Note: Descriptions are shown in the official language in which they were submitted.
'6 1 ;~
096/02839 PCT~S95/07642
cHEMILu~lINEscENT SIGNAL ENHANCEMENT
saokground of the Invention
s 1. Field Gf the Invention
The present inventiorl relates to immunoassays
utilizing chemiluminescent compounds and, more
particularly, to the use of surfactants for enhancing the
chemiluminescent signal of acridinium sulfonamides.
11~
2. sackground of the Invention
Immur;oassays which employ a chemiluminescent label as
the signal generating compourld are known. For example,
chemiluminescence generation ar.d detectior. in immunoassays
is disclosed in h'. R. Seitz, "Immunoassay Labels sased on
Chemiluminescence and Bioluminescence,'l Clinical
siochemistrv~ 17:12n-126 (]984). The use of acridinium
esters as labels for immunoassays and subsequent generatior.
of short-lived chemiluminescence signals from these labels
is disclosed in I. weeks, et, al., "Acridinium Fsters as
E~ighly Specific Activity Labels ir. Immunoassays," Clinical
Chemistry, 19:1474-1478 (1984~.
Methods of increasing the intensity of a
chemiluminescent signal generated hy acridir.iulll esters and
certain other labels in an immunoassay are also known ir.
the art. For example, U.S. Patent ~lo. 4,59,182 describes
a method for amplifying the chemiluminescent signal
generated from alkaline phosphatase-catalyzed l,2-
dioxetanes by the addition of a surfactant and a
fluorescent compound attached to it. U.S. Patent No.
4,927,769 discloses use of certain types of surfactants for
enhancing the chemiluminescent signal generated from
acridinium ester conjugates. The method uses a first
signal generation reagent under acidic conditior.s and, a
35 second signal generating reagent to increase the pH and
trigger the chemilum,inescent signal. The acridinium esters
require using this two stage process to efficiently emit
the signal. This is a problem, however, in that antibody-
conjugated acridinium esters are often unstable above
~1 91 ~1 3
Wo~ 2839 r
certain pH levels due to h~dro:lysis.
To avoid this problem, other chemi].umi.llescent. labels
have been developed. ~or example, stab]e acri.dinium
sulfonamides a.s labels for irr.munoassays i.s descL~ibed in
s commonly-assigned published European E~atent. ~pplicatio~ o.
273,115 of ~attingly et al. However, while acridinium
sulfnn~idps are useful chemiluminescent reagents, there
presently exists a need to enhance the qenerated
chemiluminescent signal of these compourlds in order to
impro1~e immunoassay sensitivity and efriciency.
Therefore, a general object of the present invention
is to provide enharlcemerlt of the chemiluminescent.. signal. c~f
acridinium sulfonalr!ides. other objects will hereinaft.er
become eviderlt to those skilled in the art.
Summarv o~ the ~nv~ntion
Tne present inventi.on concerns a mettlod for ger.erating
an enhanced chemilum.inescent signal from an acridinium
sulfonamide compound. The method comprises the step of
2~ cont.act:ing the acridinium sulfonamide compound with a
tri.gger solution in the presence of an erhancer to obtain a
chemiluminescent siynal which is about 1.5 to ~ t.imes
stronger than would otherwise he generated in the abserce
of t.he enhancer.
2S In a related aspect, the invention is a method for
generat:in~ an enhanced chemiluminescent signal from a
acridinium sulfonamide compound, the method comprising the
step of contacting the acridinium sulfonamide compound with
a trigger solut.ion in the presence of an enhancer
comprising at l.east one member selected from the ~roup
consisting of (i~ nonionic surfactants, (ii~ ~wltterionic
surfactants and (iii~ arionic surfactants.
Tne in~rentiorl further provides a trigge.r sol~!t.ic)n for
generating a chemiluminescent signal from an acridinium
3s sulfonamide compo~md. The trigger solution comprises an
oxidant and an enhancer. The enhancer comprises a
surfact.allt effective for increasirlg the int.ensity of the
chemiluminescent signal generated hy the acridirlium
sulfona.mide compound.
2 ~ 9 1 6 ~ 3
~ W096~0~839 PCT~S9S/076~2
The trigger solution of t}~e invention, containinc3 ~he
oxidant and the enhancer, can be provided as a reagent in
an immunoassay teat kit. in which the acridinium sulfonamide
compound is included as another reagent. Accordingly, the
invention is further directed to an immunoassay te.st. kit
having reagents which comprise (a) an acridinium
~ sulfonamide compound; (b) a trigger solution separate from
said acridinium sulfonamide; and (3) an enhancer ComE)rising
at least one surfactant selected from the group consisting
o of ~i) nonionic surfactants, ~ii) zwitterionic surfactants
and (iii~ anionic surfactants. The acridinium su].fonamide
compound can be provided in the form of an acridinium
sulfonamide-labeled conjugat.e for use in a variety of
immunoassay formats. In a preferred embodiment of the test
kit, made possible oy our discovery that the enhancer of
the present invention is stable in the trigger solution,
the acridinium sulfonamide c.or.jugate can be supplied as one
reagent, and a trigger solution c~nt~in;ng a mixture of the
oxidant and the enhancer can be provided as a second
reagent. Alternatively, the trigger solution and the
enhancer can be supplied separately from one another.
Further, the enhancer can be supplied in the form of a
mixture with the acridinium sulfonamide compound.
2s Det,aile~ Descrirtion
As used herein, the term ~acridinium sulfonam.ide
compound~ means the chemiluminescent compounds identified
by the formula:
21 ~ 1 6~ 3
W096to~39 PCT~IS9~076~2
'I
R"- ~1
Y-
0~/\
N - S02- R
R - X~
wherein R, R', P ", Xl , and X7 are substituents which do not
interfere ~~ith the chemiluminescent signal provided by such
s cher,ilulrlinescent compounds, with the T~ro~iso that P.n-Xl and
R-X~ may be independently hydroger.. More specifica].ly, R
and p~n may be spacer arms and Xl and Y.7 may he
indeperldently members selected from the group c.cnsi.sting of
hydrogen, carbox~, carboalkoxyl, carbox.amido, carboaryloxy,
o cyano, carboximido, isocyanato, isot.hiocyarat.o, sulfo,
s-ulfonyl halide, carbon~l halide, N-succinimidyloxyca.rbollyl
and N-succinimidyloxys-llfonyl. Y~ is an appror,riate
courlt.erior., and may be selected from the group consisting
of sulfat.e, alkylsulfate, halosulfate, haloborate,
haloacetate, halophosphate, phosphate and halide.
Preferably, the counterion is sulfate or halide
R, P~, and R" may independently include a member
sel.ected from the group consisting of alkyl, alkylene,
aryl, subgtituted alkyl, substituted aLkylene, and
substituted aryl groups, such that one or more h~drogens of
said member can be replaced by an al'kyl, aryl, alkylene,
substituted alkyl, substituted alkylene, suhstituted aryl,
alko~y, aryloxy, halo, amino, protected amino, substituted
amino hydroxy, protected hydrox~, oxo, thio, imino,
mercap~o or substituted mercapto grollp; or suc.h that one or
more carbon atoms of the member can be replaced by a
heteroat.om. The heteroatom may be selected from the group
consisting of nitrogen, phosphorus, sulfur ancl oxygen.
R and P.~ independently may also be spacer arms c,f the
21 9 1 6 ~ 3
~ WO9G/02839 PCT~ISgS/07642
formu]a -(C~12)l~- where n = 0-50.
The preferred acridinium sulfonamide compounds for use
in the presert invention are 10-methyl-ld-(2-carboxyethyl)-
N-tosyl-9-acridinium carboxamide and 10-(3-sulfoprop~ d-
s ~2-carboxyethyl~-1d-tosyl-9-acridiIIium carboxamide. The
most preferred acridinium sulfonamide is 10-(3-
~ sulfopropyl)-Id-(2-carboxyethyl~-N-tosyl-9-acridinium
carboxamide.
Also suitable for use in the present inventioIl are the
o acridinium sulfonamide compounds referenced in Molz et al.
European Patert Applicativn No. 257,541 (published March 2,
1983) incorporated herein by reference. The acridinium
sulfonamide compounds discussed in Molz et al. have the
following general formula:
R~
¦ A-
R2~R3
O = C - R4
In which p~1 stands for hydrogen, an alkyl, alkenyl or
alkynyl radical with 1 to 10 carbon atoms, a benzyl or aryl
group, R2 and P~3 stand for hydrogen, ar alkyl group with 1
to 4 carbon atoms, a substituted or unsubstituted amino
group, a carboxy, alkoxy, cyano, nitro group or halogen, R4
represents a radical in which a sulfonamide group is bound
directly to the carbonyl group via the nitrogen or a
2s thioalkyl or thioaryl radical of formula II
-S . X . R5 (II)
where X is a branched or unbranched aliphatic or aromatic
group which may also contain heteroatoms, and R5 is a
reactive group which selecti~ely under gentle conditions
W096l0~839 2 1 9 l 6 1 3 PcT/us9~/n76~2
G
can enter into a bond wi.t.h aminc, carho~ - th;ol or other
functin~r.,al groups in substances of biological interest,, and
A- i9 an anion which does not i.mpair chemiluminescence.
Preparation of the acridinium sulfonami,cle compounds
s useful in the present invention i.r. dificlosed in Mat,t..inc31y
et al. European Pat.ent. Application 273,115 published July
6, 1988, incorporated herein by reference. Molz et hL
published European Patent Application No. 257,541 also
discusses preparation of acridinium sulfonamides.
o The acridinium sulfonamide can be oxidized by any
oxidant whicll reacts with the acridinium sulfonamide to
yield a product in an electronically excited state. As it.
returns to the ground state, this product Ieleases energy
in the form of light, in a chemilumlinescent reaction.
As used herein, a "trigger solutior," means t.he
solut,ior containing the oxidant which i,nitiates or
catalyzes t,he chemiluminescer.t, reaction. A pre~erred
trigger solution comprises hydrogen peroxide in dilute
alkali.
2~ As used herein, the term ~enhancer~ means a reagent
provided b~ the present invention that increases the total
light., emission of the chemiluminescent react,ion and~or t,he
signal to background noise ratio ot the chemiluminescerlt
reaction in com,oarison to that, achieved by the acridinium
2s sulfonamide in the absence of the enhanceI- The enliallcer
is comprised of at least one member selected from the grou~,
consisting of nonionic, ~witterionic, and anlorlic
surfactantE,.
Noniorlic surfactants are discussed in Surfac~tant
Scierlce and Technolocry~ by Drew Myers, VCH PublisneIs,
Inc., ~.Y., 1988, which is incorporat,e-l b~ reference
hereill. Preferred nonionic surfactants include
polyoxyethylenated alkvlerlated alkylphenols,
polyoxyer,hylenated straight,-chain alcoho,ls,
polyoxyethylenated sorbitol esters, and alkanolamine-fatty
acid condellsates. Commerciall~ available noniorlic
surfactants suitable for use with the present invention
include T~TON X-100, T~JEEN-20 and P~ -35.
~ 1 9 1 6 1 3
~ W096/0283g PCT~S9~/07642
zwitterionic surfactants are also discussed in Myers,
su~ra. Precerred zwitterionic surfactants are of the
formula:
R2
Rl - N~ - R3 - SO3-
R4
wherein Rl is C14-Cl~ aliphatic; R2 and R4 are methyl or
ethyl; and R3 is Cl-C3 aliphatic. other suitable
zwitterionic surfactants include the formula wherein R~ is
o C8-C20 aliphatic; R2, R4, and ~.3 are Cl-C4 aliphatic.
Commercially available zwitterionic surfactants include
Tetradecylzwittergent, Hexadecylzwittergent and C~APS.
Anionic surfactants are also discussed in Myers,
suora. Preferred anionic surfactants include lithium
dodecylsulfate, sodium dodecylsulfate and cholic acid.
We have found that the cationic surfactants Merquat
and Cyastat, which are of the type disclosed in U.~. Patent
l~o. 4,927,765, provide li~:tle or nG ennancement of the
acridinium sulfonamides.
Generally, enhancers suitable for use according to the
present invention should be soluble in the reagents under
the conditions in which the chemiluminescent reaction takes
place. We have discovered that the surfactants prescribed
for use as enhancers in the present invention enhance the
chemiluminescent signal from acridinium sulfonamides such
that the intensity of the signal is about 1.5 to 8 times
greater, and preferably about 3 to 8 times greater than the
~signal intensity obtained in the a~sence of the enhancers.
These results are unexpected because not all surfactants
30 provicde enhancement, much less the sarne degree thereof, in
the acridiniurn sulforlamides. Moreover, the enhancement
provided by the surfactants prescribed for use in the
present invention, including without limitation, the degree
of response, could not have been predicted from the
3s enhancernent observed in the case of acridinium esters. For
W096~t2~3~ 2 1 9 l 6 ~ 3 F~~ Y~ ~2
examcle, the cationic surfactants ~5erquat and Cyastat
perform poorly or not at ali as enhancers for the
acridinium sulfonamide compounds used in the presellt
inverltic~n .
s 5econda~y reactior~ conditions which can affect the
clegree of signal enhancement include pH, temperature, and
reagent concentration.
As deacribed below, the preferred r-ange for the
en~lallcel~- pI-eser t iII all immulloassay chemilu~rlirlescent
lo reaction is from about 0.25% to about 5~ hy weight, baseA
on the total weight of the solution in which the
chemiluminescent. reaction takes place. ~i.e., the solutlon
containing the enhancer, the trigger sol.ution ar.d the
acridi:rium sulfonamide compound~. More preferahly, the
enrancer should be present in such solution in an arrount of
ahost :~ by weight..
B~ amplifying the signal generated by acridinium
sulfonamide compounds, the enhancers of the present
invention facilitate determination of the presence of ar
20 acridinium nulfonamide in a test sample. A particulaI-ly
useful application of the present invention involves
determination of an acridiniuro sulfonamide conjugate in an
immunoassay. Thus, an imrrunoassay test sarr,ple suspec:ted of
cor,taining an analyte can be cont.acted wi.th (i.~ the
~s acridini.um sulfonamide compound ~i.e., a conjugate oc tne
acridi~ium sulfonamide with an anti~en, hapte~l, antibod~,
nucleic acid, etc.); (ii) the trigger solution; and ~i.iil
an enhancer accordi.ng t.o the invention, sc as to generate
an enhanced signal. The trigger and the enhancer can be
combined before addition to tne acridinium sulfonamide.
The acridiniurr. sulfonamide soluti.on and t.he
trigger/enhancer soluti.on can ne brought in contact with an
analyte either sequentiall~ or simultaneously. Techrliques
for preparing acridiniurrl sulfonamide conjugates for use in
~s imm,llnoassay.s are desc.ribed in 15attingly et al. published
European Patent Application 273,115, incorporat.e-l by
referenc.e herein.
~ rhe present invention can be employed in varic~us
heterogeneous and horr,ogeneous immunoassay system format..s
2 1 9 ~ ~ ~ 3
~/02~9 rCT/US9~/07~42
known in the ar.. SUCh immunoassay system formats incluc~e,
but are not intended to be limited to, competitive and
imrr.urometric techniques. Generally, such immunoassay
systems depend upon the ability of a binding member, such
as, for example, an immunoglobulin ~i.e., a whole antibody
or fragment thereof) to bind to a specific analyte from a
test sample, wherein a labeled reagent comprising a binding
member lahelec1 with a chemiluninescent compound, such as
the acridinium sulfonamide described herein, is employed to
lG determine the extent of bir.ding. Typically, the extent of
binding in suc:h immunoassay system formats is determined by
the amount of the chemiluminescent compound present in the
labeled reagent which either has or has not participated in
a bindinc~ reactiorl with the analyte, wherein the .signai
which is generated by the chemiluminescent compound as
described herein is detected and correlated to the amount
of analyte present in the test sample. The test sample can
be any material suspected of containing the analyte The
test sample can be used directly as obtained from the
source or following a pretreatment to modi.fy the character
of the sample.
Elomogeneous immunoassays typically are performed in a
competitive immunoassay format involving a competit.ion
between an analyte from a test sample and a labeled reagent
for a limited number of receptor binding sites on an
antibody to the analyce. The labeled reagent comprises the
analyte or analyte-analog labeled with a chemiluminescent
compound wherein the concentration of analyte in the test
sample determines the amount of the labeled reagent that
30 will specifically bind to the antibody. The amount. of the
labeled reagent-antibod~ conjugate produced by such binding
may be cluantitativel~- measured and is inversely
proportional to the amount of analyte present in the test
sample.
3s Heterogeneous immunoassay formats involve a labeled
reagent or tracer comprisirg an analyte, analyte-analog, or
an antibody thereto, labeled with a chemiluminescent
compound. The assa~ involves formation of a free species
and a bound species. In order to correlate the amount o~
21 ql 6~ 3
~o~ 283~ PCT~S95~76~2
tracer in one c~f such species ~.o t.ht-~ amollnt of ana:Lyt.e
presen~ in t.he test sample, the free species rr,ust fi.rst be
separated from the bound specie,, which can be accomE)Iished
according to methods known in the art empioying solid phase
5 mat.erials fo.r the direct. immobili.z.ati.on of one of the
binding participants in the bindirg reaction, such as the
antibocl~{, analyte-analog, or- analyte, whereirl one of the
bindin~ participants is immobili~ed on a solid phase
material, such as a test tube, beads. particles,
o microparticles or a matrix o~ fibrous material, and the
likef according to methods known in the art, The solid
phase rrlaterials can be any solid material tc, whic11 a
binding participar.t can be irrlrnobilized and include, but are
not interded to be limited to, beads, magnetic particles,
paramagnetic particles, rnicroparticles or macro particles,
test tubes, and microtiter plat.es. Such solid phar,e
materials can be made from synthetic materials, naturally
occurring materials, or naturally occurrin3 mat.eria~.s which
have been synthetically modified, and include, but are not
2Q intended to be limited to, cellulose materials, such as
paper, cellulose and cellulose derivatives such as
cellu.lose acetate and nitrocellulose; fiberglass; natl.lra:ily
occurri.ng cloth such as cotton; synthetic cloth such as
nylon; porous gels, such as silica, agarose, dextrar., and
2s gelatin; porous fibrous matrixes,; starch ba.sed materials,
such as cross-linked dextran chains; ceramic materials;
olefin or thermoplastic materials including polyvinyl
chloride, polyethylene, polyvinyl acetate, polyamide,
polycarbonate, polystyrene, copolymers of vinyl acetat.e and
3~ vinyl chloride, combinations or polyvinyl chloride-silica;
and the like.
Hetero~ereous immurloassais can be perEormed in a
competitive imrrlunoassay format whereil~, for exarr.ple, t.he
antibody can be immobilized to a solid phase materi.al
whereby upon separation, the signal generat.ed by the
chemiluminescent compound of the bound or free species can
be detected and correlated to the arrlount of analyte present
in the test sarr.ple. Another form of a het.erogeneous
immunoassay employing a solid phase materLal is re~erred to
2 ~ ~ ~ 6 1 3
~ W096/02839 PCT~Sg~/0764
Il
dS a sandwich immuroassay, which involves consacting a test
sample containing, for example, an antigen with a protein
such as an antibody or another substance capable of binding
thc- antigen, and which is immobili~ed on a solid phase
- s material The solid phase material typicall~ is treated
with a second antigen or antibody which has beer labeled
with a chemiluminescent compound The second antigen or
antibody then becomes bound to the correspording antigen or
antibody on th.e solid phase material and the signal
o generated by the chemiluminescent compound in the bound or
the free species can be detected and correlated to the
amount of analyte present in the test sample.
~ s used herein, the term "test sample~' means ar.y
sample derived from any biological source, such a.s a
physiological fluid, including, blood, saliva, ocular lens
fluid, cerebral spinal fluid, sweat, urine, milk, ascites
fluid, mucou.s, synovial fluid, peritoneal fluid, amniotic
fluid or the like. The test sample can be pretreated prior
to use, such as preparing plasma from blood, diluting
viscous fluids, or the like; methods of treatment can
involve filtration, distillat.ion, concertration,
inactivation of interfering components, and the addit.ion of
reagents sesides physiological fluids, other liquid
samples can be used such as water, food products and the
2s like for the performance of environmental or food
production assays in addition, a solid material su.qpected
of containing the analyte can be used as the test sample.
In some instances it may be beneficial to modify a solid
test sample to form a liquid medium or to release the
30 analyte. The analyte can be any compound or composition to
be detected or measured and which has at least one epitope
or binding site.
As used herein, the term '~analyte" means any substance
for which there exists a naturally occurring binding member
3s or for which a binding member can be prepared. Analytes
include, but are not limited t:o, toxins, organic compounds,
proteins, peptides, microorganisms, amino acids, nucleic
acids, hormones, steroids, vitamins, drugs (including those
administered for therapeutic purposes as well as those
2~ q 1 6 ~ 3
W096/0~39 r~111S95/07G42
12
adnr,inisteKed tor il.licit purposesi, virus particles and
metaboi.i.tes of or antibodies t.o any of the above
subr,tances. In particular, such ana:lytes include, ~.nlt: are
not ir.tended to be limited to, ferrit.i.n; crearir1ine ki.na.se
s Ms tc~-~s?; digoxini phenytoi.n; pher.oba.rbital;
carbarr,azepine; vancomycin; gentamicin, theophillirLei
valproic acidi quinidille; luteinizing hormone ~LH~
follicle stimulating hormone ~FSH); estradiol;
progesterone; IgE antibodies; vitamirl sl2 micro-globulin;
glycated hemoglobin (Gly. Hb); cortisol; digitoxir~
acetylprocainar1lide ~NAPA~; procainanide; antibc,dies to
rubella, such as rubella-IgG and rubella-Ig~; arltibodies to
toxoplAsmosis, such dS toxoplasmosis IgG (Toxo-IgC,i and
toxoplG~smosi.s Ig~ ~Toxo-Ig~l); test.osterone; s.alicylates;
lS acetaminopheni hepatiti.s B virus surface ant.igen l~lssAg)i
antibodies to hepatitis B core art.igen, s~lch a.s ant.i-
hepatit.is B core antigen IgC and IgM ~.nti-HBC')i hum.ar
imrr.une deticiency virus l and 2 (E~IV-'1 and HrV~:23i hurnarl T-
cell leukemia virus l and 2 ~HT~V-l and ~T~V-2'1; hepatiti6
2~ Be antigen (HseAg); antibodies to hepatitis Be ant.igen
~Anti-~Be3; thyroid stimulating hormone (TSH); total
thyroxine ~total T41; ~ree thyrc~xine. (free T4~; tot.a].
triiodothyrolline ~Total T3); free trii.odothyronine ~F.'ree~
T3); carcinoembryoic antigen ~CEA)i and alpha fet.al prot.ein
2s ~AFP). Drugs of abuse and controlled substances include,
but are not intended to be limited to, amphetamir.ei
methamphetamine; barbiturates such as amobarbital.,
secobarbital, pentobarbit.al, phenobarbital, and barhit.a];
ben~odiazepines such as librium and valium; cannabir1oids
such as hashish and marijuana; cocaine; fentar.yl; ~SDi
methaoualorle; opiates such as heroin, morphir1e, codeillef
hydromorphone, hydrocodorle, rmethador1e, o~ycodorle,
oxymorphone and opiurr,i phencyclidine; and propoxyhene. ~he
term analyte also includes an~ antigenic substances,
haptens, anti.bodies, macromolecules and combillatiorls
thereof. The foregoin~3 is not intended to be a lirniting
definition.
As usecd herein, the term "analyte-analog/~ mear!s any
substance which cross-reacts with an analyte-specific
~' 1 9 1 ~ 1 3
~ WOg610283g rcrJusss/076~2
binding memher, although it may do so to a greater or
lesser extent than does the analyte itsel~. The analytc--
analog can incLude a modified analyte as well as a
fragmented or synthetic pGrtion of the analyte molecule, so
s long as the analyte-analog has at least one epitope site in
common with the analyte of interest. An example of an
analyte-analog is a synthetic peptide sequence which
duplicates at least one epitope of the whole-molecule
analyte so that the analyte-analog can bind to an analyte-
lo specific binding member. The binding member is a member ofthe binding pair, i.e., two different molecules wnerein one
of the molecules specificall~.~ binds to t=e second molecule
through chemical or physical means. In addition to antigen
and antibody binding pair members, other bindir.g pairs
include, as examples ~lithout limitation, biotin ancl avidin,
carbohydrates and lectin~" complementar~- nucleotide
sequences, complementary peptide sequences, effector and
receptor molecules, enzyme cofactors and enzymeC.~ erzyme
inhibitor.s and enzymes, a peptide sequence and an antibody
specific for the sequence or the entire protein, pols~meric
acids and bases, dyes and protein binders, peptides and
specific protei.n binders (e.g., ribonuclease, S-peptide and
ribonuclease S-proteirl), and the like. Furtherrmore,
binding pairs can include members that are analogs of the
original binding member, for example, an analvte-analog or
a birding member made b~ a recombinant techniques or
molecular engireering. If the binding member is an
immunoreactant it.. can be, for example, a monoclonal or
polyclonal antibody, a recombinant protein or recombinant
antibody, a chimeric antibod~l~, a mixture(sl or fragment(s)
of the foregoing, as well as a preparation of such
antibodies, peptides and nucleotides for which suitability
for use as bindint~ membe2s is well known to those skilled
in the art.
The above-described in~ention is useful for enhancirg
the chemiluminescent signal provided by acridinium
sulfonamides. Immunoassays assays in which the invention
can be used include, by way of example, and not by way of
limitation, assays for thyroid hormones, cancer markers,
~96~02839 21 9 ~ 6 1 3 14 PCT/~SY5~7~2
vira1 antigerls c~r their antibodies, therapeutic drugs" et.c.
The inve1ltion provides a simpler, more sensitive ancl
more convenier1t chemiluminescent imm.1lnoassay t.han
previously described. Simplification result.s in that the
enhancer reagent can be addecl in a si.ngle .e.tage for an
effective chemiluminescen~ immunoassay.
A further benefit of the invention i8 the atta;nment
of markedly improved assay efficienc~. ~mproved efticier1cy
makes it possible to achieve desired assay results usillq
lD less acridinium sulfonamide conjugate. This benefit.
results in a substantial reduction in assay cost without.
sacrificir.g assay sensitivity.
~he following Examples are set Eorth for purposes, of
illurtl-atiorl and are not intended to limi~ the present
imrention.
EX~MP LE
Materials
Antibody to human thyroid stimulating hormor.e ~h-TSH)
was labeled wit.h 10-(3-sulfopropyl)-N-(~i-carboxyethyl)-N-
tosyl-9-acridinium carboxamide. For convenLence, t.his
sulfopropyl substituted acridinium sulfc~rlami.de will
hereafter be referred to as ~'acrldinium su1foT-1a[r,ide
2s lsulfopropyl~." Similarly, prostate specific an~..igen (~:~SA)
goat ant.ibody was separately labeled with lO-me~hyl-N- ~.2-
carboxyethyl)-N-tosy].--9-acridir1ium carboxamide and with lO-
13-sulfopropyl)-N-[2-carboxyethyl)-N-tosyl-9-acridinium
carboxamide. This methyl substituted acridinium
sulfonamide will hereafter be referred to as ~acrid1inium
sulfonamide (methyl). n ~he preparation of the acridinium
(methyl)- and acridinium (sulfopropyl)-labeled antiboclies
was carried out as described in commonly assigned ~atting].y
et a~. published European Patent Application 273,ll~i,
3s incorpora~ed herein by reference.
The enhancers tested included: ~er~uat (a cationic
~ surfactant dimeth~-ldiallylammonium chloride) purchased from
Calgon Corporation, Pittsburgh, PA; TWEE~-20 (sorbitan
monooleate polyoxyethylene) and BRIJ-35 ll.aur~l alccihcil
2 1 9 1 6 ~ 3
~ wOs6/02839 PCT~595/076~2
1~
ether- polyoxyethylene) both obtained from Fisher
Scientific, Fairlawn, I~J; T~.ITON X-100 la-[4-(1,1,3,3,-
Tetramethyl-butyl)phenyl]-w-hydroxypoly(oxy-1,2-
ethanediyl), CHAPS (3-[(3-cholamidopropylldimeth~lammonio]-
1-propane-sulfonate) and l.DS (lithium dodecylsulfa~e)
obtained from Sigma Chemical Company of St. Louis, MO; SDS
(sodium dodecylsulfate) obtained from Bio-Rad Laboratories,
~ichmond, CA; CYASTAT (a cationic surfactant 3-
lauramidopropyltrimethyl ammoniummeth.ylsulfate) purchased
0 from American Cyanamide Company, Charlotte, NC; and cholic
acid purchased from Aldrich Chemical Company of Milwaukee,
WI.
A Model Ls 9501 Luminometer was obtained from
Laboratorium Berthold, hlildbad, Germany.
The trigger was an alkaline peroxide solution of 0.25
1~ NaOH containing 0.6% hydrogen peroxide (H~02) which also
contained 0.01% antifoam agent. The antifoam agent was
Antifoam C Emulsion Sigma No. A-8011 obtained from Sigma
Chemical Company, St. Louis, MO. We have not observed any
effect of the antifoam agent upon signal intensity output.
~ethod
The acridinium sulfonamide conjugate was diluted with
distilled water to give approximately 250,000 count.s and
~s was added in the amount of 100 ul to a vial and placed in
the luminometer. While the via]. was in the measuring
position, 300 ul of trigger containing 1% of the selected
enhancer was injected into the vial. A control sample was
also measured which used a trigger containing no enhancer.
The light emitted was measured in the luminometer for two
seconds.
Results
Table 1 shows the ratic~s of the signal intensity
3s enhancement exhibited by the chemiluminescent reaction of
the acridinium sulfonamide (sulfopropyl)-labeled h-TSH
antibody, the acridinium sulfonamide (sulfopropyl~-labeled
PSA antibody, and the acridinium sulfonamide (methyl)-
labeled PSA antibod~ over the two secorld measuring time in
2 ~ 1 3~096~2839 PCT/US9~l07~2
l6
t.lle presence ancl ahsence of CHAPS, l~terauat ~cationic1. S~S,
LDS, TWEE~-20~ Bp~rJ-35l ~EITOI~ x-100, CYASTAT ~catiorlic)
and cholic acid.
TABLE 1
~tio of Sianal ~ntenSitV Enhancement.
Acridinium Sulfon~mide Antihod~ Coniucrc~tec.
SulEopropyl- Sulfopropyl- Methyl-
Lahe]ed h-TSH Laheled PSA LabeleM PSA
Control 1.0 1.0 1 0
CHAPS 1.6 1.7 1.5
Merauat 0.9 0.g 1.0
SDS 1.2 1.3 3.0
LDS 1.3 1.3 3.0
TWEEN-20 1.2 1.2 2.1
Brij-35 1.6 1.6 2.3
TRITOM X-'100 2.3 2.3 3.1
CYASTAT 1.8 1.6 1.5
cholic acid 1.5 1.4 1.7
EXAMPLE 2
~5 Materials
The enhancers tested included TRITOI~ X-lQ0, SDS, PRIJ-
and TWEEN-20, as well as Hexadecyl~wittergent (N-
hexadecyl-~,N-dimethyl-3-ammonio-1-proparlesulfollate) and
Tetradecyl~wittergent (N-tetradecyl-N,N-dimethyl-3-ammonio-
l-proparlesulfonate which were both obt,ained from ~:;igma
Chemical Company. Folate labeled with acridinium ester was
obtained from CIBA Corning Diagnostics, Corp., ~ledfield,
M~. Acridinium sulfonamide (sulfopropyl~-labeled h-TSH
antibody was prepared, as described in Example 1.
Simila-rly, acridinium sulfonamide (methyl)~labeled h-T.5r.
antibody was prepared. The trigger reagent was as
descrihed in Example 1.
2 1 9 1 6 1 3
~ W096/02839 17 PCT/USg~/076~2
The acridinium sulfonamide conjugates were diluted
with distilled water to produce approximately 7,000 counts
when triggered without an enhancer. 50 ul of the diluted
s conjugate and 50 ul of 0.03 N l~2SG4 ~ere added to a vial.
The acid was necessary to obtain light emission from the
ester conjugate. The triggering process was as described
in Example 1.
o Eesults
Table 2 shows the ratios of the signal intensity
enhancement exhibited by the chemiluminescent reaction of
ester-labeled folate, the acridir.ium sulfonamide
(sulfopropyl)-labeled h-TSE, antibody (Ab), and the
acridinium sulfonamide (methyl)-labeled h-TSH antibody (~b)
over the two second measuring time in the presence and
absence of TRITON X-100, SDS, sri;-35, TWEEN-20,
E~exadecylzwittergent and Tetradecylzwittergent.
2 0 TABLE 2
E~tio of Siqnal Intensitv Fnh~ncement
Eolate h-TSH Ab Sul- h-TSH Ab
Enhancer Ester fooroovl ~ethvl
Control 1.0 1.0 1.0
TRITON X-100 10.4 4.0 6.4
SDS 2.6 1.0 2.8
srij-35 3.8 1.8 3.0
TWEEN-20 2.6 1.1 2.3
E~exadecyl-
zwitterger.t10.1 4.7 6.1
Tetradecyl-
3s zwittergent 23.0 5.6 7.6
EXAE!PLE 3
~ateri~ls
Magnetic microparticles were coated with PS~
monoclonal antibod~- using EDAC (1-ethyl-3(3-dimethyl
aminopropyl)carbodiimide hydrochloride) coupling as
_ _ _ _ _ . _, _ _ _ _ _ _ _ _ _ ... . ... . . ... . . . .
W096l()283~ 2 ~ 9 1 6 1 3 l8 PCT~5~5~76~2
previGusly described in U.S. Patent 3,~57,g3l tHagar~
lg74~ which is hereby incorporated by reference. q~ e.y were
then diluted to a concentration o~ approxirmately P.15
scllids.
s A standard containing lQO ng/ml PSA (Abbott.
Diagnostics, Abbott Park, IL) was used. Acridinium
sulfonarmide ~sulfopropyl)-labeled goat PSA ant,i.bod~
described in Example 1 was used to test two enhancers. ~he
enhancers included TRITON X-100 and Tetradecylz.witt.ergerlt.
o The wash solution used was a micropa.rticle enzyme
immunoassay (MEI.A) diluent buffe.r a~ailable from L~bbott.
Diagnostics. The ~ri.gger solution used is described iII
Example 1.
lS Method
The following protocol was then used for a ~L~SA assay.
A suspension containing 50 ul of the magnetic mic.rop,art.icle
solid-pllase and 50 ul of the PSA standard was, incubat.ed at
37 '~C for ten minutes. The microparticles were washed two
20 tirnes by subjecting them to a magnetic field. The
rr.icroparticle pellet, was resuspended in 150 ul of the
acridinium sulfonamide-labeled PSA antibody solut.iol-l. TLIiS
susperlsiorl was incubated at 37 ~C for 20 minutes after
which t.he microparticles were subjected to a magnetic fielc1
2s and washed four times. The washed rricroparticles were then
mixed with 400 ui of distilled water and placed in the
luminorlleter in contact wlth trigger solution whereupon the
signal was read as described in Example 1.
Trigger solutions containing 0.0%, 0.5%, 1. n~ and 2.0~
3~ T~ITON X-100 and Tetradecylzwittergent were used to
determln.e the effect of enhancer concentration on light
emission.
~esults
3s Table 3 shows the effect of T~ITON X-10~ ar,d
~et.radecylzwittergent enhancer concentration on the light,
emission from the PSA assay. The effect is demons.t.rated by
the ratios of signal intensity enhancement.
21 9 ~ 6 ~ 3
W096/02839 PCT~S9~/076J2
1~
TABLE 3
Ratio of Sinn;tl Inten~.itv En~,tncement
s
Enhancer Concentration
Enhancer 0% 0.5% 1.0% 2.0%
TRITON X-100 1.0 1.68 2.28 3.50
10 Tetradecyl-
zwittergent 1.0 3.72 3.94 4.46
EXA~PLE 4
Materi;tl~
Magnetic microparticles were coated as described in
Example 3, with PSA antibody, hepatitis B surface antigen
(HBsAg~ antibody, h-TSH antibody, and triiodothyronine
antigen ~T3) antibody. The calibrators used were as
follows: the calibrator for HBsAg was recalcified human
plasma spiked to 0.5 ngiml with surface antigen, PSA as
described in Example 3, and the h-TSH and T3 calibrators
are both from Abbott Diagnostics.
As described in Example 1, h-TSH antibody was labeled
with acridinium sulfonamide (sulfopropyl~. Similarly, the
25 PSA antibody, HBsAg antibody and T3 were also separately
labeled with the acridinium sulfonamide (sulfopropyl~.
~ he enhancers tested included Tetradecylzwittergent
and TRITON x-100. The trigger reagent was an alkaline
peroxide solution of 0.25 ~ NaOH containing 0.45~c H2~2
which also contained 0.01% antifoam agent as described in
Example 1.
Method
The optimal range for the enhancer concentration and
3s the trigger was det.ermined for four different assays. The
same standard assa~ protocol described in Example 3 was
used, with the following exceptions. The sample size for
the HBsAg and h-TSH assays was 200 ul instead of 50 ul.
The second incubation period for the T3 assay was shortened
to ten instead of twenty mintltes. Tne concentrations of
21 q 1 6 1 3
W0-)6/02839 r~ 76~2
'2C
t.he enhancers used were 0.5% 2.3~ and 4.0Y.~.
P~esults
The l~elative Light Units ~RI.U .s! or the
s chemiluminescent reaction of the four assays with varying
concentrations of two enhancers are exhibited in Table 4.
~ RT.li! 4
Effect of T~ITON X-100 on Chemilllminescence ~RLUsl
Calibrator ~asa~0-5% 2 3~ 4. 0~d
0.5 ng/ml HBsAg 440 788 810
100 nglml PSA 173 000 270 000 23 000
100 ~llJiml h--TSH1 108 0001 790 000 1 940 000
0 pg/ml T3 25 000 43 000 46 000
~fec~ of Tetradec~lzwitteroent on Ohemill~min~c.cence t~nu-s!
Calibrator ~ssaY D.5~ 2.3~
0.5 ngiml HPsAg 860 940 1()30
100 ng/ml PSA 283 000 342 000 327 000
100 ullJ~ml h-TSH1 850 000 1 gl4 000 1 946 000
0 pg~ml T3 36 000 37 000 35~ 000
2s ~SAMP~ 5
~aterials
The acridiniu.nl sulfonarmide conjugate solutions
magnetic microparticles and calibrators were prepared in
the same manner described in Example 4. The enhancers
tested included Tetradecylzwittergent and TRITON X-100.
The trigger was an alkaline peroxide solution of 0.25
NaOH containing 0.3~ H2~2 which also contained 0.01
antifos.m agent and 2~ of the er~Lancer.
Method
The same standard protocol for the assay describcd in
Example 3 was used. The only difference was that the
assays were performed with the conjugate concent.ratiorL at
0.25X 0.5X and lX the concentration chosen for the
2 ~ 9 1 6 1 3
~ WO96l02839 PCT~'S9~/07642
'2
unenhanced assay.
Table 5 shows the RLU's of the cherniluminescent
reaction of sulfopropyl-labeled conjugates of h-TSH, HBsAg
and PSA antibodies and ~'3 at varying concentrations over
the two second measuring time in the presence of TRITON X-
100 and Tetradecylzwittergent. A control contained no
enhancer.
TABLE 5
Effect of TRITON X-100 or Chemiluminescence IRLUs~
Assav Control 0.25X 0~5X 1.0X
HBsAg 266 390 500 610
PSA 86,000 84,000 154,000267,000
h-TSH 600,900 668,000 1,064,0001,562,000
T3 10,800 12,400 21,000 31,000
Effect of Tetradec~lzwittercent on Chemiluminescence (RLUs~
Assav Control Q~ Q~ 1.0Y
HBsAg 266 460 700 910
PSA 86,000 106,000 197,000409,000
h-TSH 600,900 931,000 1,411,0001,926,000
T3 10,800 12,000 15,600 29,100
EXAMPLE 6
Materials
The effect of pH on the conjugates was tested by
preparing the four different assays described in Example 4.
The enhancer tested was TRITON X-100. The trigger was an
alkaline peroxide solution of 0.25 N NaOH containing 0.3~
H2~2 which also contained 0. Ol~o antifoam agent and 2~ of
the enhancer.
Method
The same standarcl protocol for the assay described in
Example 1 was used~ The only difference was that the four
assays were performed with the same trigger and the pl-l was
wo g.r,~2839 2 1 9 1 6 1 3 2~ PC~I~S95/il7ii~2
vari.ed from 12.3 t.o 13.2.
Results
Table 6 shows the ratlo oc tne trigQer wlth the
5 enhancer to t,he trigger without the enhancer at different,
pH values. ~'he sulfopropyl acridinium sulfondmide
conjugat.es of h-TSH, HssAg and PSA antibodies and T3 were
n,easured o~er the two second time period in the preaence
and absence of 2% TRITON X 100.
T~B LE 6
Ratio of Sian~l In~..ensit.v With ~ W/C! Enh~ncer
~ PSA h-TSH ~rBsAa T 3
:LS 13.2 3.88 ~.57 4.55 4.48
13.0 3.83 3.51 ~1.36 4.~5
12.8 3.78 3.77 4.27 4.63
12.fi 3.69 3.61 4.08 4.3g
12.3 3.66 3.31 3.97 4.08
As a matter of convenience, ~he reagenta necessary for
practic:ing the chemiluminescence enhancement method of the
present. i.nvention in an immunoassay can be provided irl the
fornl of a reagent kit where the reagents are in
, predetermined ratios, so as to opti.mi~e sensitivity of the
assay in the range of interest, Wet or dr~r reagents may he
used. If dry reagents are used they can be reconsti.tuted
priGr t,o use to obtain a desired concentration of the
reagents fo.r a particular assay.
3n rhe reagents may be mixed with various ancillary
materials such as me.nbers of the signal producing sysr.em,
buffers, and the like. In a reagent kit accordir,g to the
present. invehtion, the trigger is kept separate frorm the
acridinium sulfonamide. The enhancer ma~r be kept separate
35 from or added to either the trigger reagent or t.he
acridir.ium sulforamide reagent, The chemi.lurnine~.cerlt
reaction is thell triggered by combining the trigger reagent
with the acridinium sulfonamide.
While particular embodiments ar.d applications of the
2 1 9 1 6 1 3
09610283g PCT~595/07~2
2~
present invention have been illustrated and described, it
is to be understood that the invention is not limited to
the precise construction and compositions disclosed herein
and that various modifications, changes, and variations
which will be apparent to those skilled in the art ma~ be
made ir. the arrangement, operation, and details of
construction of the invention disclosed herein without
departing from the spirit and scope of the invention as
defined in the appended claims.
