Note: Descriptions are shown in the official language in which they were submitted.
W O 93/21346 2 1 3 3 8 2 6 P ~ /US93/03224
ASSAY FOR DErECTlON OF HIV ANTIGEN AND HIV ANTIBODY
This application is a continuation~in-part of U.S.S.N. 787,710 filed
November 4, 1991, which is a con~inuation application of U.S.S.N. 071361,733
5 filed June 2, 1989 tabandoned), which is a continuation-in-part application ofU.S.S.N. 07/320,882 filed March 9, 1989 (abandoned), which is a continuation
application of U.S.S.N. 07/020,282 filed February 27, 1987 (abandoned
which is a continuation-in-part application of U.S.S.rJ. 06/811,240 filed
December 20, 1985 (abandoned), which enjoy common ownership and are
1 0 incorporaled herein in reference.
BACKGROUND OF THE INVENTION
1 ~ This invention relates generally to immunoassays, and rnore particularly,
relates 2O an immunoassay and products for the simultaneous detection of HIV-1
antigen analyte and HIV-1 anlil~.~y analyte and/or HIV-2 antibody analyte in a
test sample.
Acquired immunodef~ciency syndrome (AIDS) is a disorder of the immune
2 0 system ~ss~ Pd with oppOnunistic infections and/or neoplas",s which has
reached epidemic pr~po.lions in the United States as well as in Europe and in
central Africa. The epidemiobgical data suggest that AIDS is caused by at least
h~o types of human immunodefic;~.)c" viruses, collectively designated as HIV.
I IIV type 1 (HIV-1) has been isoi~te~ from patients with AIDS and AlOS-related
2 5 complex (ARC), and from healthy pe,~Gns at high risk for AIDS. See, for
exa,.")l~, F. Barre-Sinoussi ~t al., Science 220:868-871 g1983); M. Popovic
et al., .S~ ence 224:497-500 (1984); and R. C. Gallo et al., Science 224:500-
503 (1984~. HIV-1 is l~ans".illed by sexual contact. exposure to blood and
certain blood product~,,or from ~n infec~ed mother to her fetus or child. P. Piot
et al., Science 239:573-579 (1988). The prevalence of HIV-1 antibodies in
AIDS and ARC pa~ients and ~r~ons at risk is high, and the virus can be isol~ted
from nearly 90% of all se,uposi~ e individuals. See, for example, M. G.
Sa...gaJha.~n et al., Science 224:505-~08 (1984); and D. Gallo et al., J. Clin.
Micro. 25:1291-1294 (1987).
In 1986, a second human immunodeficiency virus, HIV-2, was isolated
from ~)atienls with AIDS in west Africa. F. Clavel et al., Science 233:343-346
t1986). HIV-2 infections also have been identified in individuals from several
WO 93/21346 ; PCI'/US93/03224
21~3~26 - 2 -
coJntries outside of west Africa. See, for example, A. G. Saimot et al., L:anceti:688 (1987); M.A. Rey et al., Lancet i:388-389 (1g86); A. Werner et al.,
Lancet i:868-869 (1987~; G. Brucker et al., AIDS 2:141 (1988); and IC.
Marquart et al., AIDS 2:141 (1988). Although at the present time HIV-2
appears to be endernic only in west Africa, it appears likely that, based on the
-
experience with HIV-1, HIV-2 will spread to other parts of the world. ,
:
HIV-2 virus is similar to HIV-1 virus in its morphology, cell tropism,
interaction with the CD4 cellular receptor, in vitro cySopathic effect on CD4
cells, overall genomic structure and its ability to cause AIDS. F. Clavel, AIDS
1 0 1:135-140 (1987). However, HIV-2 differs from HIV-1 in several respects.
See F. Clavel, Ibid and R.A. Weiss et al., AIDS 2:95-100 (1988).
Serological tests indicate that HIV-1 and HIV-2 share muitiple common
~ ;
epitopes in their core antigens, although their envelope glycoproteins are much
less cross-reactive. F. Clavel, supra. This limited cross-reactivi~y of the
envelope antigens may explain the failure of most currently-available
sero'~g ,,~l assays far HIV-1 to react with certain sera from individuals with
antiL,G~y to HIV-2. F. Denis et al., J. Clin. Micro. 26:1000-1004 (1988). A
cG"""e-cially available assay for HIV-1/HIV-2 antibody, recently available
from Abbon LabG.atories (Abbott Park, IL 60064), designated as the Abbott
HIVAB(~ HIV-1/HIY-2 (rDNA) EIA, uses recombinant antigens corresponding to
the two viral p,-~teias, HIV-1 envelope and HIV-2 envelope. The use of fhese
recombinant anligens allows for the improved detection of anti-HlV-1 andlor
anti-HlV-2 containing test samples, while mini~,.kil)g non-speci~ic reaclions
largely due to cross rea~;tions with whole virus or viral Iysate. The usa of at
2 5 least one recombinant HIV protein to detect HIV antibody in a test sample with the
use of l~ "~d recombinant HIV anligens is described in the parent patent
applications previously inco",orated herein by reference. -~
Based on the nucieotide analysis of the viral genome the HIV geno",~e RNA
encodes (beginning at the ~' end):
3 0 ( i ) a gag gene extendirig belJ:een nucleot;ldes 310 to 1869 and encoding for
the internal structure core or nucl~ocP~r~ proteins including p24, the most
antigenic core protein;
( i i ) a pol gene exlending ~et~een nucleotides 1,629 to 4,673 and encoding for -
the e~ ,e, reYerse ~,anso,i~tdse; and
3 5 ~ i i i ) an env gene extending between nucleotides 5,781 to 8,369 and encoding
for the envelope glyc~protein including gp41, the most antigenic envelope
protein. Ratner et al., Nature 313:277-284 (1985).
WO 93/21346 2 1 3 3 8 2 6 PCr/US93/032~4
One of the challengés faced by today's medical community is the protection
of blood produots from contamination by HlV, which has been found in blood
products (as well as other human body fluids), and which reportedly has been
transmitted in the blood supply. Several assays are available to date, includingthe assay described in U.S. Patent No. 4,520,113 to Gallo et al.
Also, other assays which can detect HIV antigen or HIV antibody are
known. Such assays include the anti-HlV-1/HlV-2 assay described hereinabove
and disciosed in the previously referenced parent arpliG~tion, as well as those
taught by U.S. Patent No. 4,748,110 to D. Paul, U.S. Patent No. 4,983,52~ to J.
Stewart et al., and U.S. Patent No. 5,173,399. How3ver, all known federally-
approved assays for detection of HIV antigen analyte or HIV antibedy analyte areonly capal~lc of separately delecti"g either HIV antigen analyte or HIV antibodyanalyte in a test sample. No known commercially available, federally-approved
assay is available for deteotion of both HIV anligen analyte andlor HIV antibody1 ~ analyte in a single assay using a test sampl~.
The detec~ion of more than one analyte in a test sample usually involves
the separate ~ete~l~n of each analyte in a sepa-tte assay. Such detection methods
have been prefer~ed since they a~low ~or s~ingen~ quality assurance
dele.".;,~dtions to be pelf~,r--,ed for each analyte to be tested.
Advances in medicine have brought a ,ec~,-ilion of new markers for many
d:se~ses and clinicai conditlons, along with the d~"-and for clinical tests for these
marlcers. Labor~to-ies are faced with the problem of providing increasing
amounts of tests in a timely manner while atle",plil)g to keep costs down. For
example, the testing requirements of blood banks have increased dramatically dueto the addition of Human T-L~ukemia Virus Type 1 (HTLV-1), I~IV and Hepatitis
C Virus (HCV) to the panel of agents tested in these labo(atones on donor blood
for the p~ese"ce of or e~posure to these a~ents.
One possible solution to reducing the lal~l~tory workload brought about
as a result of testing reqlJirements, especi~'ly in blood banks. is to find ways to
3 0 combine assays. I low3ver, combining assays without compromising their
individual pe,f,.--,ance sldncJa~tJs is difficult and more importantly, the
problems involved in manufacturing and quality control can be insurmountable.
Several in~es~ig~to,~ have dcve'~ or have a~e,..pled to develop assays
to simùltaneously detect more than one analyte in a test sample. Such an assay
3 5 would be ad~anlageous since the time involved in de~ec~ing mor~ than one analyte
in the test sample would diminish considerably, and the cost of each assay would
WO 93/2134~ PCr/US93/03224
2133826 4
be lowered since less technical time, reagents, and equipment would be required
to perform such an assay.
For example, U.S. Patent No. 4,315,gO7 to Fridlender et al. teaches"a
heterogeneous specific binding assay system wherein separation of bound-species
5 from a free-species form of the labeled reagent occurs.
U.S. Patent No. 4,378,344 and EP 0270n8 to Zahradnik et al. teach a
solid phase device for determining the presence of each analyte comprising a
recept~la and an ins~rt wherein the presence of each analyte is determined by
the ~a;,ncd assay method. .
Great Britain Patent No. 2188418 ~eaches an assay tray assembly having
reaction wells each with openings in the top surface from which a projection is
e~lended and wherein the inner surface of each well sidewall and the outer
surface of each projection may be incubated simultaneously for detecting two or
more sp~ilic substances present in a specimen which has been introduced into
the reaction wells.
EPA No. 0 351 248 to applic~nt IDEXX Co"uoralion discloses a
simultaneous immunoassay for feline viruses or HIV in which an antigen and/or
art;luoJy "-erlber of a single binding pair are d~lec"'~l~. Also, U. S. Patent No.
5,039,604 to Pa,~sidero teaches an immu-,oassay which simultaneously detects
2 ~ two HTLV or I ilV aniibodies by adding two dif~erent anl;gens and then a single
anlil~dy which is reactive with both antigens. In ad~it;on, U.S. Patent
No. 4,870,D03 to Kortright et al. discloses a solid phase immtlnoassay for
~let~tion of an antigen and/or antil)ody of a single binding pair utilizing an
antigen ~spike~ of inactivated antigen.
Also, the detec~ion of one or more analytes using two or more solid phases -
is the subject matter of co-~enJing U. S. Patent ~pplica~ion Serial No. 574,821
(EP-A-473 065, published March 4, 1992).
The critical factors which have been identified to the development of
simultaneous assays are !hat the two assays to be performed simultaneously must
3 0 have the sarne sample volumes, identical incubation times and identical cut-off
- calculations. Such a simultaneous assay also should be capable of being
separately quality cor,l~ d for each analyte, both al the manufacturer and at
the la~ratory using the assay, to ensure the sensitivity, specificity and
reproducibility of the immunoassay.
3 ~ It ll-e~fore would be advantageous to provide an assay wherein thepres~ of more 1han one HIV analy~e could be simultaneously deter ted, yet each
s~:~d.ate analyte to be detected could be individually quality controlled. Such an
WO 93/21346 PCr/US93/03224
5 - ~13~326
assay would be an improvement over other known assays since the simultaneous
determinations of the presence of either HIV antigen analyte and/or I IIV antibody
analyte would be performed in one well, separation of solid phase components
would not be required if more than one solid phase was utilized, and the assay
5 could be quality controlled for individual analytes which were to be detected in
the simultaneous assay.
SUMMARY OF THE INVENTION
1 0
This invention provides an assay to simultaneousiy detect the presence, if
any, of an HIV antigen analyte andfor an HIV anlibody analyte which may be
present in a test sample. The assay comprises simultaneously contacting the testsample with (i) at least one HIV antigen analyte capture reagent specific for said
15 HIV antigen analyte attached to a solid phase, and (ii) at least one HIV antibody
analyte capture reagent specific for said HIV antibody analyte attached to a solid
phasa, to form a first mixture; incubating said first mixture for a time and
under col-titions sufficient to form HIY antigen capture reagentfHlV antigen
analyte complexes andfor HIV an1iL~ capture reagentfHlV antibody analyte
2 0 co"-l~lExes; contacling said complexes wi1h (i) an HIV antigen analyte indicalor
rea~enl which co,-,pri~es a ..,e,.-l~er of a binding pair spedfic for said HIV antigen
analyte labe"Ed with a signal gener~ting compound, and (ii) an HIY ar,liL~d~
analyte ir.~icdtor reagent which ~,--prises a member of a binding pair specific
for the I IIY anliL~Jy analyte l_Lel'ed with a signal generating compound, to form a
2 5 second mixture; incubating said second mixture for a time and under conditions
sufficient to form HIV an1igen capture reagenVHlV antigen analyte/indicator
reagenl complexes andJor HIV anlil,Gdy capture reagentfHlV antibody
analyte~ Jicator reagent complexes: and determining the presence, if any, of
said HIV antigen ana!y~e andfor said HIV anlil~dy analyte in the test sample by
3 0 delecti-lg a signal genert~ed an indication of the presence of either or both
- analytes in the test sample.
The presenl invention also proYides an assay to simultaneously detect the
p-~sence, it any, of an HIV antigen analyle andfor an HIV anlil~d~ analyte in a
test sample, co-.-~,-isi..g simultaneously contacting the test sample with: (i) at
3 ~ least one HIY antigen capture reagent spec 7f;e for said HIV antigen analyteatl~ched to a solid phase, (ii) at least one HIV anliL~dy capture reagent specific ~;
for said HIV antil,ody analyte attached to a solid phase, and (iii) an HIV antigen ~
W O 93/21346 P ~ /US93/03224 . .
213~826 (-' ~
- 6 ~
:
analyle indicator reagent which comprises a member of a binding pair specific
for said HIV antigen analyte lab~lle~ with a signal generating compound, to form a -~
first mixlure; incubating said first mixture for a time and under conditioas ;,
sufficient to form HIV antigen capture reagenVHlV antigen analyte/HlV antigen -
5 indicalor reagen~ complexes and HIV ar,lil,od~ capture reagen~/HlV antil)ody
analyte complexes; conlacting said complexes with an HIV antibody analyte ;
indicator reagent which cG,-,~.rises a member of a binding pair specific for said
HIV antibody analyte IzLel'ed with a signal generating compound, to form a second
mixture; incubating said second mixture for a time and under conditions ~- ~
sufficient to form anliL~Jy HIV anliL~-ly capture reagenUHlV antilGdJ ~ ;
analyle/Hlv antil~JJ~ analyte indicator reagent complexes; and dete.-.. ;ning the ;
presence, if any, of said HIV antigen analyte and/or said HIV antibody analyte in
the test sample by detecting a signal gener;~ted.
The HIV antibody capture reagent and said antigen capture reagent can be
all~.;l.eJ (bound) to the same or ~ lerenl solid phase. r~efe.-ed solid phases
indude magnetic beads, non-magnetic beads, wells of a re~lion tray,
microparticles, nylon strips and nitrocelluhse strips. The HIV antigen analyte ~ ~ -
is HIV p24 gag antigen and HIV antigen analyte capture reagenl is anli-HlV p24
antibody, eithér .-.GnoclQnal or polycbnal or reco-..~ 1antly derived, or
2 0 f~-enls~ thereof. ~lel~r~ , the HIV antigen capture reagent is a mixture of
,,~rn~lonal antibodies secret~l by ~.yl.-i~---a cell lines ATCC Deposit Nos. HB
9725 and~iHB 9726, while the HIV anlibody capture reagent is tlIV-1 p41 -~
proteh, or a mixture ol HIV-1 p41 protein and HIV-2 p41 env protein. Most
preterably, these antigens are recombinantly produced The signal geneidting
2 5 ~ com~ound of the HIV antigen analyte indicator ~eagtnl and the signal generating
compound of the HIV antibody analyte indir ator reagen~ is s~ le~ from the groupc~n~ 1ing ot enzymes, luminescent comrounds, chemiluminescent compounds and
radbactive~ elements. Add~tional~y, either or both indicator reagents may further
comprise a hapten. ; , I ~
3 0 The abo~e~es~il,ed assays can be ~cl~ed for the detection of both HIV-1
an~en anakte and HIV-1 and HIV-2 antibody analytes separately. In this assay, :~
the steps comprise (a) contacting the test sample with a solid phase to which at h ~ ,
least one anti-HlV p2~ antibody and at least one recombinant HIV-1 p41 antigen
and at least one HIV-2 p41 antigen have been attached, to form a mixture; (b)
incubating said mixture for a time and under conditions sufficient to form anti- ~
HIV p24 antibody/HlV-1 p24 antigen complexes and/or HIV-1 p41 anligen/HlV- ~- '
1 antibody and/or HIV-2 p41 antigen/anti-HlV-2 p41 antigen complexes; (c)
.
WO 93/21346 PCI'/US93/032~4
7 213~826
detecting the presence of HIV p24 antigen in the test sample by: ~i) contacting
said anti-HlV p24 antibody/HlV p24 antigen complexes with an anti-HlV p24
antibody capable of sp~cifi~lly binding HIV p24 antigens for a time and under
conditions sufficient to form anti-HlV p24 antibody/HlV p24 gag antigen/anti-
5 HIV p24 antibody complexes (ii) contacting said anti-HlV p24 antibody/HlV
p24 antigen/anti-HlV p24 antibody complexes with an indicalor reagent
co",plising an anti-species anti~ody labeled with a signa~ generating compound
and (iii) de~ecling the signal generated as an in~lical:~n of the presence of HIV
p24 antigen in the test sample; and (d) detecting the presence of anti-HlV-1 p4110 antibody and/or HIV-2 p41 antibody in the test sample by: (i) con~&c~ing saidHIV-1 p41 antigenlanti-~llV-1 p41 antibody complexes and/or HIV-2 p41
antigen/anti-HlV-2 antibody complexes with an i--d;ea~or reagent comprising
HIV-1 p41 antigen labeled with a signal generating compound and HIV-2 p41
antigen labeled with a signal generating compound; and (ii) detecting the signal1~ generated as an indication of the presence of anti-HlV-1 p41 antibody an~or
HIV-2 p41 antiLody in the test sample. This assay also can be pe.h.r",ed using
two (or more) solid phases.
Test kits for pe.Iu",.i.,9 the assays of the present invention also are
provided.
DETAILED DESCRIPTION OF THE INVENTION
An assay for the dete~t;~n of HIV antigen analyte andlor HIV antibody
25 analyte in a test sample is u.oJided. Th~ assay p,~fc,ably is pe,lu-,,,ed as an
imml",oassay although the prese,-~ invention is not limited to immunoreactive
assays. Any assay utilizing spe~ific binding ~,.e,-~be,~ can be pe-lu,,,,ed. A
~p~;~;o binding member~ as used herein is a n.e..,ber of a Spe~if;G binding
p~ir. That is, two different molecules where one of the molecules through
3 0 chemical or t)h~ical méahs specifically binds to thè second moiecule Therefore ~ '
in addition to antigen and ar,t;~ specific binding pairs of c~,.. -,-Gn ;~
immunoassays other spec;fic binding pairs can include biotin and a~idin ~ '
carbsnyJr~les and lectins complementary nucleotide sequences effector and '
f~pt~r ~ lscu1es cof~ctof~ and enzymes. enzyme inhil,it~r~ and en~-"es. ~nd ~;
the like. Fu.ll,e-,-lore spec;fic binding pairs can indude members that are '~
analogs of the original specifi~ binding member for example an analyte-analog.
Imml,n6(eacti~e specific binding members include antigens. anligen fragments;
WO 93/21346 2 1 3 3 8 2 6 PCI /US93103224
~ 8 ~ r
antibodies and antibody fragmenls, both monoclonal and polyclonal; and
complexes thereof, including those formed by recombinant DNA methods.
I'Analyte,~ as used herein, is the substance to be detected which ma~ be
present in the test sample. The analyte can be any substance for which there
exists a naturaily occurring specific binding member (such as, an antibody), or
~or which a specific binding member can be prepared. ~hus, an analyte is a
subst~nce that can bind to one or more specific binding members in an assay.
"Analyte~ also includes any antigenic substances, haptens, antibodies, and
co"-~i"alions thereof. As a member of a specific binding pair. the analyte can be
10 detected by means of naturally occurring specific binding partners (pairs) such
as the use of intrinsic factor protein in the capture and~or indicator reagents for
the determination of vi~amin B12, or the use of a lectin in the capture and/or
indicator reagents for the determination of a carbohydrate. The analyte can ~ -
include a protein, a peptide, an amino acid. a hormone, a steroid, a vitamin, a
15 druy including those administered for therapeutic purposes as well as those
a.J~,.;.,i~tered for illicit purposes, a bacterium, a Yirus, and metabolites of or
anti~odies to any ot the above suL,sldnces.
The test sample can be a ~la~ alian bioiogical fluid such as whole blood
or whote blood components including red blood cells, white blood cells including2 0 Iy~ hoc~te or Iy"",hocyte subset preparations, platelets, serum and plasma;
~ites: saliva; stools; cere~rospinal fluid; urine; sputum; tracheal aspirates and
other constituents of the body which may contain or be s~Jspected of containing the
an~lyte~s) of interesl. The test sample also can be a culture fluid supernatant,or a s~ ensiol1 of cultured cells. Mammals whose body fluids can be assayed for
2 5 HIV antigen analyte or HIV an~i~ody analy~e according to the present invention
includ~ humans and ,~.,i",~tes, as well as other rnammals who are suspected o1
containing these analytes of interest. It also is contemplated that non-h o'cgica
fluld samples can be utilked.
The indicator leagenl co"",lises a label conjug~ted to a specitic binding
3 0 Il~ l~r of each analyte. Each indicator reagent produces a clele.~ 'Q signal at a
Ievel relative to the amount of the analyte in the test sample. In a prefened -
enbodiment, each indicator reagent, while co~"p,ising a specific binding member -
of a different analyte. is conj~g~te~1 to the same signal generating compound
(label), which is capable of generali"g a detectablQ signal. In general. the
3 5 indicator reagent is detected or measured afSer it is cap~ured on the solid phase ;
materiai. In the present invention, the total signal generated by the indicator
rea~entls) indicates the presence of one or more of the analytes in the test
WO 93/21346 2 1 3 3 8 2 6
sample. lt is contemplated that different signal genera~ing compounds can be
utilized in the practice of the present invention. Thus, for example, different
fluorescent compounds could be utilized as the signal generating compounds, one
for each indicator reagent, and detectien could be determined by reading at
5 different wavelengths. Or, a short-lived chemiluminescent compound such as an
acridinium or phenanlhIidinium compound and a long-lived chemiluminescent
compound such as a dioxetane can be utilized to generate signals at different-times
for clifIerenl analytes. Methods which detail the use of two or more
chemiluminescent compounds which are c~pah'Q of generating signals at different
10 times are the subjec~ matter of co-pending patent ~F'ic~tion PCT Applic~tion No.
9290~80~.3, filing date December 23, 1991. Acridinium and
phananthridinium compounds are desc~ ed in co-pending U. S. patent ~pplic~tion
Serial No. 07/271,763 filed June 23, 1989, which enjoys common ownership
and is inc~r~ orated herein by referelnce.
In addiliGl) to being either an antigen or an antibody member of a specific
binding pair, the sl,ec;fic binding member of the indicator reagent can be a
~--e.~ er of any speci~;c binding pair, including either biotin or avidin, a
ca~oh~ate or a tectin, a comple"-e~tary nucleotide sequence, an effector or a
receptor ~ ~'ec~lle, an elnL~ -e cofactor or an enzyme, an enzyme inhibitor or an
2 0 enL~.-,e, and thel like. An immunoreactive specific binding member car; be an
anliL~y, an antigen, or an antiLGJy/antigen complex that is c~hlQ of binding ~ -~
either to the analyte as in a sandiwich assay, to the capture ~~agent as in a
oompetitive assay, or to the ancillary srecific binding ,-,e,--~er as in an indirect ~-
assay. If an antibody is used. it can be a lllGnGc~nal antibody, a polyclonal
2 5 a~nilJGJ~, an antibody ~ay.--enl, a recombinant anti~ody, a mixture thereof, or a
mixture of an antibody and other specific binding members. The details of the
p~e~,aidlion of such antibodies and their suitability for use as specific binding
me.. ~l~.s are well known to those in the art. --
The signal generating compound (label) of the indicator reagent is capable
3 0 of generaffng a measurable !sbnal ~el~ le by external means The various
signal generating compounds (labels) conte,..~Jla~ed include chromagens; catalysts
such as ei~lr...es for example, hG.~elaJ;sh per~xidase, alkaline phosp~t~se, and~g?'~twd~se: lumifiescent compounds such as fluorescein and rhodamine;
chemilumifiescen~ compounds such as acridinium compounds, phenanlhridinium ~ ~-
3 5 compounds and dioxetane compounds; radioactive ele.-~nts; and direct visual
labels. The sel~c1ion of a particular label is not critical. but it will be car~bl~ of
producing a signal either by itself or in conjunction with one or more additional
WO 93/21345 Z 1 3 3 8 2 6 PCI~/llS93/03224
-1 O-
substances. A variety of different indicator reagents can be formed by varying
either the label or the specific binding member.
The capture reagents of the present invention comprise a specific ~inding
member for each of the analytes of interest which are anached to at least one
5 solid phase and which are unlabeled. Although the capture reagent is specific for
the analyte as in a sandwich assay, it can be specific for indicalur reagent or
analyte in a competitive assay, or for an ancillary specific binding member,
which itself is specific for the analyte, as in an indirect ~ssay. The capture
reagent can be directly or indirectly bound to a solid phase material before ~he10 performance of the assay or during the performance of the assay, thereby
enabling the separation of immobilized complexes from the test sample. This
attachment can be achieved, for example, by coating the specific binding mernberonto the solid phases by absorption or covalent coupling. Coating methods, and
other known means of attachment, are known to those in the art.
The specific binding member of the capture reagent can be any mo~ecule
c~p~h'e of sp~cif~c~lly binding with another molecule. The specific binding
member of the capture reagent can be an immunsreactive compound such as an
al)tibody, antigen, or antibodylantigen complex. If an anlibod~ is used, it can be a
",onoclonal anlil,o.ly, a polyclonal antibody, an antibody fragment, a reco" ~ nant
2 0 ar,lil,o J~, a mixture thereof, or a mixlure of an antibody and other specific
binding members. ~-
The "soiid p~hase" is not critical and can be selected by one skilled in the
art. Thus, latex pallicles, micrc,pa,licles, magnetic or non-magnetic beads,
membranes, plastic tubes, walls of wells of reaction trays, glass or silicon chips
2 5 and tanned sheep red blood cells are all suitable examples. Suitable methods for
immobilizing capture reagents on solid phases include ionic, hyclrophob c,
c~ lenl in~e.~clions and the like. In one example of the present invention, 60-
well polysly,ene reaction trays and 1/4 inch polystyrene beads are utilized,
while in another example, a 96-well reaction tray is the only solid phase
3 0 utilized. It is conle,~ laled that all solid phases be present during the
quanlilation of signal. thus eliminating the need to separate solid phases for
detection of signal.
A ~solid phase", as used herein, refers ~o any material which is ir soluble.
or can be made insoluble by a s~bse~uent reaction. The solid phase can be chosen35 for its inl,insic ability to attract and immobilize the capture reagent.
Altsrnatively, the solid phase can retain an additional receptor which h~s the
ability to attract and immobilize the capture reagent. The additional receptor can -
WO 93/21346 ' 2 1 3 3 8 ~ 6 Pcr/us93/o3224
-1 1-
include a charged substance that is oppositely charged with respect ~o the capture
reagent itself or to a charged substance conjugated to the capture reagen1. As yet
another altemative, the receptor molecule can be any specific binding member
which is immobi~i7ed upon (attached to) the solid phase and which has the ability
to immobilize the capture reagent through a specific binding reaction. The
receptor molecule enables the indirect binding of the capture reagent to a solidphase material before the performance of the assay or during the performance of ~;
the assay. The solid phase thus can be a plastic, derivatized plastic, magnetic or
non-magnetic metal, glass or silicon surface of a test tube. microtiter well,
sheet, bead, microparticle, chip, and other ccnfigurations known to those ot
ordinary skill in the art.
It is conte"-plated and within the scope of the invention that the solid
phase also can comprise any suitable porous material with sufficient porosity toallow access by detection antibodies and a suitable s~!rface affinity to bind ~ - -
anligens. Microporous structures are generally preferred, but materials with 'gel structure in the hydrated state may be used as well. Such useful solid ~;
supports include~
Natural polymeric carLohy~lla~es and their synlhetically modified, cross-linked
or substituted derivatives, such as agar. agarose, cross-linked alginic acid,
substituted and cross-linked guar gums, cellulose esters, especi~lly with nitric ;
acid and carboxylic acids, mixed cellulose esters, and cellulose ethers; natural ; '
,~Dly---ers containing r.it,ugen, such as proteins and derivatives, including cross-
Iinked or modified gelalins; natural hy~i~oca,LGn polymers, such as iatex and
rubber; sy.,the~.c polymers which may be prepared with suitably porous ~ ~ ~
Z5 structures, such as vinyl polymers, including poly~tl.~rlene, polyprop~lene, ;~ '
polystyrene, polyv;n~lchloride, polyv;"~ cet~e and its partially hydrolyzed
derivatives, ~Iyac~yla...ides, p~ly...etl.acrylales, copoly--.ers and te.l.oly.,-ers
of the above polyoondensates, such as ~Iyeste~s, polyamides, and other
polymers, such as polyuretl.anes or polyepoxid~s; pwous i,.or~an c ~.,ale.ial
3 0 such as sul~.es or ca-~nates of alkaline ~earth metals and magnesium, including
- barium sulfate. calcium sulfate, calcium ca.lona~e, silicates of alkali and
alkaline earth metals, aluminum and ",ag,-esium; and aluminum or silicon oxides
or hydrates, such as clays, alumina, talc, kaolin, zeolite, silica gel, or glass(these materials may be used as filters with the above polymeric .--ate-ials~; and
3 5 mixtures or copGly.--er~ of the above c~Cses, such as graft copolymers o~taie~l
by initializing poly.--erization of synthetic polymers on a pre-sxisting naturalpolymer. All of these materials may be used in su;'~'e shapes, such as films,
WO 93/~1346 PCI/US93/03~24
2I3~g26 - 1 2-
sheets, or plates, or they may be coated onto or bonded or laminated to
appropriate inert carriers, such as paper, glass, plastic films, or fabrics.
The porous structure of nitroceliulose has excellent absorption and
adsorption qualities for a wide variety of reagents including monoclonal
S antibodies. Nylon also possesses similar characteristics and also is suitable.It is contemplated that such porous solid supports described hereinabove
are preferably in the form of sheets of thickness from abou~ 0.01 to 0.5 mm,
preferably about 0.1 mm. The pore size may vary within wide lirnits, and is
preferably from about 0.025 to 15 microns, especially from about 0.15 to 1
10 microns. The surfaces of such supports may be activated by chemicai processeswhich cause covalent linkage of the antigen or antiL~dy lo the support. The
irreversible binding of the antigen or anlibody is obtained, however, in general,
by adsorption on the porous material by poorly understood hydrophobic forces.
Preferred solid phase materials for fiow-through assay deYices include
15 filter paper such as a porous fiberglass material or other fiber matrix
materials. The thickness of such material is not critical and will be a matter of ~ -
choice, largely based upon the ,~,up~,lies of the sample or analyte being assayed,
such as the fluidity of the test sample.
To change or enhance the iln,i"sic charge of the solid phase, a charged -
20 s~6st~nce can be coated di-ectly to the material or onto micfopa-licles which then are telai"e~l by a solid phase support matorial. Al~ernatively,
",icn3parlicies can seme as the solid phase, by being retained in a column or
being suspended in the mixture of soluble reagents and test sampie, or the
particles the"ls~lves can be retained and i"""o~;lked by a solid phase support
25 material. By ~rel~;"ed and immobilized" is meant that the particles on or in the
suppon lnal6fial are not car~'Q of subslanlial movement to positions elsewhere
within the support material. The particles can be sel~cted by one skilled in Iheart from any suitable type of particulate material and include those composed ofpolystyrene. polymethylacrylate, polypropylsne, latex, polytetrafluoroethylene,
30 polyac,~rlonit,ile, polycarbonate, or similar ,-,at~rials. The size of the particles
is not critical. although it is preferred that 1he average diameter of the panicles
be smaller lhan the average pore size of the support material being used. ~hus,
embodiments which utilize various olher solid phases also are contemplated and
are within the scope of this invention. For example, ion capture procedures for
35 il-..l.obili,i"g an i"""obil ~able reaction cornplex with a negatively charged
polymer, ~3esc-il,ed in co-,uen-Jing U. S. Patent Application Serial No. 150,278(co,-esponding to EP Publication No. 0326100), and U. S. Patent ~pplication
WO 93/21346 21 3 ~ ~ 2 6 PCI/US93/03224
- 1 3 -
Serial No. 375 029 (EP Publication No. 0406473) which enjoy common
ownership and are incorporated herein by reference can be employed according
to the present invention to ~ffect a fast solution-phase immunochemical ~eaction.
An immobilizable immune complex is separated from the rest of the reaction
mixture by ionic interactions between the negatively charged poly-
anion/immune complex and the previously treated positively charged porous
matrix and delected by using various signal generating systems previously
described, including those described in chemiluminescent signal measurements
as described in co-pending U.S. Patent Applic~tiQn Serial No. 921979
co-,esponding to EPO Pul,lication No. 0 273,115, which enjoys common
owl,er~h4~ and which is incorporated herein by reference. ; ~
Also, the methods of the present invention can be adapted for use in ~ -
systems which utilize ",icro~a,licle techncl~gy including automated and semi-
automated systems wherein the solid phase comprises a ~-,ic,oparticle. Such
systems include those desoribed in pendi.)g U. S. Patent Ar,~l ~ ?tion 425 651 and
U. S. Patent No. 5,089,424 which co,-espond to published EPO ~plic~ions Nos.
EP 0 425 633 and EP 0 424 634, respecli~ely, and IJ.S. Patent No. 5,0û6 309
all of which enjoy c*,-~ Gn o~ rshi~. and are i.,co"~ordted herein by reference.In the pfactice of one smbodiment of the present invention a test sample
2 0 suspscted of containing any of the HIV antigen analyte or HIV ahli6ody analytes of ~;
intef~sl is simultaneously contacte.i with a solid phase to which a first specific
binding ~--e--~ber of a first analyte is attached and a solid phase to which a first
:,pec-ific binding ..-e--lber of a second analyte has been ~tlacl-ed thereby forming a
mixture. The s~ecif;c binding l-lell-ber:. serve as capture reagents to bind the25 analyte(s) to the solid phases. If the speoific binding ",e,-~er is an
immunolea~anl, it can be an artil,GJy, antigen, or complex thereof specific for
each analyte of interest. If the ~ec;fic binding ...e-"ber is an anliL,o.ly it can be
a ~.~GnG~;nal or polyclonal antibody, an ar~til,o-ly l-dg--,enl, a recombinant
antiL~, as well as a mixture thereof, or a mixture of an anli~)~y and other
3 0 specific binding ,.,e"lbefs. This mixture is ina~b~ted for a time and under
- conditions sufficient for a binding reaction to occur and which incub~tion results
in the forrnation of capture f~agenUfi-at analyte cGm~ exes of the first analyte if
it is pfeseol in the test sample, and/or the formation of capture reagent/secondanalyte complexes of the second analyte if it is present in the test sample.
3 5 Then, an indicator reagenl for each analyte is contacted with the
complexes. The indicator reagent for the first analyte col"prises a specific
binding ~"e"lber of the first analyte of interest which has been labelled with a
WO 93~21346 2 1 3 3 8 2 6 PCT/US93/03224
-1 4-
signal generating compound. The indicator reagent tor ~he second analyte
comprises a specific binding member of the second analyte of interest which has
been labeZled with the same signal generating compound as the indicator reagent
for the first analyte, thereby forming a second mixture. This second mixture is
incubated for a time and under conditions sufficient to form capture reagenVfirst
analyte/indicator reagent complexes andlor capture reagenVsecond
analytelindicator reagent complexes. The presence of either analyte is
deterrnined by detecting the signal gen~rated in connection with the complexes
formed on the solid phase as an indication of the presence of one or more analytes
in the test sample. If the indicator employs an enzyme as the signal generating
compound (label), then the signal can be detected visually or measured
spectrophotometrically. Or, the label can be detected by the measurement of -~
fluorescence, chemiluminescence. radioactive energy emission, etc., depending
on the label used to generate the signat.
1 5 The capture reagents can be attached to the same solid phase, or can be
attached to different solid phases. It is contemplated that all capture reagentscould be attached to the same solid phase, or that each capture reagent could be~lached to a sepa.dte solid phase, or that combination of capture reagents couldbe all~ched to sel~a(dle solid phases. For example, if microparticles were the
2 0 solid phase of choice, then separale micf~,pa, licles could have at least one capture
reagent(s) allached to it. A mixture of "~ic,~pa,licles (solid phases) could be
used to capture the various analytes which may be present in the test sample by - ~-
using the mixture of micropa.licles. It is contemplated thal different ratios of ~;
capture reagent~ attached to solid phases could ~e utilized in such an assay. tooptimize analytei(s) deteclion. -
In the embodiment desc~ ed hereinabove, it is preferred that the specific
binding ,-,e"lber used as a capture reagent for the HIV-1 antibody analy~e be HIV-
1 p41 antigen, and that the sp~cific binding member used as the capture reagent
for the HIV-1 antigen analyte be anti-HlV-1 p24 antibody. It is most preferred
3 0 that the Hl~/-1 p41 used be a reo~ binantly prepared antigen (protein). Also, it
- is ~)refer.eJ that ~he specific binding member for the antibody analyte indicator
reagenl is HIV-1 p41 antigen, labe'l~ with an enzyme, and that the specific
binding member for the antigen analyte indicator reagent is anti-HlV p24
anlil,oJy, l~helle~ with an enzyme. It is most preferred this HIV-1 p41 antigen
3 5 be recombinanlly produced. and that the enzyme be hofsera.lish peroxidase (HRPO).
W O 93/21346 2 1 3 ~ 8 2 ~ PC~r/US93/03224
-1 5-
ln another embodiment o~ the present invention, a test sample suspected
of containing any of the analytes of interest is simultaneously contacted with afirst solid phase to which a first specific binding member ot a first analyt~ and a
first specific binding member of a second analyte have been attached, an indicalor
5 reagent for the first analyte co",prising a specific binding member for the first
analyte labelled with a signal generating compound and an indicator reagent for
the second analyte co",prisi"g a specific binding member for the second analyte -
l~he"e~ with a signal generating compound, 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 immunoreaclan(, it can be an -
arlil,ody, antigen. or complex thereof, specific for each analyte of interest. If the
speci~ic binding member is an anlibo.ly, it can be a monoclonal or polyclonal
antibody, an antibDdy fragment, a t~cG",binant ant;L,ocly, as well as a mixture
thereof, or a mixture of an antibody and other specific binding members. The
15 indicator reagenls comprise specific binding members ot the first and second
analytes of interest which have been ;a'_elled with a signal gener~ting compound. ~-
This mixture is incu~ted for a time and under oond;~;ons sufficient for a binding
re~tion to occur and which incuba~ion results in the formation of capture
r~agenVfirst analyte/indicator reagent complexes of the first analyte and/or ~'
2 0 caplure reag~nl/seoc,nd analyte/indicator reagent complexes of the second
anal~e, if either or both the first or second analyte are present in the test
sample. The presence of either analyte is deterrnined by detecting the signal
gen~.ated in connection with lhe complexes forrned on either or both solid phases
as an indication of the presence of the first analyte andlor the second analyte in
2 5 the test sample. If the indicator employs an enzyme as the signal generatingcompound (label), then the signal can be delected visually or measured
~pe.,t~-,pl-oto,.-eDt~ically. Or, the label can be delected by the measurement of
tluG(escenoe, chemilumirescence. radioactive ener~y e".;ssilDn, etc., depen-lingon the label used. Also, it is conle~ lated that the assay can include the use of a
3 0 hapten-arili-hapten system, in which c~e ths indicator reagenl can further
co...~,.;se a hapten such as biotin. The use of a biotin/anti-biotin system for
~ assays is the subject matter of co-pending U.S. Patent Application Serial No.
687,785 which co~ a~Jonds to published Eu.vpean Patent Application No.
0160900 (published r:Dv6"~ber 13, 1985), which enjoys common ownership
35 and is inco.,~-ated herein by ref6rence.
In the embodimen~ desc-ibed here;..above, it is preferred that the specific
binding member used as a capture reagent for the HIV-1 antibody analyte be HIV-
WO 93/21346 ~2 1 3 3 8 ~ 6 PCr/US93/03224
-1 6-
1 p41 antigen, and that the specific binding msmber used as the capture reagent
for the HIV-1 antigen analyte be anti-HlV-1 p24 antibody. It is most preferred
that the HiV-1 p41 used be a recombinantly prepared antigen (protein). ~Iso, it
is preferred that the specific binding member for the antibody analyte indicatorreagent is HIV-1 p41 antigen, labelled with an enzyme. It is most preferred thisHIV-1 p41 antigen be recombinantly produced, and that the enzyrne be
horseradish peroxidase (HRPO). Solid phases preferred include a magnetic or
non-magnetic bead, a well of a reaction tray, and microparticles, either alone or
in any combination.
Positive and negative controls can be included in the assay of the present
invention to ensure reliable results. A blank solid phase(s), to which no cap~ure
reagent has been attached, can be utilized as the negative reagent control.
Positive controls can include a positive control for each analyte which control is
tested sepa,a~ely, and a combined positive control wherein the presence of all
1 5 analytes to be detected in the assay are deterrnined.
As previously stated, it is preferred lhat recombinantly-prepared
antigens be used in the assay. Although it is known to use inactivated whole virus
which has been cultured in a cell line capable of virus replica1ion as a reagent in
an assay for HIV anli~-ly, u"i-,fected per~ol1s may occAc~onally give false positive
2 0 results. - The deteclion of false positive (reactive) results in screening assays of
blood products is one of the most pressing problems acsoc:~led with many known
~"etl-ods of HIV detection. These false problems often are ~ss~ ~l~ with
nGu:".eciIic binding of immunoghbulins to cellular protein in the viral isolates.
These reagenl~ also have the .ha~ ack of potentially dangerous mànufacturing
2 5 methods, since culturing of live virus in vitro and isolation and deactivation
processes can expose workers to the HIV virus.
Although lhe most spec;fic test for HIV infection remains isolation of the
HIV virus, this is an impractical method for large ~ale use due to the
complexity, difficulty and time spent isolating HIV in culture.
3 0 The use of recombir antly-produced HIV protein (antigeh) as an antigen
- reagent in assay ll,elllods may solve these problems. Recombinant proteins are
non-in~ec1ious and therefore the production and isolation of such proteins wouldbe safer Ihan culturing the whole virus. Also, the use of pure viral protein
obtained by recombinant methods should eliminate some of the false positive
reactions due to nonspeci~ic reactions with contal,-inating proteins. Fu.-ther,
slanda~liLal;on of reagents should improve the specificity and predictive value of
the assay.
W O 93/21346 2 1 ~ 3 8 2 6 PC~rJUS93/03224
.
-1 7-
,~,.,
Expression of HIV gp~1 or parts of HIV gp41 have demonstrated the
utility of recombinant DNA (rDNA) derived HIV envelope sequences in diagnostic
assays. Wood et al., Cold Spring Harbor Symposium on RNA Tumor Viruses, Cold
Spring Harbor, New York, May 22-26 (1985); Chang et al., Biotechnology
3:g05-909 (1985); Crowl et al., Cell 41:979-986 (1985); Cabradilla et al.,
BiotechnoJogy 3:128-133 (1986). While it is general knowledge that viral
proleins expressed in E. coli or other organisms have potential utility in
diagnostic assays, development of immunoassays using these reagents, whi~h also
will have the specificity and sensitivity equal to or greater than the native viral
1 0 proteins derived from the cell culture has be~n a difficult task. Further, the
expression of HIV gag p~oteins in E. coli have indicated that the HIV gag proteins
produced by rDNA technology could have potential diagnostic value. Wood et al.,
Cold Spring Harbor Symposium on RNA Tumor Viruses, Cold Spring Harbor, New
York, May 22-26 (1985); Dowbenko et al., PNAS USA 82:7748-7752
1 5 (1985); Ghrayeb et al., DNA 5:930g9 (1986~; Steirner et al., Virology
150:283-290 (1986).
The ,~resen~ invention utilkes recombinantly-produced HIV envelope ~;
proteins as assay .eagert~. The cbning of th~ HIV genome and ex~.ression of HIV
envelope and oore protein in E. coli, the pu.ilication and characterization of gp41
2 0 and p24, and various assay fo.,.lats which utilize these rec~",binant proteins are
described in EP-A-3~6 713, published Septe,-,ber 12, 1990 (U.S. Patent
A~pl;c-~l.ion Serial No. 071020,282 filed February 27, 1987 and previously
incorporated herein by ref~rence, from which this present invention claims
priority). Briefly, HlV-infected HT-9 cells were han~ested and total cellular
2 5 DNA was iso'~'~ an subiected to digestion. The DNA sey",ents enc~-ling for ~he
core protein and for the envelope gly~prot~in were funher subcloned into
bactf~rial e~-ession vectors using well-known recombinant technology. The
priority application also teaches that, In the ¢Jete~tion of HIV-1 antibody, the use
of recombinant anli~el,s as the capture f~agenl and the indicator reagent allows
.
3 0 for the detqction of anti-HlV-1 antibodies of different immunoglobulin classes,
- These imml.. osbbulin c~ ~ses include IgG, lgA, lgE and IgM. The detc~tion of
anti-HlV-1 IgG, IgM and IgA using the Abbott HIVAB~9 HIV~1/HIV-2 (rDNA) EIA
assay has ~een des~ ed in an a~ d~l by J. L t~ d~ et al.,5th Annual Forum
on AIDS, Hep~tit;~ and Other Blood-Borne Dise~ses, Atlanta, Georgia, March 29-
3 5 April 1, t 992.
It is cGnle---plated and within the scope of the present invention that
recombinant antigens produced in heterologous sources can be utilized in the
WO 93/2134~ 2 1 3 3 8 ~ 6 PCI/US93/03224
- 1 8 -
assay and will contribute an even greater lessening of false positive results. For
example, if an E. coli prepared recombinant antigen such as p41 is used as the
capture rea~ent, then a recombinant antigen p41 produced in any suitable ~ource
- different than E. coli, such as in a suitable yeast host or other suitable host such
as B. megaterium, can be used.
Further, although the present invention preferably utilizes
recombinantly produced antigens, it is well within the scope ot the invention toutilize synthetic proteins instead of recombinantly produced antigens. Thus,
various synthetically prepared HIV peptides, of varying length, can be used.
1 0 The present invention also utilizes antibodies which specifically bind to
HIV antigen analytes. In a preferred embodiment, anti-HlV p24 antibody is used.
In a most preferred e",bodin,ent, a mixture of monoclonal antibodies, both
specific for HIV p24 antigen, is used. In this mixture, one monoclonal antibody -;
which specifically binds to an epitope on HIV-1 p24 to which epi~ope human
1 5 anti-HlV-1 p24 IgG does not competitively bind is used with ano~her monoclonal
antiL~.ly which specifically binds to a different epitope of HIV-1 p24 to which
dill~.~nl epitope human anti-HlV-1 p24 IgG does competitively bind. Further,
the ."onocbnal anlibody which does not coi"peti~ively bind human anti-HlV-1
p24 IgG also spec;fio~ binds to HIV-2 p24 antigen. These monoclonal --
2 0 al"i~odies and their use in HIV antigen assays are the subject matter of co-
pending U. S. Patent No. 5,173,39g, issued December 22, 1992. These
",onG.,l~nal antibodies are designated as 31-42-19 and 31-90-25. Hybridoma
cell line 31-42-19 producing monoclonal antibody 31-42-19 was deposited at
the American Type Culture Ccllection, 12301 Parklawn Drive, Rockville,
2 5 Maryland, 208~2 on May 26, 1988 and has been accolded ATCC Deposit No. HB
9726. Hyl"idoma cell line 31-90-25 producing monoclonal antibody 31-90-
25 was deposited at the American Type Culture Collection, 12301 Parklawn
Drive, Rockville, Maryland, 20852 on May 26, 1988 and has been accorded
ATCC Deposit No. HB 9;~25., The use of these l"on~iclonal ar,til,od;es as antibody
3 0 i-ag",enls in HIV antigen assays also has been described in U.S. Patent No.
5,1 73,399.
It is conte""~lalecl and within the scope of the invention that the detection
of HIV-2 antigen is possible with the assay of the invention. In ~his assay
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-1 p41 antigen
capture reagenl and I IIV-1 anii~y capture reagents. The solid suppon can be
the same solid support to which all other capture reagents are attached, it can be
W O 93/21346 2133826 PC~r/US93/03224
-1 9-
the same solid support to which HIV-1 reco"lLinant ant;gens have been attached,
or it can be attached to a solid phase to which no other capture reagent (exceptfor HIV-2 p41 ) has been anached. The assay procedure would be Ihe same as
described here.nabove for the various embodiments of the invention. The HIV-2
S anlil~d~ analyte indicalor reagent would comprise HIV-2 p41 antigen altached to
a clelect~kle label. In a preler,ed embodiment, it is preferred that recombinantly
prepared HIV-2 p41 is utilized. The sequence for the HIV-2 virus (including
p41 antigen) is des~ribed in EP-A 347,365, published Dece-,-ber 20, 1989 to
Diagen CG",GraliGn, which is il,co.~,oraled herein by reference. A most ~'
10 prefe..e.l HIV-2 recombinant antigen encodes the first 104 amino acids of theHIV-2 p41 antigen. The resulting plasmid designaled as pJC104 ex~.resses the
HIV-2 env protein as a fusion with CKS protein. This plasmid enc~des a
recombinant protein conlaining the first 239 amino acids of the CKS protein, 13
amino acids from the pTB210N multiple ,est-icl;on site linker, 104 amino acids
15 from the HIV-2 env protein (amino acids 506-609), and an additional 15 amino
acids from the pTB210N mulliple resl~iclion site linker, f~ g the methods
rl~seJ by Bolling and Manded~ CKS Method of Protein Synlhesis~. EP-A-
475 182~ published March 18, 1992 (U.S. Patent Application Serial No.
167,067, filed March 11, 1988, which enjoys co.-.-,~on o~ ership and is
20 inco.~.a~e.l herein by reference).
- ~ ~ EXAMPLES
Example 1
Goaling Procedure Using Two Solid Phases -
This p.ocedure utilized 114 inch polystyrene beads (available from
- ~ ~ Abbott Laboratories, jAbbott Park, IL 60064) and a 60-well polysl~.ene
reaction tray (available from Abbott LabG.alo.ies, Abbott Park. IL 60064).
TWG different anli-HlV-1 p24 lllGn~clonal an~ibodies were coaled on the beads, as
' folbws. The beads were coated al a a~ncerlt~alion of 8 llg/ml (app-o~ .at~l~F 1.6
~lg/mUbead) in a 0.25 M sodium citrate buffer (pH 7.2) for two hours at 45~C.
The beads then were washed in the 0.25 M sodium citrate buffer (pH 7.2), and
then they were f~ted with a deter~ent solution containing 0.1% Triton X- ~-
1000 (pGlyox~lhtlene ether, available from Sigma Chemical Co.. St. Louis,
MO) hr one hour at 45~C. The beads next were blocked with 1% bovine serum
,
WO 93/2 1 346 2 1 3 3 !8 2 6 PCI'/US93/03224
-20-
albumin (BSA) in 0.25 M sodium citrate buffer (pH 7.2) for 30 minutes at
45~C, and then overcoated with 2% sucrose, 1% phosphate glass for 15 minutes
at 15-30~C in 0.25 M sodium citrate buffer and allowed to dry. The ~vo
monoclonal an~ibodies used are designated as 31-42-1g and 31-90-25. They
are the subject matter of a patent arplica~ion U.S. Patent Application Serial No.
071204,798 that describes their development and uses, previously incorporated 'herein by reference. Their use also has been desc-ibed in U.S. Patent No.
7,204,798, which enjoys common ownership and is incorporated herein by
reference. Hybridoma cell line 31-42-19 producing monoctonal antibody 31~
1 0 42-19 was deposited at the American Type Culture Boll~ction, 12301 Parklawn
Drive, Rockville, Maryland, 20852 on May 26, 1988 and has been accorded
ATCC Deposit No. H8 9726. Hyl,rido,.-a cell line 31-90-25 producing
~.,onGclonal antibody 31-90-25 was deposited at the American Type Culture
Collection, 12301 Parklawn Drive, Rockville, Maryland, 20852 on May 26,
1 5 1988 and has been accG-ded ATCC Deposit No. HB 9725.
Next, the wells of the 60-well reaclion tray were coated with HIV
antigen, as follows. Tha recombinant protein HiV-1 p41 env protein designated
as pTB319 was added to each well at a concent~alion of 1 ~g/ml in 0.1 M 3-
[cyclohexyld~..ino]-1-propanesulfonic acid (CAPS buffer, pH 11), and incubated
2 0 for lwo hours at 40~C. The wells then were ~:ashed twice with 400 ~11 of
phGsp~ate buffered saline (PBS, pH 7.5), reacted with 0.1% Tween-20(9 for
one hour at 40~C, and then blocked with 3% BSA in PBS for one hour at 40~C.
The wells next were overcoated with 5% sucrose in PBS for 20 minutes at room
te,--pe~ture and allowed to dry.
2~ The pTB319 plas.. -W producing recombinant protein pTB319 is the
subject matter of a patent applic~l;on to Bolling and Mandecki, ~CKS Method of
Protein Synthesis,~ U.S. Patent Application Serial No. 167,067, filed March 11,
1988, previously incol~JGrd~ed herein by (efe-ence. pTB319 was produced by
ins~,ti.,g a synthetically-produced DNA fragment which encoded the carboxy
terminal 42 amino acids of HIV-1 p120 into the plasmid pTB315, as describèd
- in Bolling and Mande.,k;, Ibid.
Example 2
Simultaneous Assay fo- HIV antigen and HIV ant,~dy
'
The hHo solid phases prepa~d as desc,i~ed in Example 1 were used in an
assay for detection of HIV antigen andlor HIV an~ibody in a test sample, as
2133826
W O 93/21346 . .' PC~r/US93/03224
-2 1 -
follows. An HIV-1 seroconversion panel, which contained 65 serum samples
derived from nine HIV-1 infected individuals undergoing seroconversion, was
used in the assay. Each serum sample was diluted in a separate well of lh~e 60-
well tray previously prepared in Example 1 by adding 150 ill of the serurT
sample to 50 ~11 of specimen diluent, which contained 2% Tween 20~9
(polyoxyethylenesorbitan, available trom Sigma Chemical Co., St. Louis. MO).
Then, a bead previously coated with anti-HlV p24 anSibodies as described in
Example 1 was placed in each well containing a sen~m sample. The weils of each
tray were incubated for 60 minutes at 40~C under continuous rotation.
Following incub~tion, each well of the 60-well reaction tray was washed with 15
ml of deionized water (dH2O) in the Abbott Parallel Processing Center~ (PPC,
available from Abbon Laboratories, Abbott Park, IL). 200 i~LI of an HIV p24
antibody probe reagent (rabbit polyclonal Flab'12 anti-HlV-1 ~active
ingredient: anti-p24 antibody at a concentration of 2 to 6 ~glml] in an antibodydiluent (2.25% BSA, 7.5~~O calf serum, 73% goat serum, 25% human
rec~'Gir,ed plasma. 0.1% sodium ~ide) was added to ea~,h welUbead and then the
resulting mixture was incub~ted for 60 minutes at 40~C without rotation. Each
bead/well in the react.on tray was washed with 1~ ml of dH20. Then, 200 -11 of
conjugate diluent (0.18% Tris, 1.19 % Tris HCI, 0.38% NaCI, 9.0% calf
serum, 0.9% goat serum, t0.0% human caloified plasma, 4.5% Triton X-
100~19, 0.013% genla,-,icin sulfate. 0.009% thimerosal) which contained a
mixture of recGI~binan
HIV-1 p41 antigen l~t)elle~ with horseradish peroxidase (pTB319 coupled to
HRPO), and HRPO-late"ed goat anti-rabbit IgG antibody were added to each
2 5 bead/well of the ~eaction tray and allowed to incubate for 60 minutes at 40~C
without rotation. Each bead/well of the 60-well reaction tray was washed with
15 ml of dH2O as previously desc-i~Jed. Then, 300 ill of o-phenylenediamine-
2HCI (OPD) was added to each wel-h~ead and then was incub~ted for 30 minutes
at room te,l-~e(allJre in the dark. Ths reaction then was slopped by adding 300
3 0 of a stopping reagent (1 N H2SO4) to each welUbead. The reaction was read
using the Abbott PPC which measured the optical density of the reaction at 492
nm using a 630 nm ref~rence. The cutoff value was established as 0.1 OD + mean
OD of the nega~;~re control. Thus, serum samples were ccnsidered reactive
- ~positive) if the sample to cutoff value was greater than t.
All 65 serum samples from the 9 individuais desc.ibed hereinabove were
tested follo~;.)g this procedure. The results obtained then were ~""~a,ed lo lheresults oblained for the same serum sample when using an HIV antigen assay
W O 93/21346 2 1 3 3 8 2 6 P ~ /US93/03224
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(HIVAG~9, available from Abbott Laboratories, Abbott Park, IL) and an HIV
antibody assay (Human Immunodeficiency Virus Types 1 and 2: E. coli and B. -megaterium, recombinant antigen, Abbot~ HIVAB(~ HIV-1/HIV-2 (rDNA) EIA; ~-
available from Abbott Laboratories, Abbott Park, IL) following manufacturer's -
5 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 sample/cut-off
value, ~Result.~ refers to the interpretation of the test, "HIV-1/2 Ab HIV-1 Ag
Comb" designates the assay of the invention, ~HIV-1/2 Ab" designates the
HlVABi~ HIV-1/HIV-2 (rDNA) EIA assay and "HIV-1 Ag~ designates the Abbott
1 û HIVAG~ assay.
;
'.
,
W O 93/21346 2 1 3 ~ 8 2 6 P ~ /US93/03224
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TA~LE 1
Sample HIV-1/2 Ab HIV-1/2 Ab HIV-1 Ag
ID HIV-1 Ag
Combination
SICO Reslllt ~Q ~LII~ ~Q Resul~
SV0021 1 1.34 + 0.33 - 1.10 +
2 1.92 + 2.5~ + 1.80 +
3 3 . 15 + 6 .32 ~ 2 .20 +
4 1.79 + 3.65 + 0.60
SV0031 5 0.83 - 0.13 - 0.40
6 0.87 - 0.13 - 0.30
7 0.86 - 0.13 - 0.35
8 0.78 - 0.1 5 - 0.35
9 0.95 - 0.17 - 0.40
1 0 0.80 - 0.13 - 0.88
1 1 0.62 - 0.10 - 0.35
1 2 3.40 + 9.08 + 13.20 +
13 3.40 +1 0.09 + 8.50
14 3.40 + 3.00 +1 0.50 +
3.40 + 3.28 + 6.40 +
16 1.87 + 5.2~ ~ 3.20 ~ .
17 1.52 + 7.34 + 0.80
1 8 1.38 + 7.28 + 1.20 + ;~
SV0051 19 O.g7 - 0,40 - 1.70 +
2 ~ 2.2~ + 1 .49 +1 5.00 +
2 1 2.55 ~ 2.66 ~ 7.gO +
2 2 ~.54 + 7.30 + 2 .20 + ~:
2 3 2.57 + 5.51 + 1 .80 +
24 1.89 + 3.81 + 0.40 - .
SV0091 25 2.40 +0.24 - 18.80 +
26 3.40 +0.44 - 7~.60 +
27 3.40 +2.81 +~.20 +
2 8 3.4Q +2.75 +1 .90 +
29 2.71 +2.43 +0.65
3 0 2.23 +2.25 +0.65
3 1 1 .~4 +3.~2 +0.4g
32 ~1.35 +10.57 +0.41
33 nt-
SV0111 34 3.40 +0.12 - 32.8û +
3 5 3.io +0.45 - 42.90 + . .
3 6 3.40 +1 1 .23 +20.70 +
37 2.3~ +4.16 +1.40 +
3 8 1.82 +3.81 +1.10 +
3 g 1 .71 +3.34 +0.80
4 0 1.40 +5.49 +0.72
PCr/US93/03224
WO 93/21346 21 3382 6
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Sample HIV-1/2 Ab IV-1/2 Ab HIV-1 Ag
ID HIV-1 Ag
Combination
~0 Result S/CQ E~L~
SV0161 41 1.01 + 0.12 - 1.44
42 1.21 ~ 1.14 - 3.82
43 2.17 + 0.79 - 8.41
44 3.40 ~ 10.25 + 1 1.80
4 ~ 2.48 + 2.82 + 0.65
46 1.51 + 2.37 + 0.48
SV0061 47 1.13 + 0.20 - 2.90
4 8 1.23 + 0.26 - 3.60
4 9 3.33 + 1.93 + 10.90
5 0 3.09 + 1 .72 + 13.50
51 1.93 + û.96 - 7.10
5 2 1 .88 + 1 .39 + 4.40
53 1.30 + 1.60 + 1.90
54 1.45 + 3.10 + 0.97
5 5 1 .22 + 4.28 + 0.98
SV0071 56 û.97 - 0.13 - 0.40
57 0.84 - 0.14 - 0.40
58 3.19 + 2.97 + 2.30
5 9 1.82 + 2.37 ~ 0.52
û.97 - 2.84 + 0.42
SV0081 61 0.80 - 0.12 - 0.45
6 2 0.85 - 0. 1 5 - 0.32
63 0.83 - 0.14 - 0.49
64 1.41 + 3.40 + 0.43
1.10 + 4.96 + 0.37
6 6 1.23 + 8.58 ~ 0.37
No. Positive/ 51/65 41/65 35/65
No. Tested
~n~: This sample of the seroconversion panel was unavailable for testing.
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A
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The data from Table 1 indicates that the method of the present invention
had greater sensitivity than either the HIV-1/2 Ab or the HIV-1 Ag test when ~heresults from the three individual tests were compared separately to each o~her.
IRs expected that the assay can be optimized even further to detect both HIV p245 antigen which is present early in the course of infection and also in the final
stages of HIV infections, as well as HIV antibodies which appear later in infection
at the time of seroconversion.
Example 3
1 0 Coating Procedure For One Solid Phase
In this procedure, only one solid phase was coated with HIV antigen and
HIV antibody, as follows. Into each well of a 96-well micro~iter plate (Irnmulon4(Z~, available trom Dynatech, Alexandria VA) monoclonal antibody 31-42-19,
monoclonal antibody 31-g0-25 and recombinant HIV-1 p41 antigen desi~nated
as pTB 319 (as ~lesc,iLed in Example 1) were coated at a concel,ttd~ion ~f 1
g/ml each in 0.1 M carbonate buffer (pH 9.5) for two hours at room
temperalure. The wells next were blocked with 300 1ll of blocking reagent
(co-"p-i~;ng 5% non-fat dly milk, 10 mM Tris lpH 8.0] 150 mM NaCI and
0.05% Tween-20(~j for one hour at room temperature.
- Example 4
HIV Antigen/Antibody Assay Using One Solid Phase
2 5 The solid phase prepared as desc,iL.ed in Example 3 was used in an assay
to detect the presence of HIV antigen and~or HIV antibody in a lest sample, as
follows. Each serum sample of a 12-member seroconversion panel (Panel G
available from Boston Biomedica Inc., Boston MA) as well as positive and negative
controls were tested. 150 ~1 of each serum sample or positive or negative
control was diluted in a sep~r~te well of the ~ie~uliler plate with 50 111 of
specimen diluent (containing 15 ~11 of Tri~on X-100~9 and 35 ~11 of blocking
reagent, as des-;,ibed in Example 3) and incub~ted for 60 minutes a~ room
temperature without rotation. After incubation the wells were washed with eight
cycles of 300 ~11 of washing buffer (0.05% non-fa~ dry milk, 10 mM Tris EpH
8.ûl, 150 mM NaCI, 0.05% Tween 20~9) using a Nunc 8-channel
"Immunowashn manifold (available from Nunc, Denmark). Next, 175 ~l of an
HIV p24 antibody probe reagent (rabbit polyclonal F[ab'l2 anti-HlV-1 ~active
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ingredient: anti-p24 antibody at a concentration of 2 to 6 Ilg/mll in an antibody
diluent (2.25% BSA7 7.5% caif serum, 7.5% goat serurn, 25% human
recalcified plasma, 0.1% sodium azide) were added to each well and incubated for60 minutes at room temperature without rotation. After incubation the wells
5 were washed with eight cycles of wash buffer as previously described. Then,
150 111 of conjugate diluent (as described in Example 2) which contained a
mixture of recombinant HIV-1 p41 protein (pTB 319) labelled with HRPO and
HRPO-labelled goat anti-rabbit IgG (previously described in Example 2) were
added to each well and incubated for 60 minutes at room temperature without
10 rotation. The wells were washed with eight cycles of wash buffer (described
previously herein) and then rinsed with dH20. Then, ~25 ~1 of OPD substrate
was added to each well and the wells were incubated at room temperature for 10
minutes in the dark. The reaction was stopped by adding 125 1ll of stopping
reagent (previously described in Example 2). The absorbance of each well was
read at 490 nm with a 630 nrn reference. The cutoff value of 0.025 OD + mean
OD of the negative control was established. Samples were ~ns;dered reactive
(positive) when the samplelcutoff value was greater than 1
The data from these assays are presented in Table 2. In Table 2, ~OD"
refers to the oplical density reading, ~S/CO~ means sample/cut-off value,
20 ~Result.~ refers to lhe i--te-~,retation of the test, ~NC~ refers to negative control
and ~PC~ refers to the positive control.
WO 93/21346 2 1 3 3 8 2 6 PCr/USg3/03224
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TABLE 2
HIV-1/2 Ab HIV- HIV-1/2 Ab HIV-1 Ag ~ '
1 Ag Combination
SICO Re~ult ~ Q E~l~ p~ml~
0.8~ -
0.72
0.82
PC 1.35 +
PC 1.13 +
1.22 + 0.09 - >200 +
2 1.64 + 0.23 - >200 +
3 2.33 + 2.55 + ~200 +
4 4.û4 + 4.07 + 155 +
2.42 + 1 .92 1 4 0 +
6 1.03 + 1.95 + 5 +
7 1.09 + 4.87 + 0
8 1.57 + 7.31 + 0
9 1.61 + 7.31 + 0
1.79 + 9.40 + 0
1 1 3.76 + >17.85 + 0
12 5.62 + >17.85 + 0
TOTALS 12112 1 0112 6/12
'pglml - picograms/ml
', , ! '
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As the data from Tabte 2 demonstrate, the assay of the invention was
capable of detecting the presence of HIV antibody and/or HIV antigen in the
seroconverslon panel. Compared individually to the HIV-112 Antibody tes~. and
HIV-1 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.
When the results from Table 2 of the method of the present invention are
compared to the combined results of the HIV-1/2 antibody test and the HIV
antigen test, the method of the present invention was able to detect all specimens
that were reactive by either test.
1 0
Example 5
Coating Microparticles Simultaneously Wi~h
HIV Antibody and HIV Antigen Capture Reagents ~:
Both monoclonal anti-HlV p24 antibodies previously described (31-42-19 and 31-90-25~ and the recombinant HIV-1 p41 antigens previously
described (HIV-1 p41 recombinant protein pTB319, and HIV-2 p41
recombinant protein pJC104) are together and simultaneously coated onto a
uniform 0.5% suspension (wVvolume) of polystyrene microparticles (available
20 from Seradyne Inc., Indianapolis, Indiana) at concent,alions of 150 ~g/ml each
in 0.01 M carbonate buffer (pH 9.5) for two hours at room temperature (15- -
30~C). The suspension of mic,upa-~icles is briefly centrifuged and the
r~ a.licle pellet is resuspended in 0.05M Tris buffer (pH 8.0) to wash away
excess uncoupled protein This washing is repe~ted until no uncoupled protein
26 re.. a;.. s. After blocking the microparticles with 10 mgtml casein in 0.01 M
Tris (pH 8.0), 0.15 M NaCI at 56~C for 18-24 hours, the ",icroparticlss again
are washed as described herein and diluted to 0.015% suspension (wt~volume)
in 0.05 M Ttis (pH 8.0), 0.15 M NaCI, 1% BSA, 15% sucrose and 0.1% sodium
~ide.
Example 6
Simul~aneous Detection of HIV Antibody and HIV Anligen Using Microparticles
The Abbott IMX(9 Microparticle Enzyme Immunoassay (MEIA) system is
35 used, although any system which employs ~ic,upa~licles can be used. The Abbott
IMX(D MEIA system is thoroughly described in the Abbott IMX~ Opera~ion and
Customer Training Manuals (available from Abbott Diagnostic Division, Abbott
2133826
W O 93~21346 . ;, PC~r/US93/03224
; - .' ., 1. ~
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.~
Laboratories, Abbott Park, IL). In this assay, 100 ~LI of the 0.15% suspension
~crepared in Example 5 is mixed together with 100 ~lt of test sampl~ suspected of
conlaining HIV-1 and/or
- HIV-2 antibody and/or HIV-1 antigen, and incubated at 40~C lor ten minutes in
an Abbon IMX~ reaction cell to form a reaction mixture. HIV antibodies and/or
HIV antigens bind to the microparticles in an antibody/antigenlmicroparticle
complex. 150 ,ul of the reaction mixture is transferred onto a glass fiber matrix
to which the microparticles are retained in an irreYersible binding. The
antiLoJy/antigen/l"icroparticle complexes then are reacted with 50,ul of a probeconsisling of biotinylated recombinant HIV-1 and HIV-2 recombinant p41
antigens (pTB319 and pJC104, previously describedl and biotinylated F~Ab')2
anti-HlV-1 p24 in 0.05M Tris 9 pH 8.0), 2% BSA, 0.25% saponin and 0.1%
sodium azide at 40~C for ten minutes. 50 111 of an antibody conjugate consistingof goat an1i-biotin alkaline phosphatase in 0.1 M Tris (pH 8.0), 0.5M NaCI,
0.9% Brij-35~, 1.0% BSA and 0.1% sodium ~ide then is allowed to react wilh
the biotin probe/antibody/antigen/,nicropa-lic'e complexes for ten minutes at
40~C. Then, thase ~-.ic~op~rti.,le complexes are was~,ed six times wilh 0.05 M
Tris (pH 8.0), 0.3 M NaCI and 0.1 % sodium ~ide, the biotin
p-Obe/;~lltibOdy/~nti~el) ll~icl~pa~ Q complex is ~eac~ed with 50 ~1 of the
substrate methyl~-n~sl'iferyl phosphase (MUP, Abbott LabGrdtories, Abbott
Park, IL), and the fluorescence of the product, methylumbelliferon, (MU) is
measured. The rate of MU production is propG.tional to the concentration of
a-.al~fte~s) in the test sample.
2 5 Example 7
Coating Micropa.li.,les Separ~lely wilh
HIV Antibody and HIV Antigen Capture Reagen~s
In ~his example, the various analyte capture ~eagen~s are coated
30 s~parately onto polystyrene rioro,o,licles (available frorri Seradyne Inc.,
Indiana,~lis, Indiana). Each of the reagents may be coated separately from each
other or in various combinations with each othar. After each of the analyte
capture r*age-~ts is coated on their respeclive microparticlss, the various coated
microparticles are pooled together and used in the assay.
In the presenl example, the two .. -onGclonal anti-HlV p24 anlil,odies
(31-42~19 and 31-90-Z5) are coated 1Ogether onto microparticles separate
W(~ 93/2~346 2 1 3 3 8 ~ 6 PCI'IUS93/03224
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from the microparticles coated simultaneously with recombinant HlV-t and HIV-
2 p41 antigens (pTB31s and plC104). Although the exact amount may vary in
general the coating procedure will approximate tha~ described in Example 5.
After blocking the physically separaled microparticles with 10 mg/ml casein in
0.01 M Tris (pH 8.0) 0.15 M NaCI a~ 56~C for 18-24 hours the
",;c(opa,licles again are washed as described in Example 5 pooled together. and
dilu1ed to a 0.015% suspension (wVvolume) in 0.05 M Tris 9 pH 8.0), 0.15 M
NaCI 1% BSA and 15% sucrose. At the pooling step. the l"ic,uparticl~s may be
pooled at various ratios to affect sensitivity and specificity of the assay in order
to optimize their use.
Example 8
Simultaneous Detection of HIV Antibody and HIV Antigen on Mic.opa,lic:es
Separately Coated With HIV Antibody and HIV Anti~en Capture Reag~nts
1 5
The Abbott IMX~ Miclupalli..le Enzyme Immunoassay (MElA)-system is
used, although any system which employs ",ia~pa,t es can be us~d. The Abbott
IMx~9 MEIA system is thoroughly des.;,il~ed in the Abbott IMX ED Operation and
Customer Training Manuals (available from Abbott Diagnostic Division, Abbott
LaLoratories, Abbott Park IL). In this assay, 100 111 of the 0.15% suspension
p,epared in Example 5 is mixed logell,er with 100 111 ol test sample suspect~! of
containing HIV-1 andtor HIV-2 anlit~.ly and/or HIV-1 antigen, and incuk~ted at
40~C for ten minutes in an Abbott IMX~9 reaction cell to form a reaction mixture.
HIV antibodies andtor HIV anligens bind to the mic,~pa,licles in an
ar~til~J~/auti~en/micropa,~ic e complex. 150 ~l of lhe r~adion mixture is
l-ansle,.ed onto a glass fiber matrix to which the ~"icropa,~i~ ss are retained in
an irreversible binding. The ar,li~Jy/antigen/l"icrop~.lic ~ co"~plex~s then arereacted with 50~ of a probe consisli"g of biotinylated recombinant HIV-1 and
HIV-2 recombinant p41 antigens (pTB319 and pJC104, previously JescriL.ed)
and biolinjlat~J F(Ab'j2 anti-HlV-1 p24 in 0.05M Tris 9 pH 8.0), 2% BSA,
- 0.25% saponin and 0.1% sodium azide at 40~C for ten minutes. 5Q 111 of an
an~ibGdy conjugate consis~i-,g of goat anti-biotin alka,;ne phos,l~ha~ase in 0.1 M
Tris (pH 8.0), 0.5M NaCI, 0.9% Brij-35(9 1.0% BSA and 0.1% sodium ~ide
then is allowed to react with the biotin probetantibody/anligen/~"icroparticie
3 ~ cornplexes for ten minutes at 40~C. Then, these microparticle complexes are
washed six 1imes with 0.05 M Tris (pH 8.0), 0.3 M NaCI and 0.1 % sodium
~ide, the biotin probe/antibody/antigen microparticle complex is reacted with
WO 93/21346 2 1338 2 6 PCI'/US93/03224
- 3 1 -
50 ~11 of the substrate methylumbelliferyl phosphase (MUP, Abbott
Laboratories, Abbott Park, IL), and the fluorescence of the product,
methylumbelliferon, (MU) is measured. The rate of MU pro~uction is "
proportional to the concenlrdtion of analyte(s) in the test sample.
It is contemplated that the assay of the invention can be optin,i~ed even
further by varying assay conditions and/or incubation times, using various
combinations of antigen or antibody capture or pro~ reagenls, and other
methods, reagents and cor~ilions known to lhose skilled in the art. Thus, various
other antibody capture reagents can be used, including HIV p24, gp120, gp160,
pl7, and others. The variance of the anlil o.Jy capture reagent may then requirethe use of a di~lerent antigen capture reagent. All these variations are
contemplated to be within the scope of this invention. Also, while some of the
assays desc.iL,ec~ in the examples used an automated system, it is well within the
1 5 scope of the present invention that manual methods or other automated analyzers
can be used or ~lerJ to the assay of the present invention.
