Note: Descriptions are shown in the official language in which they were submitted.
Wo 92/08979 PCr/US91/08360
2~9~2~
This invantion ralates generally to immlJnoassays utilizing
chemiluminescent compoundst and more particularly, relates to
heterogeneous chemiluminescent immunoassays wherein a
ch~milurninescent signal provided by an immobilized product o~ an
immunochemical reac~ion is amplified, resulting in a more sensitiYe assay.
The generation of light as a result of a chemical reaction is known in
the art and was reviewad by Schuster and Schmidt in "Chemiluminescence
of Or~anic Compounds", V. Gold and D. Bethel, eds., Advan~in~ehy~i~l
5~1~b~ 18:187-238 Academic Press, New `~ork (1982).
1~
Immunoassays which employ chemiluminescent labels as the signal
generating compound are known. The application of chemilumin0scence
g6n~ration and detection for immunoassays has bean reviaw0d by W. R.
Seitz, "Immunoassay Labels Bas~d on Chemiluminescence and
2 0 Bioluminescence," Clini~l. ~iochemis~ 17:120-126 (1984). For example,
an apparatus and method for performing such assays are available from
Ciba-Corning Diagnostics' Magic-LiteTM system, which ernploys a
chemiluminescent label and magnetizable rnicroparticles. Because the
brown colored microparticles optically inter~ere with the chemiluminescent
2 5 signal, a very low mass of these par~icles is used. This in turn l~ads to very
slow reactions. For example, an assay for thyroid stimulating hormone
(TSH) is reported to have a three-hour incubation period. In addition, many
manipulation steps are involved, rnaking this assay configuration difficult to
automate.
An enhanced chemiluminesoent reaction in a white microtitration
plate followed by reading a generated signal in a luminome~er havin~ a
movable mask and photomultiplier ~ube are described in Lisenbee et al.,
European Patent Application No. 194,102 and are incorporated in the
3 5 AMERLITEIM system sold by Amersham, Inc. This latter reaction is
enzymatic and suffers from the limitations of an ELISA assay in a coate
plats, namely, the slow difflJsion rate of the reactants to the capture phase.
~ E3ST~TU~ SHEE;T
.
WO 92/08979 PCT/US91/08360
209~2~ 2
In addition, thc AMERLITEnd system employs a devic~, separat~ trom the
luminometer, in which the label is triggered.
A method for performing a chemilumin0scent assay involving directly
5 exciting and measuring a chemiluminescen~ signal emanating off an
immune compl~x immobilized on or in a solid, porous elemen~ that is used
as a separation means in a heterogenous immunoassay and an appara~us
for performing this measurement are described in plending U. S. Patent
Applications Serial No. 07/425,643 and 07/206,645 which enjoy common
10 ownership and are incorporated herein by reference.
The use of acridinium compounds as labels for immunoassays and
subsequent ganer~tion of short-lived chemilurninsscence signals from these
Iabels has been described by 1. Weeks ~.~ in UAcridiniurn Esters as Highly
15 Specific Activity Labels in Immunoassays," ~lin. ÇhemI~L 19:-1474-1478
(1984). The use of stable acridinium sulfonamide esters has been
described in a co-own~d and co-pending patent application by P. G.
Mattingly ~L~L., U. S. Patgnt Application S~rial No. 921, 971 t which is
incorporated herein by reference and published as European Patent
2 0 Application No. 0 273 115.
The generation of long-lived luminescent signals has been described
in the art as resulting from action of enzymes or nucleophilic agents on
dioxetane compounds containing an adamantane structure. Published
2 5 European Application No. 0 2~4 051 to A. P. Schaap; published P.C.T.
Patent Application No. WO 8906650; I. Bronstein ~,, "1 ,2-Dioxetanes,
Novel Chemiluminascent Substrates, Applications to Immunoassays," ,L
~ 4:99 (1988) and the 5th
International Conference on Bioluminescenc~ and Chemiluminescence,
3 0 Florence-Bologna, Italy, Sept. 25-29 (1988).
The use of a signal enhancer such as the use of avidin-biotin also is
known. For examplet U. S. Patent No. 4,228,237 ta H0vey ~L~I., describes
the use of a biotin lab~lled specific binding substance for a lig~nd used in a
3 5 method which also employs an enzyme labelled with aviclin. Tha use of a
biotin-anti-biotin syst~m is described in U. S. Patent Application Serial No.
608,849 file~ May 10, 1984, which enjoys common ownership and is
3Sll~ITIJTE SHEET
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.. ~ . . .
, : :
WO 92/08979 2 ~ ~ a ~ 2 j PCr/US91/0~360
incorporated hersin by reference (published on November 13, 1985 as
European Patent Application No. 160,900).
,
Me~hods of enhancing and amplifying the chemiluminescent si~nal
5 generated in an immunoassay are known in the art. Thus, U. S. Patent No.
4,927,769 describes a method of enhancing ~he chemiluminescent signal
generated from acridinium-ester labslled conjugates by the addition of
surfactants. Also, U. S. Paten~ No. 4,95g,182 describes a rnethod for
amplifying ~he chemiluminescent signal generated from alkaline
10 phosphatase~a~alyzed 1,2-dioxetanes by the addition of a surfactant and a
fluorsscent compound attached to it.
These known methods suffer from the lack of specificity. Thus, the
signal generated from the bound chemilurninescent label and the signal
15 generated from the unwashed free label are both amplified. This leads to an
increase in the -~ignal corresponding to the desired reaction as well as the
background. Thus, although the signal is amplified, no gain in the sensitivity
of the assay may be achieved.
2 0 The present invention overcom0s the shortcomings of the known art
by providing a chemiluminescent signal amplification method that uses ~he
speciflcity embodied in the inter~ction of "specific binding pairs." Thus, the
desired reaction signal is amplified to a much greater exten~ than the
back~round signal, which irnproves assay sensitivity. The pres~nt invention
2 5 further offcrs a common specific amplifying agent that can be used in a
variety of assays.
~ '
This invsntion provides a method for determining the presence of an
3 0 analyte in a test sample by specific amplification of a chemiluminescent
si~nal generated from a hetarogeneous immunoassay, which method
comprises: (a) incubatin~ a test sample containing an analyte with an
analyte-specific sp~cific binding pair member, to form a first mixture; (b)
incubating the first mix~ure for a time and under conditions sufficient to form
3 5 analytalanalyte specific binding member pair complexes; (c) contacting the
analyte/analyte specific binding member pair complexes with a probe
comprising an enhancer compound attached to an analyte-specific binding
member, to form a second mixture; (d) incubating the second mixture for a
~iVl~STlTlJTE 5HE~:ET
.
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WO 92/08979 PC~/US91/08360
2~9a82~ 4 `
tim~ and under conditions sufficient to form analyte/analyt~ specific binding
member pairs/probe complexes; (e) contacting the analyte/analyte specific
binding member pairs/probe complexes with a conjugate comprising a
chemiluminescent signal generating compound attac:hed to an enhancer-
S specific binding member, to form a third mixture; (fl incubating the thirdmixture for a time and under conditions sufficient to form analyte/analyte
specific binding member pairs/probe/conjugate ccmF)lexes; and
(g) determining the presence of tha analyte in the test sample by measuring
the detectable signal. The enhancer compound may be selectad from the
1 0 group consistin~ of a hapten, a fluorescent compound and di-nitrophenol. A
preferred enhancer compound is biotin. The chemiluminescent signal
generating compound may be selected from the group consisting of
acridinium esters, acridinium sulfonamideg, 1,2-dioxetanes and luminol. A
preferred chemiluminescent signal generating compound is an acridinium
15 sulfonamide. The analyte-specific binding pair member can be attached to a
solid pair.
A kit for performing an amplified chamiluminescent assay also is
provided.
etail~d l~es~tio~ Qf thQ InveotiQn
The chelT iluminescent properties of acriclinium compounds and their
use in immunoassays has been described. ~mmunochemical tracers with
2 5 acridinium esters or acridinium sulfonamide labels can be triggered with an
alkaline peroxide solution to produce a chamilurninescant signal that
maximizes af~er approximately two seconds. Light emission is completely
extinguished after approximately ten (10) seconds. Acndinium sulfonamide
labelin~ chemistry may be employed according to the invention ~or making a
3 0 stable tracer of high quantum yield. This method is as described in pending
U.S. Patent Application Sorial No. 371,763, which enjoys common
ownership and is incorporated herein by reference.
Chernically catalyzed, long-lived 1,2-dioxetane chemiluminescence
3 5 can be generated in a variety of ways. Thus, EP 0 254 051 (cited ~)
describes a siloxy-substituted dioxetan~ as 4-(6-tert-butlydimethylsiloxy-2-
naphthyl)-4-methoxyspiro[1,2-dioxetane-3,2'adamantane] that is triggered
with tetrabutlyammonium chloride solution to produce a chemiluminescent
~U1~3TITUTE 5HEET
WO 92/0~979 PCI'/l~S91/08360
2 ~
signal lasting for 20 minutes. Also, enzymes such as aryl ssterase and
alkaline phospha~ase react with aryl dioxetane derivatives stabilked with an
adamantane cage to produce similar long-lived chemiluminescent signals~
~i Also, WO 881 00594 (WO 8sQ66~iO, cited ~) describes long-lived
emissions from alkaline phosphatase ca~alyzed reactions of 3-(2'-
spiroadamantane)-4-methoxy-4-(3~'-phosphoryloxy) phenyl-1,2-dioxetanes
(AMPPD) and of a similar B-galactosidase substrate. Also described is the
use of these compounds in an immuneassay. Thus, alkaline phosphatase
10 labeling techniques are known and catalyzed dioxetane
chemiluminescence may ba used to generate long-lived signals.
The present inv~ntion provides an immunoassay which utilizes
specific binding members. A "specific binding member," as used herein, is à
1 S member o~ a specific binding pair. That is, two different molecules where
one of the moleculss through chemical or physical means specifically binds
to the second molecule. Therefore, in addition to antigen and antibody
spscific binding pairs of common immunoassays, other specific binding
pairs can include biotin and avidin, carbohydrates and lectins,
2 0 complementary nucleotide sequencss, effector and receptor molecules~
cofactors and enzymes, enzyme inhibitors and en~ymes, and the like.
Furtharmore, specific binding pairs can include members that ars analogs of
the original speeific binding members, for example, an analyte-analog. I
Immunoreactive specific bincling members include antigens, antigen
2 5 fragments, antibodies and antibody fragments, both monoclonal and
polyclonal, and complexes thereo~, including ~hose formed by recombinant
DNA molecules. The term "haptsn", as used herein, refers to a partial
antigen or non-protein binding member which is capable of bindin~ to an
antibody, but which is not capable of eliciting antibody formation unless
3 0 coupled to a carrier protein.
NAnalyte," as used herein, is the substance to be detected which may
be present in the tcst sample. The ana!y~e can be any substance for which
there exists a naturally occurring specific binding member (such as, an
3 5 antibody), or for which a specific binding member can be prepared. Thus,
an analyte is a substance that can bind to one or more specific binding
members in an assay. "Analyte" also includes any an~igenic substances,
- haptens, antibodies, and combinations thereof. As a member of a specific
~llBSTlTlJTE 5HEET
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Wo 9~/08979 PCI`/US91/08360
~0~582~ 6 `
binding pair, the analyte can be dste~ed by msans of naturally occurring
specific bindin~ partners (pairs) such as the use of intrinsic factor protein asa member of a specific binding pair for ~he determination of Vitamin B12, or
the use of lectin as a member o~ a specific binding pair for th~ determination
of a carbohydrate. The analyt~ can include a protein, a peptide, an amino
acid, a hormone, a steroid, a vitamin, a drug including those administered for
therapeutic purposes as well as ~hoss administered for illicit purposes, a
bacterium, a virus, and me~abolites of or antibodies to any of the above
substances. The details for the preparation of such antibodies and the
10 suitability for use as specific binding members are well known to those
skilled in the art.
A "capture reagentn, as used herein, refers to an unlabeled specific
bindin~ member which is specific either for the analyte as in a sandwich
1 5 assay, for the indicator r~agent or analyte as in a competitive assay, or for an
ancillary specific binding member, which itself is specific for the analyte, as
in an indirect assay. The capture reagent can be directly or indirectly bound
to a solid phase mat~rial before the performance of the assay or during the
pQrformancc of th0 assay, ther~by onabling thc s~paratlon of Immobiliz~d
2 O complexes from the test sample .
The "test sample" can be a sample of biological fluid, such as whole
blood or whole blood components including red blood cells, white blood
cells, platelets, serum and plasma; ascites; urine; cerebrospinal fluid; and
2 5 other constituents of ~he body which may contain the analyte of interest.
Optionally, test samples may be obtained from water, soil and vegetation.
The term "probe,~ as used herein, means a mernber of the specific
bindinçl pair att~ched to an "enhancer compoundn. An "enhancer
3 O compound" can be any compound used in th0 assay which can enhance the
signal generated by the chemiluminescent compound. Thus, enhancer
compounds include haptens such as biotin, and also include fluorescein, di-
nitrophenol, and the like.
3 5 The "chemiluminescent compound" is rneant to inolude all
compounds capable of genarating a cherniluminescant signal such as
acndinium esters, acridinium sulfor7amides, 1,2-dioxetanes, luminol, or
- enzymes that catalyze chemiluminescent substrates, and the like.
51JBSTlTlJTE SH@:~7
, .
wo 9~/08979 2 0 ~ ~ ~ 2 .:3 PCr/US91/û8360
"Conjugate," as used herein, means a chemiluminescent compound
to which a compound specific for the enhancer compound (a sp~cific
binding member of the enhanc~r) is at~ached. For example, if the enhancer
S compound utiliz~d is biotin, then anti-biotin, or avidin, can be used as the
snhancer-specific compound.
A solid phase may be used according ~o ths method of ~he invention.
A "solid phase", as used herain, refers to any material which is insoluble, or
10 can be mads insoluble by a subsequcnt reaotion. The solid phase can be
chosen for its intrinsic ability to attract and immobilize the capture reagent.
Alternatively, tha solid phase can retain an additional receptor which has the
ability to attract and immobilize the capture reagent. The additional receptor
can include a char~ed substance that is oppositely char0ed with respec~ to
1 5 the capture reagent itself or to a charged substance conjugated to the ~;
capture reagent. As yet another alternative, the r~ceptor molecule can be
any specific binding m~mber which is immobilized upon the solid phase and
which has the ability to immobilize the capture r0ag~nt ~hrough a specific
binding r~action. Th~ r~ptor mol~cula ~nablss ~h~ Indlr0~ blndlng ot th~
2 0 capture reagcnt to a solid phase material before the performance of the
assay or during the performance of the assay.
An assay device for the present invention can have many
configurations, several of which are d~penderl~ upon the material chosen as
2 S the solid phase. For example, the solid phase can include any suitable
porous material. By Nporous~ is meant that the material is one through which
the test sample can easily pass and includes both bibulous and non-
bibulous solid phase materials. In the present invention, the solid phase can
includa a flberglass, cellulosa, or nylon pad for use in a pour and tlow-
3 0 through assay device havin~ one or more layers containing on~ or rnore ofthe assay reagents; a dipstick for a dip and read assay; a test strip ~or
wicking (e.g., paper) or thin layer chromatographic or capillary action (e.g.,
nitrocallulose) techniques; or other porous or open pore materials well
known to those skilled in the art (e.g., polyethylene sheet material). The
3 5 solid phase, however, is not limited to porous materials. The solid phase
can aiso comprise polymeric or glass beads, microparticles, tubes, sheets,
plates, slides, wells, tapes, test tubes, or the like, or any other material whioh
has an intrinsic charge or which can r~tain a charged substance.
SU~S~IITUTE: S}~;E~
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WO 92/08979 PCI`IUS91/08360
-- .
Natural, synthetic, or naturally occurring materials that are
synthetically modified, can be used as a solid phase including
polysaccharides, e.g., csllulose m~terials suoh as paplar and cellulose
5 derivatives such as cellulose acetate and nitrocellulose; silica; inorganic
materials such as daactivated alumina, diatomaceous earth, lAgS04, or
other inorganic finely divided material uniformly dispersed in a porous
polymer matrix, with polymers such as vinyl chloride, vinyl chloride-
propylene copolymer, and vinyl chloride-vinyl acetat0 copolymer; cleth, both
10 naturally occurring (e.g., conon) and syn~hetic (e.g., nylon); porous gels such
as silica gel, agarose, dextran, and gelatin; polymeric films such as
polyacrilamide; and the like. The solid phase should have reasonable
s~rength or strength can be provided by means of a support, and it should
not interfare with the production of a de~eotable signal.
1 5
Preferred solid phase materials for flow-through assay devices
includs filter paper such as a porous fiberglass material or other fiber matrix
matarials. Tho thickness of such material is not critical, and will be a matt~r
of choice, largely base~ upon the properties of the sample or analyte being
2 0 assayed, such as the fluidity of the tes~ sample.
To changa or enhance the intrinsic charge of the solid phase, a
charged substance can be coated directly to the material or onto
microparticles which are then retained by a solid phase support material.
2 5 Alternatively, microparticles can serve as the solid phase, by being retained
in a column or being suspanded in ~he mixture of soluble reagents and test
sample, or the particles themselves can be retained and immobilizad by a
solid phase support material. By "retained and immobilizedr is meant that
the particles on or in tha support material are not capable of substantial
3 O movement to positions elsewhere within the suppor~ material. The particles
can be selected by one skilled in the ar~ from any suitable type of particulate
material and include those composed of polystyrene, polymethylacrylate,
polypropylene, latex, polytetrafluoroethylene, polyacrylonitrile,
polycarbonate, or similar materials. The size of the particles is not critical,
3 5 although it is preferred that the average diameter of ~he pa~icles be smaller
than the average pore size of the support material being used.
SUE3STITIJTE SHEE~
WO 9~/08979 P~/US9~/08360
8 2 ~
According to a preferred embodimen~ of this invention, a test sample
which may contain an analyte to be detected is contacted with a solid phase
to which a binding pair member specific for the analyte is attached, to form a
mixture. This mixture is incubated for a time and under conditions sufficient
5 for analyt~/analyte specific binding pair member complexes ~o form. Then,
these complexes are contacted with a probe comprising an enhancer
compound attaehed to an analyte-specific binding par mernber and
incubated again, to form a second mixture. This second mixture is incubated
for a time and under conditions sufficient for analyte/cmalyte specific binding
10 pair member/probe complaxes to form. The analyte/analy~e speciflc binding
pair msmbsr/probe complexes then are contacted with a conjugate
comprising a chemiluminescent signal generating compound conjugated to
an enhancer compound binding member, to form a third mixture. This third
mixture is incubated for a time and under conditions sufficient to form
15 analyte/analyle specific binding pair msmber/probe/conjugate complexes.
The presence of the analy~e in the test sample is determined by measuring
the signal generated by the chemiiuminescent compound.
1~ also is contemplated that a sandwich assay can be performed
2 O wherein a soluble capture reagent can include an analyte-specific binding
member which has been bound to a charged substance such as an anionic
substance.
The presen~ invention also can be used to conduct a competitive
2 5 assay. In a competitiva configuration, the soluble capture reagent agaln
includes a specific binding m0mber which has been anached to a charged
substance, such as an anionic polymer, with which to bind a specific binding
par~ner.
3 O The presen~ invention will now be described by way of examples,
which are meant to illustrat~, but not to limit, the scope and spirit of the
invention.
S~IBSTITUTE SHEE:T
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WO 92/08979 PCI/US91/~t8360
20~2~ 10
- E~eL~i
Example 1
Preparation of Polystyrene Latex Particles wit~l HTLV-1 Antigen
Carboxylated polystyrene latex particles wcre purified by stirring with
an equal w/w amount of a mixed-b~d ion-exchange r~sin for thr~ (3) hours
at ambient room temperature. The particles were isolated and then diluted
to a 0.5% solids concentration in 0.02M MES (pH 5.0) and to the suspension
10 then was added 1-ethyl-3-(3-dimethylaminopropy)-carbodiirnide in a w/w
ratio of 3:1 (EDAC:latex). This mixture was allowed lo stir for 20 minutes at
ambient room temperatura before purified HTLV-I antigen Iysate was added
in an amount to achieve a final concentration of 60 ,ug/ml. The suspension
then was stirred overnight at ambient room temperature be~ore the coated
15 particles were isolated by cer~trifugation and purified by three cycles of
resuspension-centrifugation in a pH 7.2 solution of 0.01 M phosphats and
0.15 M NaCI, containing 0.1% Tween-20~. Aft0r th0 final contrifugation
step, the solids were diluted to a concentration of 0.1% solids in the buffer .
2 0 Example 2
Preparation of Biotinyla~d HTLV-I Antigen
Purified HTLV-I antigan in 0.1 M borate buffer (pH 8.5) containing
0.1% Triton-X-100~, was treated with a 5 mg/ml DMF solution of
2 5 biotinamidocaproate N-hy~roxysuccinamide ester in a ratio range of 0.3 to
0.6:1 (antigen:ester). The r~action mixture was allowed to stir at ambient
room temperature for three (3) to four (4) hours, and then it was dialyzed into
a pH 7.5 solution of 0.01 M Tris, 0.15 M NaCI containing 0.1% Triton-X-
1 00~).
Example 3
Preparation of Fluorescein-labelled Antigens
Viral and recombinant viral proteins were labelled with
3 5 fluoresceinisothiocyanate using the procedure of Samuel et al.,
Me~llQ~ 107:217-224 (19883.
8UI~S~ITUTE: SHEET
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wo 9t/08979 P~/US91/08360
2 ~ 2 3
Example 4
Preparation of Acridinium-Labelled Anti-Biotin or Anti-Fluorescein
One (1) mg 10-methyl-9-(N-tosyl,N-(2-carboxy~thyl))acridin~
carboxamid~ was dissolved in 100 111 of DMF, ~nd then treat0d with 50 ,ul of
a 5.75 mg/ml DMF solution of N-hydroxy-succinimide and 50 1ll of 9.75
mg/ml DMF solution of ~-ethyl-3-(3-dime~hylaminopropyl)-carbodiimide. The
solution then was allowed to stir at ambient room ~emperature overnight.
The activa~ed acridinium derivative then was coupled to anti-biotin (or anti-
fluorescein) as follows. The antibody was dialyzed against o.1 M phosphate
(pH 8.0), con~aining 0.15 M NaCI, and the protein then was adjusted to a
concentration of 1 mglml in the same buffer. Tha activated acridinium
derivative at a 5 to 10 molar excess then was added to the antibody solution
a~ room temperatur~. After ten (10) minutes, the reaction mixture was
1 5 centrifuged (12,000 rpm for two (2) minutss~ to remove aggr~gates, and the
sup~rnatant solution then was applied to a TSK-250 gel fi~ration column,
which previously had been equitibrated with 0.01 M sodium phosphat~, pH
6.3, containing 0.15 M NaCI. One ml fractions were collacted, and the
absorbance monitored at 280 nm and 369 nm. Frac~ions containing the IgG
2 0 peak were pooled, and tha extend of acridinium incorporation was
calculated as follows: protein concentration was determined using
absorbance at 28Q nm corrected for the contribution made by acridinium at
this wavelength (corr~cted absorbance = A280 ~ A369 x 0.2471). Moles of
acridinium and IgG were calculatcd using a molar ~xtinction coefficient of
14,650 and 220,000 M-1CM-1, respectfully.
Exampls 5
Assay for HTLV-I
3 0 Fifty (50) ~ll of a 0.1% solid suspension of polystyrene latex particles
(pr~viously coated with tlTLV-I antigen as described in Example 1) in a pH
7.5 solution of 10% sucrose (w/w%), û.1% Bovine Serum Albumin (BSA),
0.1% Tween-20~, 0.1 M phosphate and 0.1% sodium azida was added to
100 ~,11 of sample in a reaction well. The suspension was then allowed to
3 5 incuba~e for about 20 minutes at 40C befora being transferred to the
~apture membrane by two successive 300 ~11 washes of pH 7.2, 0.01 M
phosphate, 0.15 M NaCI, containing 0.1% sodium azide.
~;U113STi71-lJTE~: SIHEET
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WO 92/~8979 PCT/US91/08360
2~382~
The washed suspension then was allowed to incubate ~or about t~n
(10) minutes at 40C before it was treated with 30 I~LI of 167 ng/ml solution ofbiotinylated HTLV-1 antigen ~prepared as in Example 2) in 0.1 M Tris (pH
8.5) 50% calf serum and 0.1 M NaCI containing 0.1% sodium a~ide. The
5 capture membrane then was allowed to ineubat~ for 20 rninutes before it
was washed with thrae (3) 100 ul portions of a pH 8.5 wash solution which
comprised 0.1 M Trjs 0.t5 M NaCI and 0.1% Triton~ containing 0.1%
sodium a~ide. Tha wash0d captura rnembrana thsn was treated with 30 lli
of a t67 ng/ml sotution of anti~biotin to whioh a cherniluminescent acridinium
10 sulfonamide moiety had bsen attached (as in Example 4) in 0.01 M MES
(pH ~.3) 0.15 M NaCI ~% BSA and 0.5% Triton~ containing 0.1% sodium
æide. A~ter a further ten (10) minute incubation at 40C the oapture
membrane was washed with three (3) 100 ~i po~tions of a pH 5.5 solution of
0.1 M MES and 0.15 M NaCI containing 0.1% sodium æide. The washed
1 5 capture membrane then was incubated for ten (10) minutes at 40C prior to
being tr0ated with an alkaline peroxide solution (0.25 N NaOH containing
0.3% peroxide). The chemiluminescence was read for six (6~ seconds and
the presence or absence of anti-HTLV-I was determined.
2 0 ~xample 6
Preparation of Polystyrene Latex Particles Coated Wigh HCV Antigan
20 ~9 1 ICV antigen was mixed with 100 mg of 2.66 micron diameter
polystyrene latex particles in 20 ml of a pH 7.0 0.1 M phosphate buffer and
2 ~ allowed to s~ir ovemight at room temp~rature. The solids then were isolatedby centnfugation (17 000 rpm for 25 minutas~ and then purified by three
cycles of resuspension~entriflJgation in 20 rnl of a pH 7.0 0.1 M phosphate
buffer containing 0.005% Tween-20~.
3 0 . Example 7
Preparation of Biotinylated HCV Antigan
One (1 ) mg of biotinamidocaproate N-hydroxysuccinamide estsr was
added to ~00 ~lg of I~C:V an~igen in 2.5 ml of 0.0~ pH 8.5 borate containing
3 5 0.1% Tween-20~. After stirring at room temperature for two (2) hours ~0 mg
of BSA was added and the solution was dialyzed overnight at ambient room
temperature a~ainst two- 500 ml changes of 0.02 M Tris buffer (pH 8.5)
containing 0.002 M dithiothreitol and 0.1% Tween-20~.
SUB$TI~T~: SHE~
WO 92/08979 PCI`/U~;91/08360
~a~s~2~j
13
Example 8
Assay for HCV
SFifty (50) 1ll of a 0.~% solid suspension of polystyrane la~ex particl0s
in 0.1 M pH 7.0 phosphate buffer, containing 0.005% 1 wean-20~
(previously coated with recombinant HCV antigen as clescribed in Example
6) was added to ~0 ~,~l of a pH 8.5 solution of 0.02 M borate buffer containing
1% Tween-20~, 0.025% e011quat, 0.01% cetylpyridinium chloride, 0.05 M
1 0 EDTA, 0.03 M NaCI and 0.1% sodium azide and then was added to 100 )11 of
sample in a reaction well. The suspension was ~len allowed to inel3bate for
about 20 minutes at 40C before being ~ransf0rred to the reaction capture
mernbrane by two successiYe 300 ~I washes of pH 7.~,0.01 M phosphat~
buffer and 0.15 M NaCI, containing û.1% sodium æide.
1~
The washed suspension then was allowed to incubate for about ten
(10) minutes at 40C before it was tr0ated with 30 ~11 of 660 ng/ml solution of
biotinylated recombinant HCV antigen (prepared as in Example 7) in 0.02 M
borate (pH 8.5), 5% BSA, and 5.0/O Triton X-100~ containing 0.1% sodium
2 0 azide. The capture membrane then was allowed to incubate for 20 minutes
before it wa~ washed with three (3) 100 ,ul portions of a pH 8.0 wash solution
which comprised 0.1 M borate, 0.15 M NaCI, and 0.05% lithium
dodecylsulfate tLDS) containing 0.1% sodium azide. The washed capture
membrane then was trsated with 30 111 of a 16~ ng/ml soiution ot anti-biotin
25 in 0.0~ M phosphate (pH 6.3), 0.16 ~ NaCI, 5% calf serum, and 0.1%
Triton~ containing 0.1% sodium azide, to which a chemiluminesc~nt
acridinium sulfonamide moiety had b0en attached (prepared as in Example
4). After a further ten (10) minute incubation at 40G, the capture membrane
was wash0d with three (3) 100 ~,11 portions of a pH 8.5 solution of 0.1 M
3 0 borate, 0.15 M NaCI and 0.02% SDS, containing 0.1% sodium azide. The
washed capture membrane then was incubated for ten (10) minut~s at 40C
prior to being treated with an alkaline peroxide solution (0.2~ N NaOH
containing 0.3% peroxide). The chemiluminesc~nce was read for six (6)
seconds and the presence or absence of anti-HCV was determined.
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WO 92/08979 PCI'/US91/~18360
2093~2~ 14
Example 9
Preparation of Polystyrene Latex Particles Coated with HIV Antigen
HIV antigen (10 mg) in 34.28 n71 of 0.5 M borate buffer (pH 8.5) is
5 mixed with 10 ml of a 0.5% solids susp~nsion of polystyrene latex particles,
and then 55.72 ml of deionized water was added. Thls suspension was then
allowed to stir overnight at room tempe~ature. The solids were ~hen isolated
by centrifugation (17,000 rpm for 30 minutes) and then purified by three
cycles of resuspension-centrifugation in 0.1 M phosphate buffer (pH 7.0)
10 oontaining 0.1% Tween~. The coated par~icles wera then resuspendecl,
allowed to stir gently at 56C for one (1 ) day, and then stored at room
temperature prior to use.
Example 10
Preparation of Biotinylated HIV Antigen
HIV antigen (1.9 mg) in 2.278 ml of 0.1 M borate buffer (pH 8.5),
containing 250 mm NaCI and 0.1% sodium æide, was treatad with 0.125 ml
of 10% Triton~ for 30 minutes. Th~n, 97 ,ul of 5 mg/ml of
2 0 biotinamidocaproata N-hydroxysuccinarnide ester dissolved in DMF, was
added. The reaction mixture was allowed to stir at raom temperature for
about two (2) hours. Th~ mixture was dialyzed ~xtensively a~ainst 0.1 M
borate buffer (pH 8.5) containing 2~0 mm NaCI, 0.1% SDS, and 0.1%
sodium æide.
Example 1 1
Assay for HIV
Fifty (50) ~,11 of an approximately 0.2% solid suspension of polys~yrene
3 0 latex particles (pr~viously coated with HIV antigen as described in Example
9) in a pH 7.0 solution of 11/~ sucrose (wlw%), 0.01 M EGTA, 0.1%
CHAPS~, 0.1 M phosphata and 0.1% sodium azide was added to 100 ~11 of
sample in a r~action w~ll. The suspension was then allowed to incubata for
about 20 minutes at 40C before being transf~rred ~o the captur~ membrane
3 ~ by two successive 300 ~11 washes of pH 8.5, 0.1 M borate buffer, ~.15 M
NaCI, and 0.01~/o lithium dodecylsulfat~ (Ll:S) containing 0.1% sodium
azide.
WO 92/08979 ~ ~ 3 ~ ~ 2 ~ PCI/US91/OX360
1 5
The wash~d suspension then was allowed ~o incubate for about ten
(10) minutes at 40C before it was treated with 30 ~LI of 750 ng/ml solution of
biotinylated HIV antigen ~prepared as in Example 10) in 0.1 M borate buffer
(pH 8.5~, 1% .E~QJi Iysate, ~00 ~lg/ml CKS, 12.~% calf serum, and 1.0%
S Cholic Acid containing 0.1% sodium a~ide. The capture membrane then
was allowed ~o incubate for 20 minutss before it was washed with thrse (3)
100 ~I portions of a pH ~.5 wash solution which comprised 0.1 M borate,
0.15 M NaCI, and 0.03% LDS containing 0.1% sodium azida. The washed
capture membrane then was treated with 30 1ll of a 1l67 ng/ml solution of
10 anti-biotin antibody to which a chemiluminescent acridinium sulfonamide
moiety had been attached (prapared as in Example 4) in 0.01 M phosphate
(pH 6.3), û.15 M NaCI, 5% BSA, and 1.0 % Triton~ containing 0.1% sodium
azide. After a further ten (10) minute incubation at 40C, the capture
membrane was washed with five (3) 100 1ll portions of a pH ~.5 soluti~n of
0.1 M borats, 0.15 M NaCI and 0.0~% LDS, containing 0.t% sodium æide.
The washed capture membrane then was incubatad for ten (10) minutes at
40C prior to being treated with an alkaline peroxida solution (0.25 N NaOH
containing 0.3% peroxide). The chemiluminescence was reacl for six (6)
seconds and the presence or absehce of anti-HlV was determined.
Other modiflcations and variations of the specific embodirnents of the
invention as se~ forth herein will be apparent to those skilled in the aJt.
Accordingly, the invention is intended to be limited in accordance with the
appended claims.
~3~E3STl~l~
