90 +
4 5 3: 3.40 + 11.23 + 20.70 +

W095/33206 2191430 ~

37 2.35 + 4.16 + 1.40 +
38 1.82 + 3.81 + L10 +
~9 L71 + 3.34 + 0.80 40 1.40 + 5.49 + 0.72 SV0161 41 L01 + O.L2 - 1.44 +
42 121 + 1.14 ~ - 3.82 +
43 2.17 + 0.79 - 8.41 +
44 3.40 + 1025 + 11.80 +
1 0 45 2.48 + 2.82 + 0.65 46 1.51 + 2.37 + 0.48 SV0061 47 1.13 + 0.20 - 2.90 +
48 123 + 0.26 - 3.60 +
1 5 49 3.33 + 1.93 + 10.90 +
50 3.09 + 1.72 + 13.50 +
51 L93 + 0.96 - 7.10 +
52 1.88 + 1.39 + 4.40 +
53 1.30 + 1.60 + 1.90 +
2 0 54 1.45 + 3.10 + 0.97 ~6 1.22 + 4.28 + 0.98 SV0071 56 0.97 - 0.13 - 0.40 i57 0.84 - 0.14 - 0.40 2 S 58 3.19 + 2.97 + 2.30 +
59 1.82 + 2.37 + 0.52 ~0 0.97 - 2.84 + 0.42 SV0081 61 0.80 - 0.12 - 0.45 3 0 ~2 0.85 - 0.15 - 0.32 63 0.83 - 0.14 - 0.49 6~ 1.41 + 3.40 + 0.43 65 1.10 + 4.96 + 0.37 ~6 123 + 8.58 + 0.37 3~
NQPosifiv~NoTes~d 5V65 4V65 3Y~;6 *nt: This sample of the S~ .,LDiUll panel was unavailable for testing.
The data from Table 1 indicates that the method of the present invention had 4 0 greater sell~ilivi~y than either the HIV-1/2 Ab or the HIV-1 Ag test when the results from the three individual tests were compared ~ ely to each other.
It i9 expected that the assay can be optimized even further to detect both HIV
p24 antigen which is present early in the course of infection and also in the ~ w09sl33206 2 1 9 1 430 f~l,.. . ..

final stages of HIV infPri;onR, as well as HIV ~ntihorlil~g which appear later in infection at the time of 5elu~,ullvel~;O~I.
EYample 3 S C~,~*n~ Pror~dllre For One Solid Ph~R.o In t-h-is ~lu~1ule, only one solid phase was coated with HIV an*gen and HIV antibody, as follows. Into each well of a 96-well _icrotiter plate (Tmmlllnn4~), available from Dynatech, Al~oY~nrlrisl VA) ~ 1 antibody 31-42-lg, ",...,~ 1 antibody 31-90-25 and 1~..."1.;"~ . HIV-1 p41 antigen ~
10 as pTB 319 (as described in Example 1) were coated at a ~ . of 1 /ml each in 0.1 M ec~llJu~ te buffer (pH 9.5) for two hours at room k--~ I e. The wells next were blocked with 300 ,ul of blocking reagent 5% non-fat dry milk, 10 mM Tris [pH 8.0] 150 rnM NaCl and 0.05%
Tween-20aD) for one hour at room L~u~ Lule.
Example 4 ~lV,Anti,~Pn/Antihl~dy ARRaY URin~J One Solid Ph~RP
The solid phase prepared as described in Example 3 wa6 used in an assay to detect the pre6ence of HIV antigen andlor HIV antibody in a test 2 0 6ample, a6 follow6. Each scrwn 6ample of a 12-member 6elu~u~ iO~I panel (Panel G available from Boston Rirlm~ Inc., Boston MA) as well as positive and negative controls were tested. 150 111 of each serum sample or posi*ve or negative control was diluted in a separate well of the IlliCI uLiLel plate with 50 ~11 of specimen diluent (r~mt~inine 15 ~1 of Triton X-100~D and 35 Ill of blocking 2 5 reagent, as described in Example 3) and incubated for 60 ~ninutes at room Lt:Lu,u~.~-Lule without rotation. After in~1h~t;~n the wells were washed with eight cycles of 300 ~11 of wa6hing buffer (0.05% non-fat dry milk, 10 mM Tris [pH
8.0], 150 mM NaCI, 0.05% Tween 20(~)) u6ing a Nunc 8-chalmel "Immunowa6h" manifold (available from Nunc, Denmark). Next, 175 111 of of 3 0 an HIV p24 an*body probe reagent (rabbit polyclonal F[ab~]2 anti-HlV-l [active ill~ l ~diellL. anti-p24 antibody at a concentration of 2 to 6 llg/ml) in an antibody diluent (2.25% BSA, 7.5% calf serum, 7.5% goat 6erum, 25% human recalcified plasma, 0.1% sodium azide) were added to each well and in~l~h~tPd for 60 W0 95/33206 2 1 9 1 4 3 0 P~ . c ~

minutes at room L~...,ut~ ,ul~ without rotation. After inrllhnti~n the wells were washed with eight cycles of wash buffer as previosuly l~srrihe~i Then, 150 111 of coluugate diluent (a6 described in Example 2) which r~ntnin~d a mixture of ~ . HIV-1 p41 protein (PTB 319) labelled with HRPO and S HRPO-labelled goat anti-rabbit IgG (previously described in E=ple 2) were added to each well and inrllhntPd for 60 minutes at room ~ U~ Ul~ without rotation. The wells were washed with eight cycles of wash buffer (described previously herein) and then rinsed with dH20. Then, 126 ~1 of OPD substrate was added to each well and the wells were inrllh~ted at room t~ lu~ for 10 10 minutes in the dark. The reaction was stopped by adding 125 ,~11 of stopping reagent (previously described in Example 2). The absorbance of each well was read at 490 n_ with a 630 nm reference. The cutoffvalue of .02~ OD + mean OD
of the negative control was ~t~ h~ Samples were ~ul~id~ d reactive (positive) when the sample/cutoff value was greater tharl 1 The data from these assays are presented in Table 2. In Table 2, "OD"
refers to the optical density reading, "S/CO" means ~S~mrl-r~u~-off value, "Result." refers to the i--~ ul ~laLion of the test, "NC" refers to negative control and "PC" refers to the positive control.

WO 9S1332~6 P~l~-J.. _A,7 ~1 -~5 Sample ID HIV-1/2 Ab HIV-V2 Ab HIV-l Ag HIV-l Ag Comb S/CO Result. S/CO Result pg/ml* Result NC 0.85 NC 0.72 NC 0.82 PC 1.35 +
PC 1.13 +
1.22 + 0.09 - >200 +
1 5 2 1.64 + 0.23 - >2û0 +
3 2.33 + 2.55 + >2~0 +
4 4.04 + 4.07 + 1~5 +
5 2.42 + 1.92 + 40 +
6 1.03 + 1.95 + 5 +
2 0 7 1.0g + 4.87 + 0 8 1.57 + 7.31 + 0

9 1.61 + 7.31 + 0

10 1.79 + 9.40 + 0

11 3.76 + >17.85 + O
2 5 12 5.62 + >17.85 + 0 *pg/ml - picograms/ml 3 0 As tbe data from Table 2 dGlllU~ .iG~ the assay of the invention was capable of detecting the presence of HIV antibody and/or HIV antigen in the sGLu~v~G~ion panel. Compared individually to the HIV-V2 Antibody test, and HIV-l Antigen test, the method of the present invention was more sensitive at detection than either test alone, based on detection of either antigen or antibody.
3 5 When the results from Table 2 of the method of the present invention are compared to the combined results of the HIV-V2 antibody test and the HIV
antigen test, the method of the present invention was able to detect all spe~m~n~ that were react*e by either test.

WO 95/33206 2 1 ~ 1 4 3 û r~ 7l ~

Example 6 CnRtin~ ~irrnnRrtirlPA ,~imlllt~nPmlcly With T~V ~ntihn~ly ~n~ ~TTV ~nti~en Ca~ture RPR~ents Both ... - ~n~l....Rl anti-~IIV p24 ""~ previously described (31-42-19 and 31-90-25) and the rernnnhin~nt HIV-1 p41 antigens previously described (HIV-1 p41~ protein pTB319, and HIV-2 p41 I~ --.-l-;.. .l. protein pJC104) are together and r~ ly coated onto a uniform 0.5%
vluJue) of pol~ y~ .v,v..-~ide~ (available from Seradyne Inc., T...l;,...~ , Indiana) at .ullL~.ILlu~ions of 150 llglrnl each in 0.01 M
10 carbonate buffer (pH 9.5) for two hours at room tf lulve~ uuè (15-30C). The suFrPn~;~n of lUi~,~ U,U<~I iicles is briêfly ~ iru~d and the Jl~ U~Val L~IC pellet is ~ d in 0.05M Tris buffer (pH 8.0) to wash away excess, 1 J
protein. This washing is repea~ed until no uncoupled protein remains. After blocking the miLIU,UCII LC1~B with 10 _g/r~l casein in 0.01 M Tris (pH 8.0), 0.15 l ~ M NaCl at 56C for 18-24 hours, the m iLI u~u~ LCI~3 again are washed as described herein and diluted to 0.015% s~cpPr~ n (wtJvolume) in 0.05 M Tris (pH 8.0), 0.15 M NaCl, 1% BSA, 15% sucrose and 0.1% sodium azide.
Example 6 2 0 Sinmllt~nPn.. ~ Detection of ~TTV ~ntihody Rnrl ~TTV ~ntiFPn Ugin~
Mi rro~Rrtirl es The Abbott IMX~D Microparticle Enzyme TmnnllnnRc~y (MEIA) system is used, although any system which employs uli~u~liicles can be used. The Abbott IMXt8) MEIA system is thoroughly described in the Abbott IMX~) 2 5 Operation and Customer Training Manuals (available from Abbott Diagnostic Division, Abbûtt T ~l.- ,.l ;rn, Abbott Park, IL). In this assay, 100 ,ul of the0.15% 7 ~ ..F- - . prepared in Example 5 is mixed together with 100 111 of test sample suspected of - .l~ HrV-l and/or HIV-2 antibody and/or IIIV-1 antigen, and inrllhslted at 40C for ten minutes in an Abbott IMX(~) reaction cell 3 0 to form a reaction mixture. HIV RntihotliPF and/or HIV antigens bind to the mi~lv~ulL~ in an antibody/antigen/microparticle complex. 150 111 of the reaction mixture is l~ullbE~ d onto a glass fiber matrix to which the microparticles are retained in an irreversible binding. The antibody/

~ WO9~i/33206 2 1 ~ 1 430 -3~ -antigen/lui~.u,ualLicle rAnnr1p~P~ then are reacted with 50i~1 ûf a probe r~AnCi~t;ne of biotinylated 1~ HIV-l and HIV-2 re~n~hin~nt p41 antigens (pTB319 and pJC104, ~Lc~iuutly APcrrihPA) and ' " ylalcd F(Ab')2 anti-HIV-l p24 in 0.05M Tris 9pH8.0), 2% BSA, 0.25% saponin and 0.1% sodium azide at 40C for tcn minutes. 50, 11 of an antibody conjugate c - . ~ of goat anti-biûtin alkalme ~ l in 0.1 M Tris (pH 8.0), 0.5M NaCl, 0.9% Brij-36!9, 1.0% BSA and 0.1% sodium azide then is allowed to react with the biotin u.ub~ ibody/antigen/l"i~lù,u~lLicle ~AmrlP~PC for ten minutes at 40C.
Then, these n: ulJal Li~lc rnnnpl~YPC are washed six times with 0.05 M Tris (pH 8.0), 0.3 M NaCl and 0.1% sûdium azide, the biotin ,ulub~'allLilJ~ e l. i~, u,ualLi~lc complex is reacted with 60 ~11 of the 8ubgtrate lUcLllyl~ hPllifPryl rhAcrh~e (MUP, Abbott Laboratories, Abbott Park, IL), and the nuul~3~c.l.,6 of the product, lucL~ l PllifPron, (MU) is measured. The rate of MU
production is prûpûrtional to the ~- -- . ~P ~ l: .. . of analyte(s) in the test sample.
Example 7 cû~*n~ r~ Licles S~ aLelY with ~TTV ~ntihAA~v and ~TTV AntiFen Câ~Ature RP~Rntc In this e~cample, the various analyte capture reagents are coated 20 sc~ Lcly onto pol~..Lyl~e microparticles (available from Seradyne Inc., TnAi~n~rAlic, Indiana). Each of the reagents may be coated bc~ LLcly from each other or in various ~ nc with each other. After each of the analyte capture reagents is coated on their ~6c~,uo~LiYc ~Iu~ lLicles, the various coated ll i.,lu~c.lLicl~s are poûled together and used in the assay.
2 5 In the present example, the two .. Afl.. ~1 anti-HIV p24 ~ntihoAiP~c (31-42,19 and 31-90-25) are coated together onto lui-,lu~c-lLicles separate from themicroparticles coated cimlllt~nPollcly with lr~ HIV-l and HIV-2 p41 antigens (pTB319 and pJC104). Although the exact amount may vary, in general, the coating ~ulucclulc will ~ uA~ a~c that described in Example 5.
3 0 After blocking the physically sc~ laLed lUil,lU~ I Licles with 10 mg/ml casein in 0.01 M Tris (pH 8.0), 0.15 M NaCl at 56C for 18-24 hours, the microparticles again are washed as described in Example 5, pooled together, and diluted to a WO9S/33206 r.~

0.015% ~ (wt/volume) in 0.05 M Tris 9pH 8.0), 0.15 M NaCI, 1% BSA
and 15% sucrose. At the pooling step, the IlPl~l V,udl Licles may be pooled at various ratios to affect ~ ;L;v;Ly and ,,I,e~.~Ly of the assay in order to optimize their use.

Example 8 Simlllt~nPm.c DetP~hnn nf ~TTV ~ntihndy An~ T-TTV Anti~en on ~ y~r S~n~ ~.1. -1V GnAtp~ With ~TTv ~nhhnf¦y ~nll T-TTV Anti~en CA~ lre RPA~ents The Abbott IMX~19 Mi~u,ual Liclt Enzyme TmmllnnAcspy (MEIA) 6ystem 0 iB used, although any system which employs ~i~.ul-~lLicles can be used. The Abbott IMX~) MEIA system is thoroughly described in the Abbott IM,~) Operation and Customer Training Manuals (available from Abbott Diagnostic Division, Abbott T~ul~u~ , Abbott Park, IL). I~i this assay, 100 111 of the 0.15% s ~ prepared in Example 5 is mixed together with 100 ~1 of test 15 sample E~rert~Pd of ~n.~lu;~;.,e HIV-1 and/or HIV-2 antibody and/or HIV-1 antigen, and inrllhAtPd at 40C for ten minutes In an Abbott IMX~ reaction cell to form a reaction mixture. HIVAntiho~liPc and/or HIV antigens bind to the Illi~.~U~a~ Licles in an n-lLil,o,ly/~lLi~ .ul.a-Lde complex. 150 ~1 of the reaction mixture is 20 L-a--~,r~ onto a glass fiber matrix to w_ich the .. i~u~dlLid~s are retained in an irreversible binding. The antibody/antigenlmi~. uludl Licle . ' - then are reacted with 50~,fl of a probe cnnr; r~in~ Of ' ~ldL~d ~ . .~,; . .-~. l HIV-1 and HIV-2 recnmhinAnt p41 antigens (pTB319 and pJC104, previously lP~hefl) and biuLillylaL~ F(Ab')2 anti-HIV-1 p24 in 0.05M Tris 9pH8.0), 2%
2 5 BSA, 0.25% saponin and 0.1% sodium azide at 40C for ten minutes. 50 Ifl of an antibody conjugate ~ e of goat anti-biotin alkaline ~llr ~l-h_t l~ in 0.1 M
Tris (pH 8.0), 0.5M NaCl, 0.9% Brij-35~, 1.0% BSA and 0.1% sodium azide then is allowed to react with the biotin ,UI ube/~ .-Libo~/~-Li~t l,/.. li~ l Ul~d. L;clc rnmrlPYPC for ten minutes at 40C. Then, these microparticle ~ nmpl^YPR are 3 0 washed six times with 0.05 M Tris (pH 8.0), 0.3 M NaCI and 0.1% sodium azide, the biotin ,ulube/a--Libody/~-L;~,~ll microparticle complex is reacted ~-vith 50 111 of the subshrate methylllmhPllifpryl phncphAce (MUP, Abbott La~ulaLuL;es,Abbott Park, IL), and the fluorescence of the product, .llt~LI.., 1 1l . , ,hPllifPron~

wo ss/33206 2 1 9 1 4 3 0 ~ u~
- -3~ -tMU) i~ ~uu-Gd. The rate of MU uLudu~.,iulL i~ proportional to the ~,ul~G.IL~Liu~l of analyte(3) in the tefit ~ample.

WO 95/33206 ~ !4 "'I

E~ample 9 PrP~aration an~ r .. ~ of RPrnnnhinant ~TV-l ,~nti~PnA
Pre~arati~n of init;al ~lv-l an~l ~TV-2 clones An HIV-1 genomic library was prepared by ligating a partial ~QRI
S digestion of genomic DNA derived from HIV-l infected HT-9 cells (obtained from Dr. Robert Gallo, National Cancer Institute, Labu.~ s of Tumor Cell Biology, Lot No. P3-21) with bart,Pri~h~ep lambda Charon 4A EcoRI arms and L n~r~ e into E. coli C600. The library was screened by l~ . with cDNA made from HIV-1 viral RNA, and a single phage (rlPPignAtpd Phage 4B) 10 was obtained c ...~n;";"e the entire HIV-l genome.
Phage 4B DNA was digested with ~zLI and ligated into the ~LnI site of pUC18 (P~ethPA~a Research Labo.llLu.;o~). A clone (~ d pcB) C - .In;- ;--r the entire p41 region of the HIV-l env gene was identified and mapped.
P_age 4B DNA was digested with EcoRI and ligated into the l~RI site of 15 pBR322. A clone (-lPAien~tPd pcR23) c~ e the entire HIV-1~ gene was identified and mapped.
A DNA fragment ~ e the/env gene from HIV-2 prophage isolate D1945 was idPntifipd within a lambda genomic library of prophage DNA. This fragment was sl~hrlr~nPd into an E;~RI site of an ~ .,u.~ on vector 2 0 aambda PL vector pKH20). The resulting plasmid was named pEIIa.
Pre~aration of ,e~-",l; ~"~ HIV-1 ~D41 anti~Pn The ~u-lDLI ucLiull of the envelope ~ U~Di(lll vector was a two step process. The first step involved the cu.lDLl U~.~U~ of an ~ ~QIi plasmid 25 ~....ln;l.;"~ a smaller DNA fragment c ---ln;..;..e env (~lPRie~at~Pd p41C). The second step involved the cullDLlu~Liu.. of an ~ vector with the ability to survive in both h RrhPrirhia ~L and ~ ~., and the introduction of the env fragment into this plasmid (-lPRienatpd pOM10).
An 854 base pair (bp) ~III/~mHI DNA fragment obtained from 3 0 plasmid pcK2 was ligated into the l~mHI site of pUC9 (Pharm~riQ). A clone c...~ a part of the env gene in the same oriPnt~tir~n as the 1~ gene was i~lPntifiP~l, mapped, and ~lPRiensltPd p41A. A 557 base pair bp ~mHI DNA
fragment obtained from plasmid pcK2 was ligated into the BamHI site of ~ WO 9S133206 2 1 9 1 4 3 0 P~ 21 plasmid p41A. A plasmid A ~ e the complete p41 sequence of the ~n~ gene in the same nriPnt~tiAn as the la~Z gene was i~lPntifiP~l, mapped, and rlP~ ed p41C.
An 13~ ~QIi plasmid ~ ; ; e the ~a~ sporulation promoter S (developed by Dr. R. Losick, Harvard University, and tlPRien~tPd pVG1) was restricted with ~m~I. This DNA fragment was ligated into the Bawllus plasmid pE194 which had ~ ;UU~Iy been restricted with ~2aI and blunt ends were formed using ~ ~nli DNA pGIy - c 1 (~enow fragment) to fill in the "sticky" DNA ends (blunt-end Lle~dLul~lt). A plasmid (~lPRien~ted pAS5) was 1 0 isolated, mapped and shown to have the ability to survive in both 1~ ~QIi and ~.
. The env gene was then inserted into pAS5. A DNA fragment from the plasmid p41C ~ ;..;..e the çnv gene was generated via EcoRV~I digestion and ,,uI,seu,u~ blunt-end l~lC diALUwlt~ This DNA fragment was ligated to plasmid pAS5 which had been l;.,P~ d with SalI and blunt-end treated. One 15 isolated clone (~ d pAS14) was rl ~ ;- Pd to have the ~rn gene fused to the ~à promoter in the proper r.. :. , I ,. ~
Finally, the t:lyllllulu.~wll resistance gene in pAS14 was replaced by the ~LIU1~ P~ 1 resistance gene from a related ~aç~ plasmid pC194 as follows. A 1107 bp DNA fragment ~- .l..;..;..~ the chlu~ h~ .; Al acetyl 2 0 I,~ r~ld&e (CAT) gene from a ~I~V~2}aI digest of the plasmid pC194 was isolated. This DNA fragment was ligated to the 6407 bp DNA fragment isolated from a Ç~LV~m~I digestion of pAS14 (a ~,. to~ which removes all of the original t~ly~ wll It ~is~cul~,e gene). The final plasmid obtained was lP~ d pOM10.
2 5 The promoter region, L ~ 1 start, and ribosomal binding site span bases 4840-4971. The coding region (bases 4972-6183) consists of derived from the spoVG region of the parent plasmid pVG1 (bases 4972-5004), ~:U~U~IlC~ derived during DNA ligations (bases 5005-5010) and ~e~lu~ rP~
derived from the HIV-1 env gene gpl20 (ba6es 5011-5145) (Ratner, L., et. ~1.
3 0 Nature 3I3:277-284, 1985). The p41 A~lu~ are from bases 5146-6180. The translation is ~ t-~d at the native tPrrnin~ti~n codon of the ~n~ gene (bases 6181-6183). The DNA sequence coding for the I. ~...,.l.; 1~.11~. protein was r~AnfirmPd by ReqllPnrin~ of the plasmid harvested after fPrmPnt~tiAn for three W0 95/33206 ~ r~/~

lots. The R~Pq~ n~Pd lots were r~l ut~ d from the same cell bank used to produce antigen for clinical mPster lots.
Plas_id pOM10 expres6es the HlV-1 envelope protein as a fusion protein c~ s.;.~;ue 11 amino acids derived from the amino t~ ..IIU8 of the spoVG
5 protein, 2 a_ino acids derived as a result of DNA m~nir~ t;~lnR during ligations, followed by the final 45 amino acids from the pl20 envelope protein and the entire p41 protein sequence. T_is protein is referred to as .~ --..~.;..-..~.
p41 (rp41).
Prenaration of r~r-.mhin~-nt ~IV-1 ~o41 filRirm grotein The construction of this ~ QIi clone ~ lC__ llg the HIV-1 CKS-120/41 fusion antigen was carried out in several steps. First the gene for the gp41 portion of the HIV-1 antigen was b.~.llL~L;~,~d and inserted into a modified pUC18 giving the plasmid pTB315. Ne~t, the DNA sequence coding for the carbo~yl 42 arnino acids of the gpl20 protein was synthesized and inserted into pTE',315 resulting in plasmid pTB316. E inally, the gpl20/41 gene was iI~ulsrel~t d to an ~ e~S; tll plasmid (pTB210) which allowed efficient of the antigen as a fusion protein. T_e resulting plasmid, pTB319, was isolated and mapped.
2 0 A gene encoding the amino acids 519-673, and 712-863 of the HIV-1 gpl60 envelope protein OE2atner et. al., ~[ah~ 313:277-284, 1985) was designed to be constructed from a series of synthetic DNA Lu~ in a pUC18 plasmid d~ . ;v~
Fourteen rI~l~;lllélllb were chemically srth~Ri7~ lu-lug a portion 2 5 of the published gp41 sequence. This sequence consists of amino acids 519-673 and 712-863 with a 38 amino acid ll ~ .. ~e region from amino acids 674-711 deleted. The 14 synthetic L~ u~ ~Ilb were subcloned into pWM500 (Ms~nfl~rki and Bolling, Gene 68:101-107, 1988), purified and ligated together to form the gp41 portion of the fusion protein. However, at amino acids 741 and 3 0 742, an A/T deletion occurred resulting in a 14 amino acid L~Lue6Lin, and ~lelui1iule translation t~rmin~t;rn in fragment 9. The resultant synthetic DNA sequence retains flanking BamHI and ~nI sites for insertion into a ~ W095133206 2 ~ 91 430 P~l~v ~r~
modified pUC18 plasmid with its ~I site destroyed by the insertion of a linker, rl~tgnAt~d pMB10.5.
A 129 base pair double strarlded DNA fragment ~ D~l.Lllg the carboxy-terminus of gpl20 was synthesized (311.3 and 311.4) and inserted into 5 the r~:... iUillg ~I site of pTB315. This fragment was inserted into plasmid pTB315 which was digested with ~I. A plasmid ~ l pTB316 was isolated and screened such that the ori~ntAt;~r, of the inserted fragment was inthe same ~ ... as the gp41 gene.
This plasmid, derived from plasmid pBR322, contains a modified 1~
10 promoter fused to a kdsB gene fragment (encoding the first 239 of the entire 248 amino acids of the ~ ~nli CMP-KD0 Synthetase or CKS protein), and a synthetic linker fused to the end of the ~ gene fragment. The synthetic linker includes multiple restriction sites for insertion of genes, tr~nFlqt;- n~l stop signals and the~ rho-in~ Ll~ls~ Lional L~ . This plasmid encodes 15 239 amino acids of CKS and 22 amino acids coded for by the synthetic linker.
Plasmid pTB316 was digested with ~amHI and ~I and a 1073 bp fragment was isolated. This fragment consisted of the original synthetic gp41 gene with the carbw~yl 42 amino acids of the gpl20 gene inserted in the proper location. This fragment was inserted into pTB210 which was pl~luuc~ly 2 0 digested with ~II and ~I. The resulting plasmid, liecignF ted pTB319, was isolated and mapped.
The promoter region, transcriptional start, and riho~mJll binding site span bases 45-125. The coding region is ~ ""'l" ;~d of s~ derived from the 239 amino acids of the CKS protein (bases 126-842) and the 11 amino acids 2 5 fro;n the synthetic polylinker (bases 843-875). This is followed by 42 residues of the pl20 HIV-1~ (bases 876-1001) and 185 residues of the HIV-1 p41 env (bases 1002-1556). The 38 amino acid deletion of the Ll~.~.-~...l- ,.,.~ region is between base pairs 1466 and 1467. Finally, there are an slrlrliti~ln~l 14 amino acids (bases 1557-1598) as the result of a frameshift due to a single A/T deletion and a 3 0 ~ "'r'iUU~ tr~ncl~t;~nlll t~rminzlt;~m (bases 1599-1601). The DNA sequence coding for the reC~mhin~nt protein was ronfirmr d by S~U,UI::11~Ug of the plasmid h~lvt~td after ~....~..lul;~-n for three lots.

WO 95133206 r~ 21 The plasmid pTB319 encodes a ,. ~ protein ~ ;..;..g 239 amino acids of the CKS protein and 11 amino acids from the pTB210 rnultiple restriction site linker. This is followed by 42 amino acids from the carbo-Ayl end of HIV-l pl20, 185 amino acids from the HIV-1 p41 protein la L- UllCdLOd proteinwith a 38 amino acid deletion of amino acids 674-711 (Ratner et. ~L. Nature Vol 313:277-284, 1985) spanning the p41 L 1 - region). Finally there are 14 amino acids resulting from a r.~ ~ and ~ ualu~c t~ due to a single A/T deletion between ,...rlP~ Li~ ,3 1556 and 1157. This protein is referred to as lF- ---.-1-;--_--1 pCKS-41 (rpCKS-41).
PICI,IAI pLi(,~. of .,.. l.;.. ~ ~IV-l D24 An~Fn The c ~llsL- u~Lion of the p24 ~ae CA~ vector was a multi-step process. The first step involved the construction of an ~ ~li plasmid, pBl, witha smaller e~E C-- IIA;II;II~ DNA fragment. The second step involved the l 5 construction of an CA1J~I vector, ~1F~ ;1 pKRR951, with the proper m~ lAr gignals to allow efficient tA~I Finally, molecular ,,.r... IIIAI -~1 wag added to the plasmid to allow regulation of gene t~
resulting in the final plasmid rKRRq55 A 949 bp ~II/~II DNA fragment obtained from plasrnid pcR23 was 2 0 ligated into the plasmid pUC9 (Pl IA I ~..Ar;A) ~ , . ;ou~ly digested with ~II
and ~amHI. A clone ...~A;I.;I~g a part of the ~ gene (inrlllrlin~ t~te p24 coding region) in the same orientation as the la~ gene was idPntifip~l. mapped, an d ~1 P ~i ~ n A t P d pB 1 The gae gene DNA fragment was then introduced into an CA~
2 5 vector pKRR810 w~tich placed the gag gene CA~ ' umder the control of the ~ ~21i lambda phage PL promoter while allowing efficient tPrminA1;nn of protein synthesis. A 963 bp DNA fragment C IA ~ E most of the ~ag gene was obtained by an ~QRI (complete)l~E~I (partial) digestion of plasmid pBl. A
synthetic oligontlrlPot~d~P DNA fragtnent of 36 bp was added to the ~ gene 3 0 fra~ment to reconstruct tLte amino-terntinus of t~te encoded protein and to place an l~QRI site immP(liAtPly upstream of the initiation codon. This modified fragment was inserted into the ~RI site of the CAy.~O~:Jll vector p~R810. A
clone (pKRR950) with the e~ gene in the same orientation as the phage PL

W0 95/332û6 r~

promoter wa6 i~lPntifiPA isolated and mapped. The size of this clone was reduced by 106 bp by ~h2aI digestion and religation of the pKRR960 plasmid resulting in a plasmid A~ 6~ d pKRR951.
To complete the ~ulll,Llu~Lion of the e~ vector, the lambda ~I~
5 regulatory gene and the ~ ~QIi la~nbda phage PR promoter were included within the construct. The addition of this t~ . . t sensitive gene allows control of the lambda ~. U~Jk~ and ~ n~ I ly of the ~ gene eA, A 2392 bp DNA fragment ~ v~ e the lambda çI$~ regulatory gene and the ~ lambda phage PR promoter was obtained via BglII digestion of a plasmid 10 called pRK248.~Its. This fragment was inserted into the BglII site of plasmid pKRR951 resulting in plasmid rKRRqv5 The promoter region, trAn~ v~l start, and ri~~_ 1 binding site span bases 7757-271. Tbis region is derived from two different lambda phage mutants and a synthetic region. The coding region is ~ d of a synthetic l 5 sequence which dllrlir~tP~ the NH2 end of the ~ gene from pUC9 (bases 272-307), seu,u,~ ,, coding for a portion of the HIV-l eae gene (bases 308-1183) including the entire p24 sequence (bases 344-1036), followed by a short sequencefrom the synthetic three frame translation ~ of the vector pKRR810 (bases 1169-1180). Translation is L~ (ed at the third termination codon in 2 0 this segment (bases 1181-1183). The sequence shows the m~1 Ll ~ls~ Li - (bases 1184-1241).
The plasmid pKRR955 produces a fusion protein ~mpricPd of 12 amino acids derived from the 1~ protein and the pUC9 polylinker region. followed by a portion of the ~ protein (inrlll~inf~ the final 12 amino acids of the pl7 2 5 protein, the entire 231 aminû acids of the p24 protein and the first 44 amino acids of the pl5 protein). followed by 4 amino acids derived from the tPrmin~t~
portion of the pKRR810 vector. This protein is referred to as l l ~ .. l .; .. ~. p24 (rp24).
3 0 P~ l.ivll of 1~ .. I.;.. ~nt ~V-2 ~o36 env ~ntiVPn The eUll~lll u~ion of this re- omhin~nt ;~ ~QIi clone ~lt s~illg the rp41 HIV-2 antigen was carried out in two steps. First a fragment of the HIV-2 env gene was isolated from a HIV-2 propha6~e and subcloned into an E,~

W0 95/33206 r~
21~1430 -~6 vector ,1~ "..l~ d pEHa. Second, a HIV-2 ~nY gene fragment was 5l1hrlnn~d from plasmid pEHa into an alL~.~Liv~ ,UI~ ;Oll vector, pTB210N, resulting in the plasmid pJC104.
A DNA fragment, - .IU;..;..E the env gene from HIV-2 (prophage isolate 5 D194.5) was identified within a lambda genomic library of prophage DNA. This fragment was -1hrlnn.qd into an EcoRI site of an E, ~ vector aambda PL vector pKH20). The resulting plasmid was named pEHa. Tis work was done by DIAGEN GmbH, r~. ~. . 1.. A~. Strasse 3, 4000 Dusseldorf (Kuhnel et. al. Proc. Natl. Acad. Sci. USA 86:2383-2387, 1989).
The cloning vector pTB210 allows the fusion of ,~.. 1.;.. 1. genes to the CKS protein. This plasmid consists of the plasmid pBR322 with a modified ~
promoter fused to a kdsB gene fragment (encoding the first 239 of the entire 248amino acids of the E ~QIi CMP-KD0 SyllLL~ 3C or CKS protein), and a syn~etic linker fused to the end of the 1~ gene fragment. The synthetic linker 15 includes: multiple l~,.,Ll;.,Lioll sites for insertion of genes, L~ ~ l stop signals, and the ~ rho-in~l~pPn~nt Ll~ Liullal t~ o .
The plasmid pTB210N contains a I~QI site in the synthetic linker and is derived from the plasmid pTB210.
Plasmid pEHa was digested with NcoI and a 314 base pair fragment 2 0 encoding the first 104 amino acids of the HIV-2 p41 protein was isolated and inserted into the NcoI site of plasmid pTB210N. This plasmid, ~ d pJC104, expresses the HI~-2 ~y protein as a fusion with the CKS protein.
The promoter region, L.all~ Liullal start, and ribosomal binding site span bases 46-125. The coding region is UUlll,Uli~ of s~ . derived from 2 5 the 239 amino acids of the CKS protein (bases 126-842) and the 13 amino acids from the synthetic polylinker tbases 843-881). This is followed by 104 residues of the amino end of the HIV-2 env (bases 882-1193) and 15 amino acids of the remainder of the polylinker (bases 1194-1238). The translation is ~ . ..; . .ù l ~d at the t~l~nin~tinn cûdon at bases 1239-1241.
3 0 The plasmid pJC104 encodes a recnnnhin~nt protein -.. lu~.. ;.. e the first 239 amino acids of the CKS protein, 13 amino acids from the pTB210N multiple ~L~;~Liull site linker, 104 amino acids from the HIV-2 env protein (amino acids 506-609 of the HIV-2 env protein), and an ~lit;~ l 15 amino acids from WO 95/332fi6 P~,~r~ Ul the pTB210N multiple restriction 6ite linker. This protein i6 referred to as rPrnmhinAnt p41 HIV-2 (rp41 HI~'-2.
pl~cmif~ host Pll gvstPm~
S The plasmid pOM10, prepared as described above, was 1""" r." ~Pd into ,lul~ll- of~ Pvn~ strainPY361(ap-ului-u,uLicd~.;`"~ of strain QMB1551 cured of native plasrnids) and viable chlu,~ ;rnl resistant cells were allowed to 1ef~ r~ EA,UI~ )~ of rp41 antigen was under the control of the ~ promoter and was observed when the cells entered the sporulation growth phase. This plasmid replicated as an in;pppn;pnt~ element, was non-mnhili7~hlP, and was ~..A ~ pd at d,UUIu~ ir~lely 10 to 30 copies per cell.
The plasmid pTB319 prepared as described above, was ~In~.~r ...PCl into E. coli K-12 strain Xll (r~;L, çn~, civrA96, thi-1, h~ll, ~a~, r~
~E~, ~Q~, l~IClzdeltAMlfi~ TN10) cells made C "l.eiv"l by the cPlcium 1 S chloride method. In this CU1rDI,I U~l,iUII the expression of the rpCKS-41 protein is under the control of the 1~ promoter. RP. -- ..~ n~ll pCKS-41 ~L,U~ was induced by the addition of IPTG to 100l1g/l. This plasmid replicated as an infiPpPnf-ipnt element, was non-mnhili7Ahl^ and was ...~ n;l~Pd at d~U,Ul u~LhllA Lely 10 to 30 copies per cell.
The plasmid pKRR955 prepared as described above, was ~In.. rr .. Pd into ~ ~QIi K-12 strain ~R136 (Dlac-pro, ~a~, thi-1, ~, ~h~fi., çn~, h~, lon-9, t~-r-462:T~lO/F'. ~a~fi, ~Q~+ ~qZ',t-iPltAMlfi) cells made C~ ..l e~v .l by the calcium chloride method. In this ~ulrDI~u~iOn the e~,uleD3;of rp24 protein was under the control of both the lambdapL and lambdapR
2 S pl uruOI~l D and the ~I~ repressor expressed from the ~I~a gene present on the plasmid. T~ePcmnhinAnt p24 expression was induced by l~r~u,uel~ule shift from 30-C to 42-C. This plasmid replicated as an inflPpPnflPnt~ element, was non-mnhili7slhlê and was mAintslinPd at d,U,UIU~ir~Ud~ely 10 to 30 copies per cell.
T_e plasmid pJC104, prepared as descTibed above, was L-~ - -r~ Pd into 3 0 _ ~ K-12 strain XL-I (r~, ç~, g~a~, ~hl~L ~11, ~i~, ~1, ~-E~, DrnAR. IArTq7:f-iPltslMlfi. ~;LQ) cells made c ~ P ~ by the calcium chloride method. In this construction the expression of the rp41 HIV-2 fusion protein was under the control of the ~ promoter. RPcnmhlnAnt p41 HIi,'-2 wo 95/33206 ~ ,r C ~

e~ was induced by the addition of IPTG to 100~1g11. This pla6mid ~e~ l,ed as an ;..~ element, wz6 non-mnhili7Ahl^ and wa6 ...n;..l~;..Pd at ~,u~lu~ ély 10 to 30 copies per cell.
The pla6mid pTB210, prepared a6 described above, was l.A~.Ar~.. ~d into S p;, ~ K-12 strain ~1 (~L çn~l, ~vrA96, thi-l, ~11, ~, ~1, ~-E~, ~Q~, ~q7.~Plt.nMlfi. ~Q) cells made ~ by the calcium chloride _ethod. In this ~. Allu~liul~ the e~yl~ of CKS protein wa6 under the control of the 1~ promoter. CKS e~ was induced by the addition of IPTG to 100~gll. This plasmid leuli~ed a6 an in~PpPn~lPnt element, was 10 non-mnkili7AhlA and was mAintAinpd at a~ulu,,i~.rAlely 10 to 30 copies per cell.
Example 10 ~TTV-l/FTV-2 Anhhodv ,~RR~ Usin~ One Solid phAAAP
One solid phase was coated with the l~.. l.;.. A.. ~. antigens rp24, rp41,15 and rp36 as described in Example 3 and u3ed im an assay to detect the pre6ence of HIV-l/HIV-2 antibody in a test 6ample. The assay ~nntlit;nnR were essentially as described in Example 3, except that sample volume wa6 decrea6ed to 60 IlL, from 150 ~lL u6ed in the licensed assay of Example 3.
Evaluation was rnn~ ted on 121 diluted HIV-2 6ample6 derived from 30 2 0 individual6, a fresh random donor pop~ll At;nn ~ of 2194 plasma and 980 serum, and 153 HIV-1 5el0~ull~G.~;ull samples derived from 19 individuals.
A6says using synthetic HIV-2 peptide I, (as described above) then were compared to the results obtained for the same serum sample when using an HIV antibody assay (Human T.. -~.n~l~f;~iPn~y Virus Types 1 and 2: ~:. coli 25 and B. ~P~,AI~-;-IIII. recQnnhinAnt antigen, Abbott HIVAB~9 HIV-lIHIV-2 (rDNA) EIA; available from Abbott T nh--- Al..l ;~ ~ Abbott Park, IL), Abbott List 3A77 or Abbott LIst 3A10 following .. IA .... fA. ~ ~ . el 's directions as provided in each product insert. The data from these assays are presented in Table 3. The absulbA,l~G of each well was read at 490 mn with a 630 nm reference. The 3 0 cutoff value of .025 OD + mean OD of the negative control was rA~AhliRh~
Samples werê considered reactive (positive) when the ~ ,uloff value was greater than 1 In Table 3, "OD" refer~ to the ophcal density reading, "S/CO"

WO 95~33206 1 ~

means ~mrl~r~ off value, "Result." refer6 to the i~ l ntion of the test, "NC" refers to negative control and "PC" refers to the positive control.

HIV-2 DILUTIONAL SE~b-llVll ~
Assay Number of samples Mean S/CO HIV-2 detected samples No. Positive/Total No.
3A77 50/121 1.87 rDNA/synthetic HIV-2 10~121 8.62 peptide I-modified 3A10 102~121 7.73 WO 95/33206 ~ 71 219l430 Individual test samples results which were D"~ d in Table 3 are given in Table 4, below.
l~ABLE 4 5HIV-2 DILI~TIONAL SE~ Llv~
Samp~e ID 3A77 rDNA/Synthe~ic HIV-2 3A10 (mean value) CO=O.lQ9 CO=0.152 CO=0.113 S/CO S/CO S/CO
261695 0.52 6.06 3.47 412378 2.59 8.16 7.98 GL99 0.63 4.82 4.15 60415K 1.80 10.99 8.57 2~310 1.25 7.77 6.48 228~4S 1.52 7.94 7.49 25017 3.93 5.40 9.38 G186 0.55 8.36 5.27 G100 1.51 13.17 10.47 25383 0.67 6.26 5.0'7`
99g.72 1.91 13.45 L2.24 G268 Q84 7.74 5.54 22681 0.95 5.52 5.41 221~4 138 9.55 9.89 18651 L99 LLll 9.29 20946 1.31 9.35 7.48 3Q~A8 0.51 791 4.47 0114~3 2.63 lQ69 lQ51 18332 L34 8.81 8.47 30533 L24 10.96 7.48 90.7A8 0.66 8.59 5.35 4371 lQ59 L2.49 14.23 41397 0.33 2.57 1.66 24436S 2.10 7.58 8.43 35L7.7 3.92 8.60 lQ91 20101 Lll 6.66 6.17 32200 L41 7.40 6.28 2~469 2.06 8.90 lQ55 N DANFU 2.77 11.54 11.05 PVINEUL 0.98 lLll 7 40 ~Dilution means of 4 6amples: 1:5, 1:25, 1:1.125, and 1:625 This data illustrates that the improved HIV-l/HIV-2 rDNA/6ynthetic HIV-2 peptide I offers increased HIV-2 sen6itivity and improved specificty, without Cuul~u~,u iDillg HIV-1 DC:IU~,~/U~D-UII Si UD;~iVily (Table 5, below). A10 reduction in the required sample volume will allow use of the assay in settings where 150 yl is too restrictive. However, either sample volume will achieve the same improved results.

W095t33~06 2 1 9 1 4 3 ~ P~ IQ41"1 ~V l SEROCONVERSION SE~blllVll 1 USING 50 IlL SAMPLE VOLUME
No. of Samples Detected Mean S/C0 Assay No. Positive/Total No. 5~ . Samples 3A77 170/249 5.10 3A10 170/249 3.36 rDNA/synthetic HIV-2 172/249 7.19 peptide I
Example 11 C~ .A.;A-- ofthP rDNA/sn-thP~ie~Tv-2peDtideformAtwith 3A10 Assay6 were ~....l... ~d as described in Example 10 using ~0 ~L sample volumes. A hr~n -' l~, - format was used. The results are pl es~t d in Table 6, below. The results show a Rigni~ nt decrease in the number of false0 positives in that assay using the synthetic HIV-2 antigen peptide I as a of the indicator reagent in contrast to a purely recnmhinAnt HIV-2 gp36 antigen as indicator.

1 5 b~PECIElCll'Y COMPARISON l~ ;~ 3AlO AND rDNA/SYNT~IC
HlV-2 ~;t'llU~; I
rDNA/Synthetic EIIV-2 Result 3A10 Result Sample ID CO=0.152 CO=0.113 S/CO S/CO
6130.86 -- 4.31 +
5140.81 -- 3.12 +
5150.67 -- 3.22 +
5160.37 -- 1.45 +
5170.42 - L76 +
5182.26 + 17.6 +
5190.86 -- 7.14 +
5200.42 -- 1.16 +
æl ~29 + 5.95 +
æ2 4.01 + 17.60 +

W095133206 21 91 430 r~ ~ o~
- ~2 -523 3.17 + 17.60 +
5~4 2.00 + 17.60 +
52~i 0.40 -- 3.05 +
526 0.40 -- 6.98 +
527 1.82 + 17.60 +
528 3.76 + 17.60 +
529 4.29 + 17.60 +
530 0.53 -- 8.62 +
531 0.53 -- 2.90 +
532 OA9 -- 3.03 +
533 0.40 -- 2.68 +
534 1.61 + 3,49 +
5~5 0.40 -- 1.34 +
536 0.63 - 6.14 +
No. samples test 9/24 24124 r.~"~.Li~ negative s~mples Example 12 DetPrt;~7n rlfH7~v Sl7htyDe SDe~;Lc;ly U~in~ a Synthetic T~lV-1 PeDtide 7~ r~7ifiPr7.
Assav The following data i7l1u6trates the ability of a H7lV-1 synthetic peptide-modified assay to detect emerging ~ uly~es of HIV strains. Assays using synthetic H7iV-1 peptide XIII were compared to the resu7,t6 ûbtained for the same serum sample when 7l3ing an HIV antibody assay (Human T... Adr~ y Virus Types 1 and 2: ~:. coli and B. I~Pl ~
10~ 7r.;~ . antigen, Abbott H71VABB) HiV-VHlV-2 (rDNA) EiA; available from Abbott Laboratories, Abbott Park, IL), Abbott List 3A77 or Abbott LIst 3A10 following ~.n.. ~ L .~,.'s directions as provided in each product insert.
The data from these assays are presented in Table 7. The absol7ut-~l~e of each well was read at 490 nm with a 630 nm reference. The cutoff va7lue of 0.100 OD
15 + mean OD of the negative control wa6 P~tfo7,rtliRhP~7~ Sample6 were con6idered reactive (positive) when the R~mp7 ~ Lurrva7lue was greater than 1 In Table 7 WO 95/33206 r~ ,.,. .v4~7~

"S/CO" means sample/cut-off value, "Result " refers to the i~ ion of the test.

D~t~Li~ of ~ ~ Samples u~ing IUV-1 synthetic petide Xm ~ ~ 1 EIA
Sample US Licensed Assay HIV-l Peptide Modified 3A77 Assay S/CO Result S~CO Result Cameroon Sample No. 5 0.60 - 8.41 +
Cameroon Sample No. 2 0.20 - L95 +
This table shows that Cameroon samples Nos. 5 and 2, which are subtype O samples were not detected in the standard assay but identified positive using the HIV-1 peptide modified assay.
HIV-1 Peptide XIII-modified ElAs on ssmples identified as Subtype 0 are 10 shown below. The results indicate an increased ~ ,iLi\d~y in the assay to subtype O antibody when the synthetic peptide is present.

of ~liv;Ly 3A10 and ~llV-I Peptidc l\T~ y on S~btype 1 5 0 Samples Ahhn~ 3A1() ~TV-i Pe~ti~ip MnAifiPrl Sample S/CO Result S/CO Result 2.00 + 15.00 +
2 0.20 -- L80 +
3 7.00 + 460 +
5 0.65 -- 10.80 +
6 3.00 + 7.60 +
7 2.80 + 12.70 +
8 2.30 + 4.70 +
No. samples 5~,7 717 tested p~ v J. ~ .
positive samples W095/33206 ~ /04'71 -s4 It i8 rnnt~nn,Al s~t~d that the assay of the invention can be optimized even further by varying assay rAntlitinn~ and/or inr-lh~ ii~n times, using various ~mnhinsltl~A~ of antigen or antibody capture or probe reagents, and other methods, reagents and ~ ..1;l - .~ known to those skilled in the art. Thus, 5 various other antibody capture reagents can be used, including ~IIV p24, gpl20, gpl60, pl7, and others. The variance of the antibody capture reagent may then require the use of a different antigen capture reagent. All these variations areC..-,~ to be v~ithin the scope of this invention. Also, while some of the assays described in the e~amples used an ~-ltA n~tPd system, it is well within I 0 the scope of the present invention that manual methods or other ~ IPd analyzers can be used or adapted to the assay of the present invention.
Therefore, the present invention is meant to be limited only by the appended claims.

Claims (19)

WHAT IS CLAIMED IS:

1. An assay to detect the presence or amount of antibodies to one or more HIV genotypes in a test sample comprising the steps of:
a. contacting the test sample with i. a first capture reagent attached to a solid phase, wherein said capture reagent is a polypeptide comprising HIV-2 env, ii. a second capture reagent attached to a solid phase, wherein said capture reagent is a polypeptide comprising HIV-1 env, and iii. a third capture reagent attached to a solid phase, wherein said capture reagent is a polypeptide comprising HIV-1 gag;
to form a first mixture;
b. contacting said first reaction mixture with i. a first indicator reagent comprising HIV-2 env antigen labelled with a signal generating compound, ii. a second indicator reagent comprising a HIV-1 env antigen labelled with a signal generating compound, and iii. a third indicator reagent comprising a HIV gag antigen labelled with a signal generating compound, to form a second reaction mixture;
c. determining the presence of said HIV-1 env antibodies, HIV-2 env antibodies and/or HIV gag antibodies in the test sample by detecting the total signal generated by HIV antibody/ capture reagent/indicator reagent complexes.

2. The assay of claim 1 wherein said first indicator reagent comprises a synthetic site-directed HIV env antigen having an immunoreactive specificity characteristic of an immunodiminant region of gp36 of HIV-2.

3. The assay of claim 2 wherein said immunodominant region of said synthetic HIV antigen is about 120 amino acids from the N-terminal region of gp36 of HIV-2.

4. The assay of claim 2 wherein said synthetic site-directed HIV-2 env antigen comprises a substantially pure peptide containing at least two cysteine residues which have been chemically cyclized to form a disulfide bridge between the two cysteines.

5. The assay of claim 4 wherein said synthetic site-directed HIV env antigen is a peptide selected from the group consisting of:
(a) (b (c)

6. The assay of claim 2 further comprising an indicator reagent comprising a recombinant HIV-2 env antigen labelled with a signal generating compound, said indicator reagent present from between about 0.014-about 1.4 micrograms per assay test.

7. The assay of claim 1 wherein said second indicator reagent comprises a synthetic site-directed HIV env antigen having an immunoreactive specificity characteristic of the immumodominant region of gp41 of HIV-1.

8. The assay of claim 2 wherein said synthetic site-directed HIV-1 env antigen comprises a substantially pure peptide containing at least two cysteine residues which have been chemically cyclized to form a disulfide bridge between the 2 cysteines.

9. The assay of claim 8 wherein said synthetic site-directed HIV-1 is a peptide of the formula a wherein a is Arg-Ile-Leu-Ala-Glu-Arg-Tyr-Leu-Lys-Asp or Arg-Ile-Leu-Ala-Val-Glu-Arg-Tyr-Leu-Gln-Asn;bis Gly or Ser; c is Ile or Leu; d is Ser or Lys; e is Ile or Val;
and f is Thr or Tyr.

10. The assay of claim 9 wherein said synthetic site-directed HIV-1 is a peptide selected from the group comprising:
(a) (b) (c) (d) (e) (f) (g) (h) (i) (j)

11. The assay of claim 7 further comprising an indicator reagent comprising a recombinant HIV-1 env antigen labelled with a signal generating compound, said recombinant HIV-1 antigen comprising the carboxy terminus of gp120 and complete protein sequence of gp41.

12. The assay of claim 1 wherein said third indicator reagent comprises a synthetic or recombinant HIV gag antigen having an immunoreactive specificity characteristic of an immunodominant region of p24 of HIV-1.

13. The assay of claim 1 wherein said first, second, and third capture reagents are bound to at least one solid phase.

14. The assay of claim 13 wherein said solid phase is selected from the group consisting of magnetic beads, non-magnetic beads, wells of a reaction tray, microparticles, nylon strips and nitrocellulose strips.

15. The assay of claim 1, wherein said first HIV-1 gp41 env capture reagent, said second HIV-2 gp36 env capture reagent, and said third HIV p24 capture reagent are recombinant or synthetically produced.

16. The assay of claim 1 wherein the signal generating compound of the HIV antibody indicator reagent is selected from the group consisting of enzymes, luminescent compounds, chemiluninescent compounds and radioactive elements.

17. The assay of claim 16 wherein said enzyme is horseradish peroxidase.

18. A test kit for simultaneously detecting the presence or amount of antibodies to different HIV genotypes in a test sample comprising:
(a) a HIV-1 antigen capture reagent comprising at least one polypeptide having an immunoreactivity specificity characteristic of the gp 41 env region of HIV-1 attached to a solid phase;
(b) a HIV-2 antigen capture reagent comprising at least one polypeptide having an immunoreactivity specificity characteristic of the gp 36 env region of HIV-2 attached to a solid phase;

(c) a HIV antigen capture reagent comprising at least one polypeptide having an immunoreactivity specificity characteristic of the p24 env region of HIV attached to a solid phase;
(d) an indicator reagent comprising a synthetic HIV-2 gp36 env antigen labelled with a signal generating compound;
(e) an indicator reagent comprising a HIV-1 gp41 env antigen labelled with a signal generating compound; and (f) an indicator reagent comprising a HIV gag antigen labelled with a signal generating compound.

19. The test kit of claim 14 further comprising an indicator reagent comprising a recombinant HIV-2 env antigen labelled with a signal generating compound.