CA2191613A1

CA2191613A1 - Chemiluminescent signal enhancement

Info

Publication number: CA2191613A1
Application number: CA 2191613
Authority: CA
Inventors: Adrian John Murray; Jonathan Grote; Sherri Jo St.John; Diethild Thut
Original assignee: Individual
Current assignee: Abbott Laboratories
Priority date: 1994-07-15
Filing date: 1995-06-16
Publication date: 1996-02-01
Also published as: EP0774121A1; WO1996002839A1; JPH10502958A; AU2775395A

Abstract

The present invention provides a method for generating an enhanced chemiluminescent signal from an acridinium sulfonamide compound, the method comprising the step of contacting the acridinium sulfonamide compound with a trigger solution in the presence of an enhancer to obtain a chemiluminescent signal which is stronger than would otherwise be generated in the absence of said enhancer. The enhancer comprises at least one member selected from the group consisting of nonionic surfactants, zwitterionic surfactants, and anionic surfactants.

Description

'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~/\

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:

Rl - N~ - R3 - SO3-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 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.

~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 ~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.

~ 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.

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~

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.

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 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 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.

Claims

WHAT IS CLAIMED IS:

1. A method for generating an enhanced chemiluminescent signal from an acridinium sulfonamide compound, the method comprising the step of contacting the acridinium sulfonamide compound with a trigger solution in the presence of an enhancer to obtain a chemiluminescent signal which is about 1.5 to 8 times stronger than would otherwise be generated in the absence of said enhancer.

2. The method of claim 1 wherein the enhancer comprises a surfactant.

3. The method of claim 2 wherein the surfactant comprises at least one member selected from the group consisting of nonionic surfactants.

4. The method of claim 3 wherein the nonionic surfactant comprises at least one member selected from the group consisting of polyoxyethylenated alkylenated alkylphenols, polyoxyethylenated straight-chain alcohols, polyoxyethylenated sorbitol esters, and alkanolamine-fatty acid condensates.

5. The method of claim 4 wherein the surfactant is TRITON X-100.

6. The method of claim 2 wherein the surfactant comprises at least one member selected from the group consisting of zwitterionic surfactants.

7. The method of claim 6 wherein the zwitterionic surfactant has the formula wherein R1 is a C8-C20 aliphatic moiety; and R2, R3 and R4 are independently C1-C4 aliphatic moieties.

8. The method of claim 7 wherein the surfactant is at least one member selected from the group consisting of Hexadecylzwittergent, Tetradecylzwittergent and mixtures thereof.

9. The method of claim 2 wherein the surfactant is an anionic surfactant.

10. The method of claim 9 wherein the surfactant comprises at least one member selected from the group consisting of lithium dodecylsulfate and sodium dodecylsulfate.

11. A method for generating an enhanced chemiluminescent signal from an acridinium sulfonamide compound, the method comprising the step of contacting the acridinium sulfonamide compound with a trigger solution in the presence of an enhancer comprising at least one member selected from the group consisting of (i) nonionic surfactants, (ii) zwitterionic surfactants and (iii) anionic surfactants.

12. The method of claim 11 wherein the surfactant is a nonionic surfactant comprising at least one member selected from the group consisting of polyoxyethylenated alkylenated alkylphenols, polyoxyethylenated straight-chain alcohols, polyoxyethylenated sorbitol esters, and alkanolamine-fatty acid condensates, and mixtures thereof.

13. The method of claim 12 wherein a chemiluminescent signal is generated which is about 1.5 to 8 times stronger than would otherwise be generated in the absence of said enhancer.

14. The method of claim 12 wherein the nonionic surfactant is TRITON X-100.

15. The method of claim 11 wherein the surfactant comprises a zwitterionic surfactant having the formula:

wherein R1 is a C8-C20 aliphatic moiety; and R2, R3 and R4 are independently C1-C4 aliphatic moieties.

16. The method of claim 15 wherein a chemiluminescent signal is generated which is about 1.5 to 8 times stronger than would otherwise be generated in the absence of said enhancer.

17. The method of claim 15 wherein the surfactant comprises at least one member selected from the group consisting of Hexadecylzwittergent and Tetradecylzwittergent.

18. The method of claim 11 wherein the surfactant.
comprises an anionic surfactant selected from the group consisting of lithium dodecylsulfate, sodium dodecylsulfate and mixtures thereof.

19. The method of claim 18 wherein a chemiluminescent signal is generated which is about 1.5 to 8 times stronger than would otherwise be generated in the absence of said enhancer

20. The method of claim 11 wherein the acridinium sulfonamide compound comprises at least one member selected from the group consisting of 10-methyl-N-(2-carboxyethyl)-N-tosyl-9-acridinium carboxamide and 10-(3-sulfopropyl)-N-(2-carboxyethyl)-N-tosyl-9- acridinium carboxamide.

21, The method of claim 11 wherein the trigger solution comprises hydrogen peroxide in dilute alkali.

22, The method of claim 11 wherein the acridinium sulfonamide compound is present in the form of a conjugate with another compound.

23. The method of claim 11 wherein the acridinium sulfonamide comprises at least one member selected from the group consisting of 10-methyl-N-(2-carboxyethyl)-N-tosyl-9-acridinium carboxamide and 10-(3-sulfopropyl)-N-(2-carboxyethyl)-N-tosyl-9-acridinium carboxamide; the trigger solution comprises a compound capable of oxidizing the acridinium sulfonamide; and wherein said contacting step results in generation of a chemiluminescent signal which is about 1.5 to 8 times stronger than would otherwise be generated in the absence of said enhancer,

24. The method of claim 23 wherein the enhancer comprises TRITON X-100.

25, The method of claim 23 wherein the enhancer comprises at least one member selected from the group consisting of Hexadecylzwittergent and Tetradecylzwittergent.

26. A trigger solution for generating a chemiluminescent signal from an acridinium sulfonamide compound, said solution comprising an oxidant and an enhancer, said enhancer comprising a surfactant effective for increasing the intensity of the chemiluminescent signal generated by the acridinium sulfonamide.

27. The trigger solution of claim 26 which, when contacted with an acridinium sulfonamide compound, results in generation of a chemiluminescent signal about 1.5 to 8 times stronger than would otherwise be generated upon contact of said trigger solution with said acridinium sulfonamide compound in the absence of said surfactant.

28. The trigger solution of claim 26 wherein the surfactant comprises at least one member selected from the group consisting of (i) nonionic surfactants, (ii) zwitterionic surfactants and (iii) anionic surfactants.

29. The trigger solution of claim 28 wherein the surfactant is a nonionic surfactant comprising at least one member selected from the group consisting of polyoxyethylenated alkylenated alkylphenols, polyoxyethylenated straight-chain alcohols, polyoxyethylenated sorbitol esters, and alkanolamine-fatty acid condensates, and mixtures thereof.

30. The trigger solution of claim 28 wherein the surfactant is a zwitterionic surfactant having the formula:

wherein R1 is a C8-C20 aliphatic moiety; and R2, R3 and R4 are independently C1-C4 aliphatic moieties.

31. The trigger solution of claim 28 wherein the surfactant is an anionic surfactant comprising at least one member selected from the group consisting of lithium dodecylsulfate and sodium dodecylsulfate.

32. The trigger solution of claim 27 in contact with at least one acridinium sulfonamide compound selected from the group consisting of 10-methyl-N-(2-carboxyethyl)-N-tosyl-9-acridinium carboxamide and 10-(3-sulfopropyl)-N-(2-carboxyethyl)-N-tosyl-9-acridinium carboxamide.

33. An immunoassay test kit having reagents which comprise:
(a) an acridinium sulfonamide compound;
(b) a trigger solution separate from said acridinium sulfonamide compound; and (c) an enhancer comprising at leaat one member selected from the group consisting of (i) nonionic surfactants, (ii) zwitterionic surfactants and (iii) anionic surfactants.

34. The test kit of claim 33 wherein the acridinium sulfonamide compound is at least one member selected from the group consisting of 10-methyl-N-(2-carboxyethyl)-N-tosyl-9-acridinium carboxamide and 10-(3-sulfopropyl)-N-(2-carboxyethyl)-N-tosyl-9-acridinium carboxamide.

35. The test kit of claim 34 wherein the trigger solution comprises hydrogen peroxide in a solution of dilute alkali.

36. The test kit of claim 34 wherein the surfactant comprises at least one member selected from the group consisting of TRITON X-100, TWEEN 20 and BRIJ-35.

37. The test kit of claim 34 wherein the surfactant comprises at least one member selected from the group consisting of Hexadecylzwittergent and Tetradecylzwittergent.

38. The test kit of claim 34 wherein the surfactant comprises at least one member selected from the group consisting of lithium dodecylsulfate, sodium, dodecylsulfate and cholic acid.

39. The test kit of claim 33 wherein the enhancer is supplied in the form of a mixture with at least one other of said reagents.

40. The test kit of claim 39 wherein the mixture comprises the trigger solution.

41. The test kit of claim 39 wherein the mixture comprises the acridinium sulfonamide compound.