CA2186463A1 - Novel functionalized hydrophilic acridinium esters - Google Patents

Abstract

Novel acridinium esters that are useful, either alone or when incorporated into liposomes, as chemiluminescent agents in binding assays (e.g., immunoassays and gene probe assays) with improved sensitivity are disclosed. In addition, the synthesis of these esters and their use in assays for detecting an analyte is described. In particular, assays for testosterone and the Rubella virus are disclosed.

Description

~vo g~l~7a2 ~ ~ ` ~ 2 1 8 6 4 6 ~

3~0VE1 F~1N~T~7~ T.TZ:T`n '~vn7~op~ TTlTc ;!~RTn~T7Jl ES~ERS
Field of the Invention The present invention relates to a novel method ~or detection of an analyte. The present invention relates to the d~er~;c~n of an analyte ~lsing .7c~ridin;117n esters as rh~m;7~nin~s~nt ~narkers which can ~e ~nr~r5~l7~ted within l;rns~ - vesicles without cjc~7n;f;c~nt lea'~cage of t'ae esters from t'~e vesicles. ~rhe present i lvention also relates- $o t'ae sy~t'aesis aI~d ~se of novel f7ln~;c~nA1;7~d-hydrophilic arr;~7;n;lnn esters ~hich are usef~l as rh~n;ll-m;n~cc~nt la'L7els, and s7rr;~;n~ly give a much higher C~uant~ yield t'aan prior ~r;d;n;7~7~1 ester The prese~lt inve~ tio~ also relates to novel hyrophilic A~ ';n;7n, esters which may be used to form direct and indirect ~;~".j uy~es . The prese~ t inve~tion 1 so relates to novel c.,~.; u~,as ~ormed ~rom such ~77n ~r~i ~nA 7; ~ hydrop'~; l; c ;~-~ri -~, i n; llm ester _ ' ~ .
The present invention fur~her relates to assays u~;l;7~;n~ these novel f7ln~in~ d hydrophilic Ac~r;c7;n;lnn esters and cw,.iuyc-Les thereof. The insta~ t invention relates to _y using t'~e _ '- of the instant inventio~.
.

'3a~L~ u-,d of t'ae InventiQn he use of 7-~rj-7;n;lnm esters as rl /~m;l~nn;n-ccent -5 labels i~ r.l;n;~l assays is ~;no~n. For example, descnbes ~ 0 082 636 ~ wo ss~7c~ 'rcr~srool~
21 ~6463 7he use Or- ~ aryl 2cridinium ester activated ~I_th an N-~
hydro~-succ niTr~idyl moiety as a chemilumi~escen.
label in immunoassays U. S . Patents No 4, 745 ,1~1 (see EP
0 263 657); 4,918,192; 5,241,070 (see EP 0 36-~
describes polysubstituted aryl acridinium esters (PAAE) which zre use~ul in immunoassays and nucleic acid hybridization assays U S. Patent No 5,227,489 ~See EP 0 353 971) and U S Pate~t 5,4~9,556 which is the parent to the instant applicatio~, describe hydrophilic polysubstituted aryl acri~in;um esters and lumisome conjugates thereo~ use~ul in clinical assays, particularly those assays involving liposomes ~P 0 273 115 describes chemiluminescent acridinium and rl~on~nt~;rl;n;1lm salts Zomer et al (227 Analytica Chimica Acta (1989)11) 1~ discusses ~eatures ~or chemiluminogenic labels ~or ~ csays .

2',6'--Dimethyl-4'--(N-s7lor;n;m;~lylu~yKu l~llyl)phenyl-lO-!lethyl-9--a~r~cl;n~ ylate ~leth~rlsulfate (DNaE-~IS) as ~crr;hP~ in II.S. Patent No. 4,74~,181, require the use of a phenoxy group substituted ~ith a benzyl~,.y~LLu.-yl group as an ;nt~ te to ~or~ l he ar~;llinP ester ~ia a long synthetic pathway. It is desirable to deveiop new and r~f;~!iPnt methods of synttP~;~;n~ the usetul acridinium ester la~els of the instant invention. ~he 1lnPYr,Prte~5 ability to f or~ 2 ' 6 ' -dimethy}--4 ' - ~-61~nr;n;m;~ylu,~y~1~lyI~phenyl 9-:7~r~;l.P. ~ ylate (D!~-~IS) by the ~:;mrl;F;P~ ~u~6du~t: of ~ ~ n;n~ a Solllt;nn Of sl~r~;n;m;~yl 3,5-dimethyl 4 hydLu~yL~ ~7~
and 4~imethylaminopyridine with g~ n~ yl 30 rhl~ r. hydrorhll~ri~l~t was not readily pr~ t~h~P in vie~ of the ~oP~-St~nre of two reactive leaving ~roups in the same rP~inn, an acid ~ P rom 9-s~rni~;nP.~ ..yl rl~lnr;~o and a sur~~n;m;~lyl ester fro~
~;llr~;n;m~lyl 3~5--dimethyl--4--lly~l~u~y~ oat~S For example, -in the case where ~--s~lrr;n;m;~yl 3(4-. .

~VO95n7~02 P~ . 'S
2t 136463 L~.lL~...yl.henyl)-propionate is reac1:ed with the 9-acri-l;n~ ..yl chloride referred to above, the cnn~l~nRAtion can be- carried out under mild conditions (approximately room temperature) due to the absence of the two methyl groups in the ortho positions. (See for example Us Patent No. 4,946,958, cols. 4 - 5, where the reactant does not contain the ortho-substituted methyl groups. ) On the other hand, when the two methyl groups are present, much more dra5tic conditions (100 degrees C. for 2 hours) are required for the c~ cltion reaction to take place, due to the steric hindrance caused by the methyl groups. It .should be noted, though, that there is a benefit provided by the presence of the methyl groups, namely the added stability of the resulting acridinium ester, as ~ c~ ecl, for example, in US Patent No. 4,745,181.
The l ;rorh~ nature of the prior art acridinium esters and other rh~mi 1 inP~C'--nt _ , _ '~ render them unsuitable for ~nr~r~l~lAtion within l i~ -- -- because of their rapid leakage through the l i, ~- - wall.
Additionally, the limited water solubility of prior Art acridinium esters and other chemill~min~ c~nt only allow the ~nr~r~llAtion of a few marker molecules per l ;, -~ - vesicle, resulting in relatively low signal amplif ication .
The novel func~;nnAliP~:l hydrophilic acridinium esters of the instant invention are useful in -- y, yield results that are superior in sensitivity to prior methods, and do not need to use dll.uelvuD r~liol;~h~l~, or organic enzyme/substrates. The use of rh~mill-min~ nt labels of this type resulted in an u..~"~euLed 1 _ .,v. t over prior methods and can lead to the fllnr~l onAl 1 ~ of previously inoperable or in7 ~ UL -te assay methodologies . The novel discovery that hydrophilic acridinium esters of the W095127702 ~ 21 8 6 4 6 3 r~
in~tant invention could be used to label biomolecules and ~ directly without reducing the solubility of the complex allows for many applications in the field of ~ - r~-gy. The novel ' of the instant invention will allow for sensitive i - --ay without the need Of exce5sive use Of reagents in the reaction mixture, and are thus ~l~ci~nPd to reduce nu" L~ecific interactions, while not interfering with desired crerifi~ interactionS. --Another llnPYrPc-ted benefit of having an PAAE
carrying a hydrophilic moiety, particularly as a substituent at the nitrogen atom of the acridinium nucleus, is the significant i ~ u~. L ûf the l-hPmi~ inpcc~pnt quantum yield relative to that of acridinium esters which simply have an alkyl group (U. S .
Patent 4,745,181) or ~,alLu~y L' yl group (G. Zomer et al., 1989, Anal. Che~n. Act~ 2i7~ 19) substituted at the same position.
The area Or ~ ~-?y is well developed, and it is the discovery of unique lAhel ;ng systems that can ~pur a quantum advAr - in the f ield of ~ ?y .
In the field of 1 y it is desirable to have assays which are highly specific, and highly sensitive to low ~ ations, and yet still yield lP~ectAhl e and distinguishable signals for mea2iuL. L. The instant invention allows for the specific detection of analytes at low ~ullc~l~LLation, without 1088 of solubility. The lnstant invention provides a means for making many speci~ic 1 AhPl i n~ agents which can be used to detect low levels of analytes. The instant invention also teaches one with ordinary skill in the art, a novel and useful means of perf orming i - - ~y that lends itself readily to automation and commercialization a6 assay kits and reagent Xits.
The application of the no_l acridinium esters of WC~ gsm702 r~ '4 21 ~36463 the instant invention in a.",; u~ s with bioactive proteins such as avidin, an~;hod;Pe, DNA binding proteins, hi6tones, and r;~- ~s, and others, is made p~;hl~ by the hydrophilic properties of the syn~hQR; ~sd ~ ' - . These novel acridinium esters are also useful for the lAhQl ;n~ of i~olated, or intact, RNA, DNA, proteins, peptides, inactivated protein, neuL~ lDluitters~ h: F, viru~es, viral ~n~ nll, bacteria, bacterial Ant;~Qn~, toxins, cytokines, antibody r. ~5, receptor proteins and other such targets both in vitro, and in vivo. The sampling of tissue samples, serum samples or other such biological samples, and the detection of previously difficult to measure specif ic analytes in rapid assays, are made possible by the compositions and methods of the instant invention .
One specif ic application of the instant invention is in the field of pe.U,ogel- detection. While there have been many assays developed to detect viral pathogens, such as Rubella, the instant invention provides a unique and highly sensitive assay which is u-,e..~euLedly superior to previous ~hQm; 1 ;r- _ L methods. The instant methods are comparable with convPnt; ~mA 1 assays in sensitivity yet are more efficient in time required to perform the assay.
Another application of the instant invention i5 in the area of detection of hl -- or haptens or other small biologically active molecules in biological samples. Where the hormone or hapten levels are low, and transient, it is useful to have a sensitive and rapid methodl of testing for, and measuring the levels of, h. -- or haptens in such biolo~ical samples. One Or the po~;ble examples is the ~QtQc~ of steroid hormone levels such as testosterone. Detection of testosterone using homologous and heterologous hapten Wo9s/27702 21 8 6 4 6 3 r~l . !5,1 ~ ~
con~ugates is known. In this me~ h~ 10gy, antibodies are generated to one f or_ of testosterone i , . . Then ~aid an~ ~ ho~l ~ P~ are u~ed to detect testosterone in a sample, while a cc,~.ju~lted competitive hapten tracer, th~t is of a different form than the ~ - ,e , is added. r logol~ assays are burdened with unacceptably high cross-reactivity such as when using C4-testosterc,.~c B ~1 conjugate haptens, and T-3-0-CN0-glucoamylase .u-lJuy.,tes. Somewhat lower ULUS:~LC aCtiVity has been obtained with lla-substituted hapten-HRP, and T-3-0-CM0 hapten-penir-; 7 l in~P (Rao et al., 1992, Steroids 57: 154-162). The instant invention provides for superior ~l~tecti on over prior heterologous hapten assay in that the hydrophilic acridinium ester labels of the instant invention contribute to reduced .o~ ecl~ic interactions.
Accordingly, it is the purpose of the instant invention to provide novel fl~nrt;-~n~li7s:1 hydrophilic acridinium esters and cunj~ es thereof for use as rhPm; 1 inP~Ir-~nt tracers. It is also a purpose of the instant invention to provide for novel methods of detecting an analyte using fllnrti~n;~l~z~d hydrophilic acridinium esters and cu..j u~tes thereof . It is also a purpose of the instant invention to provide a new i _ u~d synthetic EJ~uc--luLe for the efficient pro~ tion of acridine esters.
~I~E rV Or the Inventiûn Novel acridinium esters that are useful, either alone or when inciuL~uLc-~ed into 1 ;L ~ ~æ, as rh~-m;lllm;n~C-~nt agents in binding assays (e.g., ;ay~ and gene probe assays) with; _ u._l ~ensitivity are ~ rl cFsd . In addition, the synthesis of these esters and their use in assays ~or detecting an analyte is described. In particu~ar, assays for WO 95127702 P~ S

testosterone and the Rubella virus are ~ losed.
ief Descri~tion of ~ hD Drawin~
FIG. 1 is a diagram illustrating the i u~d synthesis of DMAE-NHs.
FIG. 2 is a diagram illufitrating the old method of ~ynthesis of DNAE-NHS.
FIG . 3 iB a standard curve f or a testosterone ~-hDm;ll~min~P~_...!e i ~y using NSP--DMAE--19--HCNT
tracer using the ' of the instant invention.
FIG. 4 is a comparison of bindi~g curves for heterologous and ~: logol~ tracers in a ~h~milll~in~ c~ e; ~~~ y for testosterone using the _ '~ of the instant invention. The figure also shows a comparison between using hydrophilic vs.
rl~,.. hy lL~ ilic acridinium esters.
FIG. 5 iB a plot showing the correlation of results from an ACS testosterone i ~ y uDing the _ _ '- of the instant invention with DPC Coat-a-Count r~ ioi - y (Diagnostic Products Corp., Los Angeles, Q).
FIG. 6 is a bar graph showing a population study comparing the positive to negative ratio of assay result6 using the _ '~ of the instant invention as compared with DNAE rubella assay.
FIG. 7 shows the assay architecture for an ACS
TL~L~DLeL~Ille assay, which is run on the Ciba Corning ACS-18 0 ill:. LL ~ ' .
FIG. 8 shows the assay architecture for an ACS Estradiol assay, which is run on the Ciba Corning ACS-180 ill~ LL L .
FIG. 9 shows the assay architecture for an ACS Specific IgE assay, which is run on the Ciba Corning ACS-180 - inD LL

Wo 95127702 2 l 8 6 ~4 6 3 ~ ~ . 11 ~
Descri~tiQn of the Invention The following terms as used in the specification and claims shall have the following ~ inj~
Analyte - the _ ' or composition to be ~~~ -1 which can be a ligand that is mono- or polyepitopic, antigenic, or haptenic. The analyte can be a piece of DNA or RNA . The analyte can be f ound in auid or on solid support.
CO~ lya~:3 -- the combination of Ch_m; ll~minD8c--n~
. _ -' with a second _ _ ' or molecule. Such conjugates can have many chemil~m;nP-7cont molecules per second ~ _ '.
Indirect Col,juy~e - the combination of ~h_m; 1 -m i n_C~C-~t " with a second _ ~ or molecule. The inPd ~h_m;lllm;n__c_nt __ ' with a ~econd ' or molecule is then capable of further ; n i n-j with a third _ _ ' or ~ l - . Such .ja~teD can have many -h_m; 1 ;n~ t molecules per complex.
Homologous - the same _ _ ' linked by the sa_e linker, at the same position.
Heterologous - the - ' linked by a different linker at a different position~ a different linker at the same position, the same linker at a different position.
Linker - a physical or ~h-mic~l bridge, tie, connection between two _ - or molecules;
bifl~n~ n~ l or multifunctional linker. The binding of a linker to one _ ' can allow for a reactive site to be made availible for further reaction.
Hapten - an incomplete antigen, ;n~~r~hle by itself to provoke an immune r~D~ol~se, but when suitably attached to another molecule becomes capable of producing an~ihot7;--~ which will sr-cific~lly reco~n;7e 3 5 the hapten .

~ W09s/27702 2~86463 /lL ~s The acridinium e~ters ~ d by the instant invention, and useful in the methods of the instant invention, can be any acridinium ester which is a hydrophilic acridinium ester, a fllnr~ jon~l;5.od form of such _ ', or conjugate thereof, and which can generate a rh~-m;l ;n~'~C~nt signa~. The hydrophilic PAI~E
of the instant invention, with an additional electrophilic or leaving group is useful for aol.Juy_tion to many targets. The hydrophilic moiety of such a PAAE
(comprising an alkyl spacer carrying a highly ~nn;7~hle group such as sulfonate, rl~ te, sulfate, phosphate) is r~-cp~n~;hle for ~nhAnr;n~ the aqueous 80lllh;1;ty~ hence reducing the ll~dLu~ })ic nature of the tracer6, particularly those formed from ou~uy-t.ing the PAAE with small organic biomolecules such as steroids, t~._L~uLic drugs, and other controlled rh~m;c~l ,iub~al.aes. When applied in lAhel l ;n~ macro-bi~ lec~ c such as proteins, nucleic acids, and polysaccharides~
the hydrophilic PAAE permits the increase in incuL~uL~-tion ratio (defined as number of PAAE molecules covalently attached to a single macro-biomolecule1 without any resulting solubility problems. Thus the increase in specific activity and ~ Ah;l;ty of the tracer would be a factor in F~nhAnr;n~ the binding assay sensitivity and i u~d signal/nûise ratio. The l; rorh i l; C nature of the prior art acridinium esters and other rh~milllmin~sc~-nt _ - render them difficult to use when ao~.ju~-ted to certain biological since there was a signif icant amount of nc,l- ~eaif ic binding due to the l~y-lLu~hObic nature of the tracer cu~juy~l e. Thus the prior art acridinium esters and other rh~m;lllm;n~ec~nt _ a_ were not Pffici~nt for use when detection required fine distinction between signal from specific vs. non-~p~a;f;c~ binding, and resulted in an increAsed risk of false positive or false wo ss/27702 r~

negative results.
Preferrea functie~n~ od acridinium esters include acridinium esters of the following formula:
Rl R2--~ R3 R~ Rs ~ :=o ~
1--~// ~ R6 Re R7 wherein Rl is alkyl, alkenyl, alkynyl, aryl or aralkyl, haYing up to 2 4 carbons and up to 2 0 heteroatoms E~Pl eci Q~ from the group consisting of nitrogen, oxygen, r~ 1. - VUS and sulfur;
Rl, R3, R5, and R~ are l-~lcv~ , amino, hydroxyl, halide, nitro, -CN, -SO3H, -SCN, -R, -OR, -NHCOR, -COR, -COOR, or -CONHR, wherein R i8 alkyl, alkenyl, alkynyl, aryl, or aralkyl, having up to 24 carbons and up to 20 heteLvat 8O1 ectocl from the group consisting of nitrogen, oxygen, ~ ---- vu8~ and sulfur;
R, and R~ are alkyl, alkenyl, alkynyl, aralkyl or alkoxyl having up to 8 carbons, with no branching wherein the side chain groups have more than 2 carbons;
R6 ~ ~Le5e lL~ the following substitutions:R6 = R9 -Rlo where R~ is not required but optionally can be an alkyl, or aralkyl group, having up to 5 heteIvat 80lectod from the group of ~ho,,~l-v u8, Sulfur, Nitrogen, Oxygen, and Rlo is an electrophile, a leaving group or a group with theS~e two i no i natures, as shown in the following . l~oc ~ wO95127702 21 86463 ~ o ~ 3 J~ N,~N
o o J~oJ~o~ OR
~C -N3, ahalid~
~ R~ ~ ~tb~R~S~5~
25 where Y i8 a.halide and R is alkyl, aryl, aralkyl group, ~nd;
where R5, R6, and R~ sustituel~tt positi~n~ on the phénoxy ring are il-~e~ A- ~-Ahlr~.
Nost prefer~bly, Rl is a sulfopropyl or sulfoethyl 30 group; R2 is a 1~1.~ , methoxy, ethoxy, nitro or halog~n; R~, Rsr and R, ~re l~y~u.~wl; R~ and R~ are methyl, ethyl or isopropyl groups; R6 i8 N--s~t~ ~; n i m i ~ylu~y ur Lv~- y 1 r - N-s~l~ r;n;m;dyluA~ rL~ lalkyl, or carboxylate SUBST~ E SHEET (RU~.E 2 Wo s5n7702 ~ 1 8 6 4 6 3 P~
It was u.-_A~e_Ledly found that PAAE with the ~ubstituted hydrophilic moiety (e.g., 2',6'-Dimethyl-4'-(N-sl~C;r~m;~yl~y~ Lv~ yl)phenyl 10-(3'-Sulfopropyl)-acridini~ ~ cc.lLvAylate (NSP-DNAE-NHS) ) is capable of generating about 1. 5 times higher l~h~-m;l ;n~13CDnt quantum yield as _ ._d to the C~ ....1; n~ PAAE with gimple alkyl substitution at the l~ame po~ition (e.g., 2' ,6'-Dimethyl-4'-(N _ -;n;m~-~yloxycarbonyl)phenyl 10-Nethyl-9-acri~l;n~ LcAylnte Nethyl~ulfate (DNAE-NHS) ) . (See Table I) . The increase in t~h~m111lm;n~ c~nt quantum yield of PAaE i~ a very d~sirable and ~ ;7~ factor in improving the signal lL~
SUBYITUTE SHEET (RULE 26) wo 95127702 2 l 8 6 ~ 6 ~ r to noi6e (S/N) ratio of the rhPmilllmint~ nt tracers and the sensitivity of rht~mi 1 ;nt~ç:ct~nt binding assays and tlt,tt,~-t; on schemes. In particular, where the signal t~nh~~~ L of the tracer is mediated through multiple PAAE lAhellin~ of a c-,.,Juytlte of ligand/carrier b~t lPr~le such as a protein, a 1.5 fold or greater L in PAAE rht~m; 1 ; nt~Gct~rlt quantum yield will mean that less PAAE is reguired to be bound to achieve the same signal amplitude. Also, i~ the same levels of 10 binding of PAAE to carrier Cc,.. j~ates is achieved, the dt~1 t~t-~ ;tln of lower L;o~ l.LLt~Lions of an analyte may be p~ ; hl e .
TABT,l~ I
Relative Ouanf~lm Yield of l)MAR--N~T~ vs. Nsp--nM~R--NHs AE Tv~e Nol. Wt. 12TT1/4 . 2 fmol l~R R L p Ratio DNAE--NHS 595 690,187 1.0 644, 587 743 . 587 mean 692, 787 SD 49, 551 % C.V. 7.2 NSP--DMAE-NHS 591 1,122, 017 1. 7 1,204,017 1, 122 . 017 mean 1,149,350 SD 47, 343 % C.Y. 4 . 1 In table I, D~AE-NHS was dissolved in acetonitrile, while NSP-DNAE-NHS in 50% (v/v) acetonitrile in water to generate stock solutions of 1. 0 mg/ml . Serial dilutions 35 were carried out to 10-7 with flashing buffer (10 mM

wo 95n7702 Phosphate, 150 mM NaCl, 0.1~ BSA, 0.05% NaN3, pH 8.0) diluent. The dilution method consists of three 100-fold ~liltlt;~n steps using 50 uL transfer volumes and on 10-fold dilution step using 100 uL LLa--..r~ volume. The light Rsi~n of the samples were ~Q~PrminPd on Ciba Corning Diagnostics NIA1 Analyzer under -Lard~- I conditions (2 sec light collection time) using 25 uL of the 10'7 dilution EOlU1 j onR .
There are many modifications which one with ordinary skill in the art will reCo~n; 7e are within the scope of the instant specif ication . A number of spacers can be attached which will facilitate the connection of the hydrophilic group to the nitrogen atom at the acridinium nucleus, and an electrophile or leaving group that will facilitate the covalent att~l L of a hydrophilic PAAE
to other c _ ' or biomolecules.
The novel acridinium esters of the present invention are highly soluble in water and can be PnrAr~ Ated in l~ro~ - at high c~l-c- l a~ions. Once inside 1;~-- -F at high . ;- l~aLions, the novel acridinium esters remain DnrAr--llAted for ~ Lel~ed periods of time and do not leak appreciably .
It will be appreciated that while the novel acridinium esters of the present invention are use~ul for Dnr~lp~-llAtiOn within 1;~ o_ - -, the novel acridinium esters of the present invention are also useful in other applications where acridinium esters are utilized, such as l~hPl;n~ ligands or analytes (sUch as antigens); lAhe7;n~
the specific binding paLLI~clD of ligands or analytes (such as the OULLC'D~ ;nl~ an~;ho~;PR); or lAhPl;n'J nucleic acids and molecules comprising nucleic acids.
In particular, the novel fl~nr~;nnAl;7D~l hydrophilic acridinium esters of the present invention will be useful for lAhPl ing biological materials for rhPm; lllm;nPRcPnt assays where it is preferable to reduce the non-specific w09sl27702 2 1 8 6 ~ 6 3 F~./ ~1 binding of tracer to biologicai m~terial. The novel fl7nrt;rn;~ hydrophilic acridinium esters of the present invention will allow for a reduction in signallnoise tS/N ratio) interference, which will allow for greater accuracy in making - - c and for diagnostic assays, since the llydrophilic nature of the novel '- of the instant invention will be less prone to non-srec; fiç interactions in comparison with other rh D~n i l l-~ 1 no ccPnt - .
Synthesis of acridinium es~ers have been described as in US. Patent No. 4, 745 ,181 and the process is shows-s in FIG. 2. This method involved the use of a phenoxy group substituted with a benzyloAy~;~LL~s-yl group to form the acridine ester. The desired E~trrin;m;Ayloxycarbonyl was then i~-LLo-~su~ed through the removal of the benzyl protecting group and the ~ tion of the product with N-hydroxysuccinimide in the presence of dicyclohexylcarkoA;; m ~ AD
In addition, the instant illvention provides for a new and i ~v~:d method of synthesis. This method takes alvcl..L~,~e of the ~ e~ l DA discovery that the acridine nucleus, under properly 8DlDr~t~DA conditions, can be directly ~ e~A~ with the phenoxy group carrying sl~rç;n;m;Ayloxycarbonyl, without any problem, this is illu-LL~ted in FIG. 1. By using this new and simpler process, two synthetic steps of the older process are eliminated, for the yLe:s~lLc~ion of hydrophilic PAaE with the proper electrophilic or leaving group. The generalized reaction scheme of Figure 1 also appliec to the sJL~s,.. L~sLion of substitute~ acridine acid chlorides, such as ~2--~ - R3 C

~'0 SS.'~, 10~ 9~40_~
. .
arc ~-~ydro~ysuccin~midLe ester o-' substi .utec p_ hycro~yber.zo2tes, suc~ 2s f~5 0 HO~
where R2,~3,P~, R~ are hydrogen, halide, ni~ro, -R, -OR, --CN,--N~ICOR, ~CORr --cOoR, or --CON~R; where R is al~yl, alkenyl, al~nyl, or ar~l~cyl, and ~here R4 ~ R8 ~re R, and l~ ~ is as def ined above ~hus the i~stant invention provides for hydrophilic P~E with an additio~2Ll electrophilic or leaving gro~p, which are uceful for ~/lLjuycLL;ng with mono- or poly-n~ or~ i c c~ o~ c or biomolecules. $he instant ~'~vention th~s provides for the covalent att~Lchment of the func~ l; 7.ed hydrophilic PAA~3 with nn~ r~
~- ~ ' such as hexyl-1,6--dia~Li~e, ethylene diY~;~e, and ~r;n~ rroic acid. q~e instant ~nve~tio~ also provides for 2~ the co~lent . ~. ~ L o~ the ~n~;~ 1;7~ hydrophilic PA~E with h;~ nl~c such as a~inoglycoside-c~ proteins, vir~l antigens, peptides, and :~m;nn~ un~;~m~l ;7.~.
ol;gonucleotidés. $hese co~juydLes will be use~lll for sensitive ;- ~ Ly~ and n~:Lcleic acid binding assdys.
Pll~+h, ~, all of the attributes of the insta~t invention are c;m;l~r~g :~r~17;~ h~D to related ;~ l;n;n-~-~ ;n~7~uA;n~ ~ut not li ~ted to, ~n7~idin;nm esters, such as those clai~ed in ~J~rDn~in~ application E;P O 61 7 288 An exa~ple of a - useful sensitive ~;hDm~ m; n~cCDn~
_y ~;1;7;n~ the ~ _ '- Llnd W.,Ljuy,L~.as of the iIIstant in~ention is one which can det~.ct s3?~ steroid in ser~m ~ c, An ; ~,v~a assay using 1:he ~1; uycLtes and ~nethods of the instant invention is a speedy assay for ~ . vl.e. This assay ~ n-~c the use .: .,. ~_ . - . . A~DED S~T

~ Wo gs~7~02 2 1 ~3 6 4 6 3 r "
of a highly specific antibody raised to testosterone and a heterologous tracer formed by conjugating a novel Cl9-mono- or Cl9-bi-substituted testosterone with a functionalized, hydrophilic PAAE. The benefit of using the heterologous ch~ ml; n~occ~nt tracer is observed through the; .,v~ -nt of testosterone assay sensitivity to o. 1 ng/mL, which cannot be otherwise achieved rapidly (ie.
about 5 min. ) with the use of the ~.ull~,uul.ding homologous tracer, or using hydrophobic P~AE.
Thus the instant invention also provides for the utilization of functit~n~l;7~ed hydrophilic PAAE which were formea as conjugates by novel Cl9-C-linXed type of testosterone derivitization (as described in the Examples to follow). These novel heterologous tracers can be formed, optionally with further olefinic lin~age at C19, or ~r~/l;t;-~n7~1 heteroatom-linked substitution at Cl9, such as the following Cl9 - O linked derivative of testosterone (NSP--DMAE--HD--19--CMET) .
SOs~
¢~ 0~
2 5 ~ H ~_ ~
3 o Thus the instant invention also provides f or t~D~o~ e derivatives of the following SLLU~;~UL~:S~
(Seven ~ are 8hown below, along with a template identifying the n~ system used for ~ ture.) SUBSTITUTE SHEEr ~RULE 2~) W095127702 , r 5'l 'S

1~ OH 1~ O
' S 1'` S
0~ 0 ~ ~A~
m I I IV I
HO/~ HO/~

1'' ~~S X ~"
HO HO~--/

X--B~ / ~\~< 18 ~/ ~2~ ~7 9\~5~7 SUBSrlTLlTE SHEET ~RULE 26~

~ wogsn770z 21 86463 wherein A i8 a funct;~nAl;7ed group selected from the group consisting of -OH, -N(Rl)2, -NHRl, nitro, halide, -ORl, -OCORl, -Tosylate, -mesylate, -azide, -isocyanate, -thioisocyanate, -SRl, nitro, -SH, -SSRl, -phn~phrn~te, -sulfonate, -NHCOORl, -NHCORl, -NHCONH2, hydrazines, cyanide, and -Rl; where Rl is an alkyl, alkenyl, alkynyl, aralkyl cf~nt~;n1ng Up to 10 carbons and up to 5 heteroatoms; where B is - (CH2)l, where n = 1-4 or -C=; when B is -C=, A is preferably omittedi where X is a carboxylate, -COOR2, -CONHR2~ (where R2 is an alkyl, alkenyl, alkynyl, aryl, aralkyl crrt~;n;nr~ up to 15 carbons and up to 5 heteroatoms), or a carbonyl ~tt~rh~ with proper leaving groups, including but not limited to halide, N-lly~iL~Ly~lcr;n;m;~ and imidazole.
The Pnh~nr~tl utility of the hydrophilic PAAE of the instant invention in; oz~say of therapeutic drug8 or hormones such as testosterone illustrates one aspect of the invention e~m~bodied in the instant specification.
Specifically, the use of testosterone derivatives wherein the C19-B linked bridges (as shown in the above figure) are C19-C and an important element to the effective use of the compound~ of the instant invention. The use of a C19-C
linkage has been found to balance the reduction of tracer SU8Sr1TUrE S~E~ (RUl 2B) WO 95/27702 P_l/~ 1 1 binding to the Epe~ anti-testosterone antibody, and the ease of its disp~ from antibody by competing sample testosterone.
A preferred: -ir L of the testosterone tracers of the instant invention to be utilized for conjugation to the func~ionll;7sd hydrophilic PAAE, via ~ifunctional cros~l ;n~ rs, ~ L the forms Lé~L~SéllLed by formula I above. Nore specifically, it is preferred that A i5 hydroxy, B is -CHz-, and X is carboxylate.
These '- are novel, being distinguishable from those known from the prior art. For example, those - cited in Rao (U.S. Patent No. 4,197,286) contain C19 - O ( i, e ., C19 to oxygen) 1; nlr:-gol::, Using the t~rh;n~ of the instant specification one with ordinary skill in the art will r~co~n; 7e the use of bifl~nrt;-~n~l linkers for ~o.ljuy-Lion~ A preferred : ' ~; L ~ a funct;~n~l;7~.d hydrophilic PAAE
i8 attached to n-hexyl-1,6-diamine at one end through the ~ormation of amide linkage, and 6ubsequently to the target bi~ l~cl~le-, such as the testo-,LeL~.Ie derivative by the formation o~ another amide linkage with the other end, as l;fied by the following (NSP-DMAE-HD-19-HCNT).
f so,-~ H ~H
o Another preferred: ' ~'; of the testosterone tracer is a ~orm of the ~ _ ' of formula I above, where A is omitted, B is -CH= form ng an olefinic linkage with .
.

wo ssl2Mn2 r~ . s 21 8~463 Clg, and x i8 carboxylate, as shown below (NSP--DN~E--HD--19--CVNT) .
fJ' ~ o~
~N ~;~
A further aspect of the in~tant invention ~
the ~ ed i ~ved results of using functinnAl i 7?d hydrophilic PAAE for lAh~lin r viral antigen as l~tili7~d in various infectious disease h~ lmin~c~nt i - ~s~Ay8.
For example, in an IgG capture assay format, where mouse anti-human IgGFc i - i 1 i 79~ golid phase and viral antigen tracer are the èaye..L~ used for assaying anti-viral ant;ho~ in the serum sample, it was found that great v~ L in the signal to noise ratio of the assay can be achieved when a functjnnAl;7~d hydrophilic PAAE is used in place of the convont i ~nA 1 D~IAE-NHS in preparing the viral antigen tracer. These results most likely occur via better binding activity resulting in ~nhAn--ocl Y f ormation between tracer and target ant;ho~4;e~, and lowered n~,.., fiC binding due to the use of hydrophilic acridinium ester tags.
Thus one with ordinary skill in the art could take the t~Ar~h;n~ of the instant invention and apply them to the ~lat~ct; on of a variety of infectious agents or ~e~LI~Gy`êll. ~hese infectious agents such as Rubella, Hepatitis of various classes and ~-uLLy~es~ Toxoplasma, Cyl -, lnvirus (C~SV), HIV, chlamidya to name only a few, are targets for early ~ tect;nn i~hich will reguire assays W095/27702 }'.,1/~,.,.. 'S ~

of great sensitivity and low inridonre of ral6e positive indication .
A typical architecture for such an assay consists of, but is not limited to, three -, a tracer, a sample containing the analyte, and a means for partitioning bound from unbound analyte. For eYample, such assays could combine the functi~nAl;77ocl hydrophilic PAAE
with suitable antigen of or specific binder for an analyte to form a tracer, incubation with a biological sample (containing the analyte), and capture of the tracer by a solid phase i hi 1 i ~ l antibody. Other architectures will become practical with the application of the te ~ ~h i n~ of the instant invention, and are readily a~art,lt to those with ordinary skill in the art of bioassay.
The instant invention ~ the use of the compositions and methods of the instant invention for allergen; ~~~ys. The hydrophilic PAAEs of the instant invention will be useful for the detection of FrPo;f;t~- IgE Ab to spPc;fic allergens. In this application, hydrophilic PAAEs are linked to "L ~L~.vidin, and can be used to detect specific allergen-biotin -lec~lP~ bound to specific an~;ho~ to the allergen.
( See an example f or the architecture of an ACS specif ic IgE as~ay, Fig. 9. ) Thu5 the instant invention ~~~ ~~ -~ methods of measuring the amount of analyte comprising a sample and detecting an analyte specifically and in proportion to its c ~ L-c.tion in a sample, and measuring a signal which is generated by a hydrophilic acridinium ester hPm;lllm;nPf~cont label that i5 directly or indirectly proportional to the ~-.,..r_"l ~,tion of the analyte in a sample.
Such methods of detecting an analyte in a sample comprise contacting a sample with a hydrophilic acridinium ester labeled detector molecule, sequestering bound w~ 95127702 2 i 8 6 4 6 3 r~ t ~
A~t~ and analyte, washing away excess lP~Pctor, and measuring a signal from an analyt~ bound detector.
Further, methods of ~Ptec1 ;n~ an ~nalyte in a sample comprising, c~ntAc1 in~ the sample with a hydrophilic acridinium ester labeled competitive tracer, and a spec;fic binder for an analyte, retrieving the specifir-binder, and clPtPrm;n;nq a sign7l1 generated by the bound tracer. (See an example for the assay architecture of an ACS testosterone assay, Fig. 7. ) Also ~ ~~~ ' are methods of ~lPtec~;nq an analyte in a sample comprising contacting a sample with a hydrophilic acridinium ester labeled competitive tracer, and a specific binder for an analyte, retrieving the specific binder, and cletPrm;n;nq a signal generated by the unbound tracer.
Tnr~ Pd are methods of detecting an analyte in a sample comprising, contacting a sample with a first specific binder, and a second hydrophilic acridinium ester labeled specific binder, and ~lPtec~;nq a signal. These assay architectures can be either in the form of sandwich assays or assays in which the second binder (or antibody) i8 reactive with the f irst binder . Many variations of these archit6~;Lu.- ~: will be aJ~arc~ to those with ordinary skill in the art.
The instant invention C5PC methods of detec~;nq an analyte in a sample comprising, contacting a sample with hydrophilic acridinium ester 1 AhPl 1 Pcl detector and a competitive binder to the ~Dtectnr, sequestering the detector bound to the competitive binder, washing away excess ~Pt Pctrr and the ~Ptect~r bound analyte, and measuring a signal from the ~P~ect~r bound to the competitive binder. (See an example for the assay architecture of an ACS estradiol a6say, Fig. 8. ) The methods of the instant invention can be practiced with or without the addition of r~lPAc;nq agents prior to detection of a signal, or other agents which will enhance wo 9~in7702 21 ~ 6 4 63 r .~ c ~
the signal generated.
A ~urther aspect of the instant invention ~ -~s"Q
the use of the composition8 and method8 of the instant invention f or gene probe assays . For example, low level ~ tion of target nucleic acids (the analyte) can be detected by hybridizing to a first nucleic acid probe, which is cvl.juy~lted with a macromolecular carrier, such as bovine serum albumin, glycoproteins, etc., labeled with multiple hydrophilic PAAEs and optionally multiple hydrophilic polymers such as polyethylene glycol IPEG), and capturing with a second nucleic acid probe, which i8 -~ i Pod on a solid phase such as p~L " ~ æt i particles. The two nucleic acid probes can be preferentially ligated with ligase after the hybridization step, but prior to the ~eparation step twhich removes the unbound probe tracer ~ ;nin~ in the ~uy~ &tant) to enhance assay specif icity . Because of the greater number of PAI~E i~.VL~VL~l~ed in the tracer due to the hydrophilic nature, good 501 llh; 1; ty is r-; nt:l-; n-~d . Such nucleic acid detection can be a~ h~d using several schemes that one with ordinary skill in the art can conceive, and using the t~a~h;n~ of the instant invention, develop the proper hydrophilic PAAE tracers for sensitive detection.
The following ~ 1P~ are presented to illustrate the present invention and are not meant to limit in any way the scope of the instant invention.
B~ p1B lA
- _ ~.JC ~ynth-3i~ of 3 0 2 ', 6 ' -Dim~thyl-~ ' - (N n; m ; lyloxyc-rbonyl ) ph~nyl 9--~Gr~;r- ~l~t~ ~ '~~ , F,. _ Or D21aB--NH8 ana N81?--D~15--NH8 ) ln;ml~l 3 5~ 1--4 ~ v.~ybal.zoate A solution of 3,5-dimethyl-4 ~ylLv~.yL~IlzOic acid tl2.0 g, 72.21 mmol) in 400 mL of anhydLvus wo ssl277o2 2 ~ ~3 6 4 6 3 ~ s tetrahydrofuran and 200 mL of anhydrous N,N-dimethylforr m;dP was cooled in an ice-water bath and treated with N h~drux2.,~ ;n;m;~a (8.31 g, 72.21 mmol) with stirring for 15 minutes, followed by addition of dicyclohexylcarbo~; ;m;llP (17.88 g; 86.65 mmol) . After 16 hours of stirring at 25-C under nitrogen, the mixture was riltered; and the wet cake was washed with 10 mL of anhydrous N,N-dimethylformamide. The _ ' inod filtrate was evA~.,L~Led to dryness under reduced ~L~=8Du~-~. The residue, after washing with ~I~L~ ous diethyl ether (2 x 50 mL), was ~ d in 80 mL of boiling ethyl acetate for 5 minutes. Cooling the s~pP~ir~n to 25-C gave 11.95 g(63%
yield) of the off-white product, which was h. , - on TLC (Rf 0.6; silica gel, ether).
9 -A rr itl; nP~ '.h 1 oride hv~ roch lor ide A mi-xture of 9-acri~l;nP ArLo.~ylic acid hydrate (lO.o g, 44.80 mmol) in 80 mL o~ thionyl chloride was refluxed at lOO-C under nitrogen for 2 hours, and then cooled to 25 C. The resulting solution was reduced to about half the original volume under reduced pLaDDuLa~ and then poured into 500 mL of a.~y-lLuus diethyl ether. The yellow precipitate was ~ollPrtP~ washed with ether (3 x 100 mL), and dried under reduced ~L~:DDULa for 2 hours, to produce 12.95 g (~100% yield) of the product, which was used immediately f or the next reaction .
2 ' . 6 ' -Dimethyl-4 ' - (N-succininidvloxYcarbonvl ) ~henvl 9-acr;~l;nP~ ~. IJuxvlate rDMAeE-N}}S~
A 8 o 1 u t i o n o f 8 u c c i n i m i d y 1 3,5-dimethyl-4 hyll-Jxyl,e--zoate (11.29 g, 42.88 mnol) and 4-dimethylaminopyridine (2.19 g, 17.93 mmol) in 200 mL of dLU~I8 pyridine was cooled in an ice-water bath, followed by addition of g-acr;~l;n~_Arl~.,,yl chloride hydrochloride (12.95 g, 44.80 mmol) . The solu~;c n was W095/27702 21~6463 P ~ ss heated with stirring under nitrogen at lOO C for 2 hours, and then at 25-C for 16 hours. After removal of the solvent under reduced ~ L a, the residue was rlash~ L I vyL~hed on a silica gel column (Baker silica gel, Cat#7024-1; F7 x 50 cm) packed with 50~ diethyl ether/hexane, and eluted with 75% diethyl etherthexane (2 . 5 L), diethyl ether (2 L), 5% ethyl acetate/diethyl ether (3 L) and 25~ ethyl acetate/methylene chloride (5 L) . Fractions containing the product (Rf 0 . 4; silica gel TLC plate, ether), col 1 rctD~ from the 25% ethyl acetate/methylene chloride eluent, were ' in~ and e.c.~u.~ted to dryness under reduced ~L~ u~a, to collect the light-yellow product (DMAeE-NHS) in 10.03 g (50% yield from 9-acri cl; n~- ~ r LVJ~ylic acid hydrate) .

15SaNPLD lB
~v~ ~ ~ of 2 ', 6 ' -Dim-thyl-~ ' - (IJ - ~ ' n 1 m ~ ~lylv,,~ ~ _rl ~1 ) ph~nyl 10-lI-thyl-9---cri~ y lat- H~thyl~ulf~t-~DHA15-N118), U~ing ~olu-n~J a~ ~ol~-nt This ~LoC~,~uL. uses toluene as a solvent in methylation of DNAeE-NHS, to replace the OZvllE de:~leting reagent 1,1, l-trichl~Lue LllaI~e, which was previously used as the solvent in the methylation process aR described in U. S . Patent t4, 745, 181.
A ~r~nRi~n of D~AeE-NHS (1.0 g, 2.13 mmol) in 50 mL
of anhydrous toluene was heated to 110 C to give a 5 - - solution, which was cooled to room t~ _ ~LuLa followed by addition of 11.0 mL (116.25 mmol) of distilled dimethyl sulfate. After 20 hours of stirring at llO-C
under nitrogen, the sOlUtion was cooled to room t~ ~ ~,l.uLa, and then at 4 C for 1 hour. The mixture was filtered, and the yellow wet cake was washed with toluene (2 x 5 mL) followed by an~.yd.~,us methylene chloride, .

~ WO 95127702 - P.~
21 ~6463 heated to boiling for 2-3 minutes, and filtered. The volume of filtrate was reduced to about 50 mL on a hot plate. Cooling the c~ te to room t~ ~-u--~ gave yellow crystals, which were collected and washed with diethyl ether (3 x 20 mL), to afford 500 mg (39%) of the pure product. FAB/NS (483 ,M+) .
l5sallpLls 2 8ynth~ of 2 ', 6 '--Dil~ t_yl--4 '--~1~ ~ ~ ' n ~ yl~A~ _~LvA~ ph~nyl 10- (3 ' 8l~opropyl) -acr~; n l 9 __~LVAY lat-. lZ--N~8 ) DMAeE-NHS (500 mg, 1.07 mmol) and 1,3-propane sultone (6.5 g, 53.3 mmol) were heated at 150-C under nitrogen in a sealed tube for 20 h. After cooling, excess 1,3 ~ al.e sultone was removed by trituration with toluene ( 3 x 5 mL) . ~he crude product was purif ied by RP-HPLC using a C18, 30 x 500 mm, 10 ~m, ~:~a.e.tive column, and eluted with a mixture o~ 65% solvent A and 35% solvent B.
Conditions: solvent A: 0.059~ (v/v) TFA/H20, solvent B:
o. 05% (v/v) TFA/C~3CN; flow rate 25 mL/min; W detection at 260 nm. The product was eluted at 26.8 min; yield: 60 mg (25~); FAB/MS (591, M+H).

IS~WPI.l~ 3 8ynth-~is of 19-C_~L~.A~ hyl-17,B,19-dihl~l.. Al 4-- ~ ~ L-n-3-on-tl~ 19 _.. L~AI ~hyl-testost~rone, l9-lECltT~
3 .17-Bis ~ethY~n~l; oxY~ -l9-eth.".Y-,c.rb~"~YlmethYl-l9-hyd~o xY-5-androstene A 50 mL 2-neck flask equipped with a rubber septum, magnetic stirring bar and N2 inlet was charged with 35 lithium bis(trimethylsilyl)amide (12.9 mL of 1.0 M

Wo 9~/27702 2 1 8 6 4 6 3 P~.. . 11 solution, 12.9 mmol). The olution was cooled to -78 C
under N2 in drY ice-acetone bath and ~ hydL vu~ ethyl acetate ~1. 2 mL, 12 . 9 mmol) was added dropwise from a ~yringe. The reaction was 5tirred at -78-C for 30 minutes a n d t h e n a 8 o 1 u t i o n o f 3 ,17-bis (ethyl~nP~l; nYy) -5-androsten-19-al (1 g, 2 . 58 mmol) (Lovett, J.A. et al., 1984, J. Ned. Chem. 27:734-740) in y Iruus tetrally-lLurllLc.~- (15 mL) was added dropwise to the sol~lt i ~n o~ the enolate . After completion of the addition, the reaction was stirred for an additional 1. 5 hours in the dry ice-acetone bath under nitrogen. The reaction was then yu~ ~ ~d with the addition of saturated ammonium chloride solution (-30 mL) and the resulting a~ pPnci~m was extracted three times with ethyl acetate (3 x 25 mL). The ;n~rl ethyl acetate extract was washed once with brine (~30 mL) and was then dried over r-gnPcillm sulfate. After filtration, the solvent was removed under reduced ~ uLe to yield an oil which was used as such in the next reaction: yield 1 . 67 g (crude); mass D~e~,L~
(~hPm;c~l ionization) mtz 477 (M+ H+).
3 .17--Dioxo-l9-~-l,u--Y -~ Lv-.v~- 1--19 hYdLV~vl--5--l~n~~ost ene The crude b;~tPt~l from above (1.67 g) was dissolved in tetral.ydLurùL~n (20 mL) and 3N perchloric acid (10 mL) was added. The reaction was stirred at room temperature for 3 hours and was then diluted with water (40 mL). The resulting s~lcpPncion was extracted twice with ethyl acetate (2 x 40 mL). The inP~ ethyl acetate extract was washed once with water (~40 mL) and was then dried over _ -~illm sulfate. After filtration, the solvent was removed under reduced presDure to afford the crude product which was purified by ~L"~Lc.tive TLC u5ing 1:1 ethyl acetate/hexanes a5 eluent (Rf s 0.17): yield 0.46 g (46%
two steps) white foam; ma55 ~ye.~Luu (~hPm;~:~l ionization) W095127702 I~ '~~ 1~

m/z 389 (N + H+).
lq-hUIv~ ,Ll~u~ 1-17B.l9-d;hvdroxY-4-androsten-3-one In a 50 mL round bottom rlask eql1; rped with a magnetic tirring bar and a nitrogen inlet, was placed 3,17-dioxo-19 eU.-,A~ ~LLu~ylmethyl-l~ ~Iy~L~ y 5 andLo~en (50 mg, 0.129 mmol) and al~lydL~u~ methanol (5 mL). The l;olution of the steroid was s~irred and cooled in ice under nitrogen and sodium ~uL.~ dLide (6.4 mg, 0.168 mmol) was added as a solution in methanol (1 mL). The reaction was stirred in ice for 8 minutes and was then quenched with the A~ ;tion o~ acetone (1 mL) and acetic acid (2 mL). The reaction mixture waæ then diluted with water (-25 mL) and the resulting 5~roncion was extracted with ethyl aCQtate (~35 mL). The ethyl acetate extract was dried over ~--gnosil~- sulfate, filtered and then c;~,llc~ L~IL~d under reduced pressure to afford a white powder: yield 51 mg (quant. ) which was ~ , on TLC
(1:19, MeOH/CHCI3) (Rf = 0.26). This material was used as such in the next reaction.
19-Carboxvmethvl-17b. 19-dihvdroxv-4-androsten-3-one ( 19--T~r) The ethyl ester from above (51 mg, 0.129 mmol) was dissolved in methanol (5 mL) and treated with KOH (0.25 g). The reaction was stirred at room t~ c.LuLa for 1 hour and was then ~ d with the addition of water (25 mL) and acetic acid until the pH of the solution was adjusted to 6. The resulting mixture was further acidified with the addition of 0.5 N HCl (1 mL) and the resulting s-~pon~ n was extracted three times with ethyl acetate (3 x 15 mL) . The ; nod ethyl acetate extract was dried over magnesium sulfate, filtered and ~ Led. The crude acid was purified by ~Lt~al~tive TLC using 2~ acetic acid in ethyl acetate as eluent (Rf = 0.4) and was W0 9~n7702 2 1 8 6 4 6 3 ~ s ~
isolated as a white powder. yield: 10 mg (22%); mass r~u (rh~ l ionization) 363 (N + H+).
~pl-8ynth~ o~
10-Car~o~yvinyl-17p-hytlro~y-19-nor-~-anl~ro~t-n-30n~
(19-C~rboYyvinyl ~ -t~ L--- ~; 19-CVN'r) 3 . 17 -8 is f ethvl ~-n~ YY ~ -1 0-etho.. y ~ bUl . ~ lv i nYl -l9 -nor-5 -An~rostene A 50 mL 2-neck flask equipped with a r~gn~tic stirring bar, reflux ~ f r with a nitrogen inlet and a rubber septum was charged with triethyl~h~ <J~retate (0.35 g, 1.7 mmol) and ~I~.Y-IL~U8 tetral.~lLuruL,,n (5 ~).
The solution was cooled in an ice-salt bath to ~ -10 C
under nitrogen and n-butyl lithium (1.24 mL of 1.6 M
solution, 1.7 mmol) was added. The reaction was stirred at -lO C for 30 minutes and then a solution of 3 ~ l7 -bis ( ethyl ~n~ yy) -5-androsten-l9-al ( 0 . 22 g, 0 . 57 mmol) in llnl~lLI UD tetral.ylL~ru~ (5 mL) was added dropwise from a syringe. The reaction was then warmed to room temperature and was then refluxed under nitrogen ~or 24 hours. After cooling to room t ~ltULe, the reaction mixture was then partitioned between a 1:1 mixture of water and ethyl acetate. ~he ethyl acetate layer was separated and washed with brine. It was then dried over r-gnPc~ m sulfate, filtered and ~u-,cel-L-~ILed under reduced ~,~S~u~t. The residue was purified by p~ Li~tiVe ~LC
using 2:3, ethyl acetate/hexanes as eluent (Rf = 0.34) and was isolated as a white fluffy solid: yield 0.11 g (4396);
mass ~e~;LLULI (rh~ l ionization) 459 (M + H+).
3.17-Dioxo-10-e~ hnYyrArbonvlvinyl-l9-nor-4-An~ro6tene A solution of the bisketal (0.11 g) from above in acetone (10 mL) was treated with p-tol~ P~ onic acid ~ ~o 9s/27702 2 1 8 6 4 6 3 (20 mg). The reaction was 5tirred at room t~ lLUL~ for 16 hours and was then ~;ul.c~--LL~-Led under reduced ples~-uLe.
The resulting residue was partitioned between ethyl acetate and water. The ethyl acetate layer was separated, dried over r-gn~i sulfate and then c ~ L~ ~Led under reduced ~LOC~uLc:. The product was purified by ~LC:1J Lat,iVe TLC using 1:1, ethyl acetate/hexan(as as eluent tRf = 0.51) and was isolated as a white powder yield 20 mg (23%).
This material was used as such Por the next reaction.

lo-Ethv~v~:albvll~lvinvl-l7B ~.~,ILv,.Y-l9-nor-4-androsten-3-one A solution of 3,17-dioxo-10 ~Ulu~y~:~rbonylvin nor-4-androstene (19.5 mg, 0.053 mmol) in methanol (3 mL) was cooled in ice under nitrogen and was treated with sodium `vû~v~.y-lLlde (2.7 mg). The reaction was stirred in ice under nitrogen for 30 minutes and was then quenched with the addition of acetone (1 mL) and acetic acid (1 mL). The resulting solllt;~n was cv..-e..LLated and the residue was partitioned between ethyl acetate and water.
The ethyl acetate layer was separated, dried over r-gn~;l-m gulfate, filtered and then ~vl.c.~..LL~lted under reduced ~L~'DUL~=. The residue obtained (19 mg) was dissolved in ~ IYILVUS chloroform (5 mL) and treated with activated MnO2 (100 mg). The blaclc ~ p~-nirn was stirred at room t~ c~Lur~ under nitrogen 30 minutes and was then diluted with ethyl acetate and filtered. The filtrate was c _~.L. ated under reduced P1~~UL ~ to afford a white powder which was ~ , ~ on TLC ( 1:19, 30 - -~~~l/chloroform, Rf ~ 0.28): yield 18.2 mg (92%).
.

10-Carboxvvinvl-17~-hvdroxv-l9-nor-4-andro6ten-3 -one ( 19~CaL bV~ v inyl-nor-testosterone: l9-CVNT) 35 A solution of 10--ethv,.yvaL l v.. ylviny~--17B ~Iy-lL VAy--19--2 1 ~64~3 ..
nor-4-~n~rosten-3-cne ~17 ~g) in methanol (3 ~) was trea~ed with S~6 ~0~ (2 ~L). The reaction was stlrred ~t ro~ temDerature ~or 30 r,inutes and was then acidi,ied N ~3:Cl. The resulting suspension was extracted twice with ethyl acet~te (z x 25 mL~. The c~ hin~ ethyl acetate e~act ~as dried over Ica~nesium sulf2~e, ~i~ter~d and con~ Lated under reduced pressure to af~ord a white powder which was hamogenous on TLC ~2% acetic acid in ethyl ac~t~t~, R~ = 0.25~; ~ield 15.2 ~ 97%); mass s~e.;LL~ (~h~ l ioniz~tion) 345 ~1 f ~) Esample 5 sy~thesis o~ 31~ ~ lg-E~C!I~ Co~ugate Cr~ iniced lrith 3~ exyl-l, 6-dia~i~ (~S~ s-~c~r) First, ~SP-D~2.i;--NHS was crosslir~ced with n-hexyl-l, 6-di~Li~e to yield NSP-D~aE-~D ~hus, to a y~llow ç:olution of ~SP-r~ S ~60 mg, 0.1 ~mol) in an~.y~ Ls D~ ~ C~o~ 4, 10 hII~ was added n-hexyl-l, 6-diarine (116. 2 3~g, 1 ~mol~ . rhe resulting colorless solution w~s stirred at rooll t~ ~ - cL~ under nitrogen for 15 hcu~s, and conceI~trated to ~ L. The c3~ud~ product ~ias puri~il3d by ~C using a pr~par~tiv~
silica gel pla~e ~2000 ~, 20 x 20 c~) developed ~ith CE~Cl~, / C~;10~ ~ ~2 ~55 / 40 ~ 5~. The product had Rf =
0.5; yield: 50.2 mg ~&4%~; F~B / MS ~593, ~Y+H~.
SUbsequent~y, a sQlution o~
19-hydr~ c;a~ vmethyl-testosterone (ls-HCI~, 4 ~;Lg, O . 011 m~nol~ in anhydrous DM~ ~0.1 ~ was diluted with arhydrou~: C~Cl3 ~0.4 mL), cooled in an ice-bath, and 3 0 act~ vated with a sol Ution of DCC ~ 7 mg, 0 . 013 m~ol) in ~Ly~l~ous C~ 0.1 nL~. ~e reaction Dlixture was stirred at O~C for 10 ~inutes, ~ollowe~L by t~e addition ~f NSP-DN~ 6 ~g, 0.01 m~ol) in anhY.l~ous ~ (0.2 mI) and the new re2ct:ion ~nixture ~as stirred at roo~
t , - ~t~ e or t S hours . The crude p~-oduct was p~ ied ~ W0 95127702 P~ S
21 86~63 by RP-HPLC u8ing a C18, 7~8 x 300 mm, 10 I~m, semi~.L.~L<ltive column, solVent A: 0.05% (v/v) TFA/H20, solvent B: 0.05% (v/v) TFA/CH3CN, flow rate: 2.5 mL/min, W detection at 260 nm. Initially 30~ solvent B was used for 15 minute followed by a linear gradient to 509~ solvent B in 5 min. The product was eluted at 24 . 5 minutes;
yield: 2.3 mg (259~; FAB t MS (936, M+H).
ISY~pl- 6 8~ of N8P--DHal5 / 19--CVNT Co.~J ~.~t-- Cro~----linlcd ~ith n--H~l--1, 6--~i~min~ ~N8P--DlLa}9--HD--19--CYNT) NSP-DMAE-HD-l9-CVNT was ~Le~ared from l9-CVNT (2 mg, 0 . 006 mmol) and NSP-DMAE-HD (3 mg, 0 . 005 nmol) a8 described in example 5. The crude product was purified by RP-HPLC using a C18, 10 ~Lm, 7.8 x 300mm semiple~aL~.tive column. Initially 30% solvent B was Used for 15 minutes followed by a linear gradient to 60% solvent B in 5 minutes, where solVent A: 0.05% (v/v) TFA/H20 and solvent B: 0.05% (v/v) TFA/CH3CN; flow rate 2.5 mL / min; W
dotec1 ion at 260 nm. The produ~t was eluted at 23.2 minutes. Product yield: 0.72 mg (16%); FAB / MS (918, N+H) .
~c~pl- 7 8r ~ of ~ / T~to~t~lron~-19-CNl!: C J__ t~
Cro~--linlc~ rith n--H~lcyl--1, 6--di~ninH ~N8P--DNAl~--HD--19--CNIST) NSP-DMAE-HD-l9-CMET was ~Le~ared from testosterone-19--CME (4 mg, 0.011 mmol) and NSP--DMAE--HD (3 mg, 0.005 mmol) a8 described in example 5. The crude reaction mixture was f irst separated on a ~L e~L c~tive TLC plate (8ilica gel, 1000 ,um, 2096 MeOH/CH2Cl2) Two bands at Rf 0.2 and Rf 0.3 were collected, combined and further purified by RP-HPLC using a C18, 10 ILm, 7.8 x 300mm semi~L.~I~tive column. Initially 30% solV nt B was used for 15 minutes ..
~ollowed by a linear qradier.t to 60~6 solverl~ B in 5 r~nutes, where solvent A: 0. 05~ (vtv~ ~Aj~I2C and sol~rent ~: O . Q5~ ~Ytv~ ~/CX3CN; ~w r~te 2 . 5 ~ ~ ~ini U~
de~ection at Z60 n~. The product ~as eluted at 24.1 5Illnu~es. Product yield: ~.3 mg (6.4 9~); ~AB / !~s ~936/
~+~ .
~ 8 108ynthesi3 ~f D}5A~/l9-~c~r con~ugal~ ros~-li~ed Yith ~-hyl-1, 6-~iamine ~ lS~
First, DNA~:--N~S wa~ derivatized with n-hexy1-1,6-dia~ine to yield D~ ~D as de~cribed in Exa~plc 5. Thus to ~ yello~ solution o~ DMP,E-N~5 (50 3~g, 0 . 084 mmol) in anhydrous D~F~C~30~ 4, 10 ~T.~ was 2dded r~-hexyl-1, 6-diamine ~'~8 rlg, 0. 84 nmol~ . rhe resulting solution wa6 stirrt~d at roorQ tt~perature u-sder nitrogen S'sr about 4 hours and evapor~ted. ~e crude product was puri~ied by Tl,C using prepar2tive sili~:a gel pla'ces ~000 ,1l~, 20 x 20 c~ developed ~-ith CH~13fCE~OH~}I20 ~65 t 25 ~
4~ . ~e produot ~iand - at ~ = O . 4 w2s strip~ed, eluted with the same solYellt syste~ he el.a~t was evap~rate~
to 2 residue w~t~h was triturated with c~lorol~cr~
f il~ered arld the f iltrate eYaporate~ ~o gi~e puriried D}~ 20 mg, 41~.
D~AE--~ID--l9~ r was prt3par~d frolrL lg--~C~ (4 . 75 ~ng, 0.013 Dn~ol~ and D~ 7 ~g, 0.012 ~olj as described in Exa~ple 5. O~e third Or the crude product was purif7ed by RP-E~LC usirlg a C18, 1~ 7 . ~ x 3 00 3~ se~nipreparative co~umn. Initially 30~6 solvent B was used E~r lS minutes follo~ed by a linear gr2d7ent to 60S Qlvent B ln 5 ~inutes, Where solvent A: 0. 7% ~v~v~ A~E~O ~nd s~lvent B: O. lS (v/v) T~A/CE~3CN, i~low rate 2 . 3 n~/~ir~
detec~ion at 26~ ~zu. q~Qe product was el-~ted at 23 ~7rl.
Product yield: 1. 5 ng (45 ~; FAB~ 28 . 5 M~) .

~ W095l277~2 2186463 --lS2allPLE 9 A~y For Tusto~t~ron--This example teaches a sensitive and rapid competitiYe rhDm; ll--in- ~c- .. e i -.~ay for testosterone using the hydrophilic NSP-DMAE-HD-l9-HCMT tracer, that is heterologous to the antibody i - ~ at the bridge. The data shows the clear adva~lLa~ of this new hydrophilic tracer ~d to the conventional 11YdL . ~I.obic DMAE--HD--19--HCMT tracer by allowing the dev~-l, t. of a rapid, high sensitivity teDL6DLe~ol.e assay.
A) Pro~ tion and characterization of polyclonal and ::lon~ 1 Ant ~ ho~ i C .;ially available, rabbit polyclonal ~ntihorl~3P~
that are anti-testosterone-l9-o-ca~ b-~LY thylether-Human Serum Albumin (RAT-l9), was purchased from OEM, (Tom's River, NJ). The pro~itlc~;nr of this antibody was previously described by Rao (U.S. Patent # 4,197,286). A
mouse ~ lAnAl antibody against rabbit IgG was ~ lu.. e~
by a modified version of the tP~-hnique described by Rohler and Milstein (Nature (London) 256, 495-497 (1975)). Mice were i 1~A~3 with purified rabbit IgG to produce a mouse ~ --lnn:~l anti-rabbit IgG (MAR-IgG). Standard antibody screening te~-hn;qll~ were used to select a hybridoma with high specificity for rabbit IgG. Ascites was ~l~duC~
using clone HlqFl-llB5-3Fl-5H7. 3~fAR-IgG antibody was purified from tLte ascites using Protein A-sepharose (1--- -jA Fine ~hF~micAlF~, Pis ~l _y, N.J.) affinity 3 0 chromatography .
B) DevPl~, L of a Testosterone `h~-m~ in~sccl~ne T ~:ay 35 Optimal Assay performance for a rapid testosterone ~ 21 86463 ~;r~scence asszLy was achi~red with the ~ IgG
(describ~d a~ove~ cova~ently c~tupLed to a solid ph2~Q and p~-ln~-that~d with the R~T-19 antibody using the NSP-3~-~jD-19-~C~r as the tr2cer. The ~ssay used paramagnetic particles (P~P) tAdvanced ~agnetic~ Inc., Ca~ridge, MA) ~5 the solid phase.
The ?~aR-IgG ~160 ~g per gra~ ct P~) in 0.1 M sodiur pho~phate, p~ 7.4, was covalently linl ed to the ~tKP ~fter acti~ation Or the P~P with 0.5~c glutar~ldehyde in ~.1 N
sodium phosphate ~rethod modi~ rom Gro~an et al., 1985, BioTechniques 3, 156-160). This was followed by incubation with R~.T-l9. ~xcess RAT-19 W2s reqLoved by additional washes of the reactio~ IlLix with phosphate bu~fer .
~wenty-five mic~oliters ~4L~ o~ a seru~ sall~tle or testosterone s~andilrd (0.1 to 24 ng/~L or 2.5 tct 600 pg~tube), 50 f~ of NSP-D~E-HD-ls-HCr~r tracer (6 x 7 o6 Relative ~ight Un$ts/test) and 300 ~ L o~ ~AR-IgGIRAT-13 ~nti~tody-sctlia phase were added simultaneously, incubated at 37 C fo~ 5 tinutes, ~.ashed and the che~llt~min~ r~ e me~sured on an AC~:180 (Ciba Corning Diagnostics, ~edfield, Na) automatQd che~i~tttn;nF~ i2runo~ssay syste~ he -17~mi lllm1n~5r~n.-e 5ignal (Relative Light Units) is inv~rsely proportional to the testosterone ~ ~ation. Assay standards consist~d of pure testoster3ne in steroid-~ree, cl~arcoal 5tripped human plasma. ~he testosterone standard binain~ cur-~e ~percent 0~ of ~;tandard to RL~T o~ z~ro 7tandard) ~ar the described assay is sho~n in FIG. 3. Testosteron~ dos from sen2n sa~ples were generated on the ACS:180 using two pQint calibration off a ~aster curve. Three other testo~ trac2rs, n~6lly, ~SP-D~E;-EID-19-CV~il (haterologoUS to t~e ~ntibody im~unogen), NSP-DMAE-EID-19--c~r tho3 1ologous to the ~ntibody ~ ~ n), and D~AE-~-19-i~ a non ~ aL,hilic vcrsion AMEN~E~ SHEET

of th~3 NSP-Dr~E-Hr:-13-~c~r~ were evaluated in the sa~e ~ssay ~onnat ~or their effect on th-a ~ind~ng cur~re 2n~i the 5~ imll~ binding (~50) of the standard binding curve.
~he DM~ lg-E~CNT trac~r was evaluated to de~ons~ra~e the advantaqe of empl cying a hydrophilic PAP~ (~S~-C~E~
tas versus a h~dropho~ic PAAE tag (DM~
C) Conventional Radioimmllno~cf:~y for Testosterone T~e resuits (n-145~ of a con~entional testos~erone radioimmunoassay ~lZ5Iodine) were c~pare~ in selec~e~ ~le and female serum samples with the ~.CS:180 chemill~m;nocc~nr~, assay using ~he neterologous tt~:to~terone tracer, NSP-D~AE-~-lS-~CMT rhe Coat--A--Count Total ~estosterone (DPC, Los Ang~les, CA~
c- cia~ system ,ras us~d This assay has a t}Iree ho~r incubati~n time with a 50 A~l. s~ple ~:ize. The reported mSn;~num deteGtablt dose iS approxi~ately 2 picograms per tube as calculated ~rom the ~nu~acturer8 packagD insert Y115 dated January 27, 1992 This assay was perfor~ed according to the ~anufacturer's instructions.
D~ C~mpetitiv~ ~ht~m; l-lm;~D~¢onre ~ss~y q~he ~s n;~~-m detectable dose ~Rodbard, 1978 Ana~.
F~io~h~. 90, 1-12~ of t_e r~pid automated test~
say u~ ing the heterolcgoT~s tc~;tof~terone tracer NSP-D~faE-~D-19-~ICI~ is 1.25 pg~tu~7e~ a l. 6 fold increase over the conve~tional 3 hour radioim~ln~ cay ~ethod 3 0 described above . As ~hown in FIG . 5, patient sample result~; ~or thc ch~n;lTTmint~70t~nt assay oarrelate very well to the radio;mTT~-7nn:~ssay. me crossreactivity profile of th~ a7say is shown in ~able 2. These ~ata suggest that the new che3~ilT7TT; T~ nt assay is of useful value ~n the analyt;ical det~ T~tion of testosterone in hu~nan serum :~5 WO95/27702 21 8 6~ 63 r~.,L '~
sample& .
TABLI~ 2 S~ecificitY of ACS Testosterone ~!hPmi lllm;nPRrnrc-~Lml~n~ccs~v ll~lin~T
NSP--CIMA~--I~--l9--HCNT tracer cross-rPA~ nt DoRe % Cro8s-reactivity Sl)eG ~ f icat; ~nR %
10 AndrostAn~P~ nP 100 ng/mL 1. 55 <1 An.lLv~LeL~.. e 1 ug/mL 0. 03 ~o .1 Cortisol 1 ug/mL 0 . 03 <0.1 Corticosterone 1 ug/mL 0 . 02 <o .1 ll-de~.. y-,uL Lisol 1 ug/mL 0. 05 <0.1 15 5a-dihydrotestosterone 100 ng/mL 8 . 3 <15 Estradiol-17B 100 ng/mL 0.12 <0.1 Estrone 100 ng/_L o . 07 <0 .1 Progesterone 1 ug/mL 0. 09 <0 .1 20 Danazol 1 ug/mL 0. 06 <0.1 Dexamethasone 1 ug/_L 0 . 01 <0 .1 A comparison of the ,jL~I...la ~ binding curves and calculated ED50's for each ~h~m;lum;nPRcpnt tracer under the same conditions are shown in FIG. 4. The highest ED50 is for the NSP-DNAE-HD-l9-CNET tracer that is ~ ls~o~-to the antibody ~ ~, . The ED50's, for both heterologous tracers, NSP-DNAE-HD-l9-CVNT and NSP-DNAE-HD-l9-HCNT, are lower than for the h~ 1 o~o--c tracer indicating that the heterologous tracers are more easily ~li cpl~c~d by testo~Le~ e than the ~ lo~o--c tracer. Little ~ pl~ ' is seen with the lly~lLu~l~obic DNAE-HD-l9-HCMT tracer indicating the clear advantage of the hydrophilic NSP-DMAE-HD-l9-HCMT version of the tracer in testosterone i --- y.

W09~127702 2 1 8 6 4 6 3 ~ -`A 11 IS~aNPI,15 10 II~IBISL~ IgC~ CaP~rllR15 A88AY
A rubella IgG capture assay wa8 used to evaluate NSP-DMAE-NHS and DMAE-NHS as reagents for the direct 1 Ahl-l 1 i n7 of rubella . Intact rubella virus was labeled with either NSP-DMAE-NHS or DMAE-NHS as described below.
Both of th~se tracer pL~aLations were 5ubjected to a titering study and a population study. The peLruLl~ la~ of each rubella tracer can be det~rmin~ by analyzing the positive sample/negative sample ratio ~el~eLaL~ by each tracer for a given sample population. In this rubella IgG
capture assay, the Positive signal/Negative signal (P/N) ratio also reflects on the ability to accurately dist;r~;h small differences in the level of rubella specific IgG. The preferred rubella tracer is one that generates appreciable differences in P/N ratios for ~amples spAnn~n~ a wide range of IU/mL levels, since this will lead to greater ~ensitivity in rubella specific IgG
detection.
A) Preparation of Rubella virus Tracers Rubella virus (grade IV, 1 mL at 0.5 mg/mL) purchased from Viral Antigens, Inc (Memphis, TN) was dialyzed into lAh~ll;ng buffer (10 mM ~hoa~lla-e, 0.125 N NaCl, pH 8) using a centricon-30 mi.l.cc,~ La~or (Amicon, Danvers, MA). The virus sample was brought to 2 mL volume in l~h~ll;n~ buffer, then ~o~ L ated 10-fold by Sp;nn;
the centricon-30 in a tabletop centrifuge for 30 minutes at 2600 rpm. The ~ t.e wa~ again brought to 2 mL
35 with lAh~l1;n~ buffer. This c ~ l ation and W095/27702 2~86463 r 1S
reconstitution process was repeated 3 times. The sample was then ~plit into 2 x 1 mL aliquots for use in .lh~q~ nt IAh~l 1 ;n~ ~, uc~llu~
To each Rubella vlrus aliquot (0.25 mg in 1 mL
buffer) was added NSP-DMAE-NHS (50 ~g in 25 ~LL
dimethylf- ;~1~) or DNAE--NHS (50 llg in 25 ~L
dimethylf-7r~~mid~) solution separately. The lAhellinq reaction mix was vortexed brief ly and in~ uL~ted at room t~ ~LuLe for 15 minutes and stopped by adding 5 mg of lysine (Sigma, St. Louis, MO) in 50 ,~LL of water. The re~ction mixture was vortexed briefly, incubated at room t ~UL~ for another 5 minutes and subjected to ~ollowing purif ication steps .
The reaction mixture was first purified through PD10 S~-rh~Y G25M sizing column (Pharmacia, Pi~-:at ~y~ NJ) preequilibrated and eluted with lAholl;n~ buffer containing 0 .1% BSA . The f low rate of the elution was maintained at 1 mL/min and 20 x 0.8 minute fractions were collected. The tracer which c~ ea~ in the void volume as the first RLU peak (fractions 6-9) was located by taking a 10 ~L sample fro~ each fraction, dillltin~ it 200-fold with lAhellin~ buffer, and counting 10 ,uL of the diluted sample on a MLA II 1~ r ter (Ciba Corning Diagnostics, Medfield, MA). The desired pooled ~ractions were cu.lc~lltL.lLed with a centricon-30 to about 300 ,uL.
The col.c:el.L~ e was sUbjected to further HPLC gel f iltration .
Waters HPLC system (Waters Associate, Nilford, MA) mounted with a Protein Pak 300SW HPLC gel filtration column (Waters ~11787~ was first equilibrated with HPLC
column buffer containing 50 mM Tris pH 8, 0.25 M MgCl~, 0 .1 % BSA, and 0 . 01% Triton X-100 . The labeled virus u~ Le from the PD10 column was loaded onto the 300SW
column. The elution was maintained at a flow rate of 0.5 mL/min and 40 x 0.25 mL fractions were collected be~inn;n~

~ W095/27702 2 ~ 86463 r ~
from il minute after sample injection. The labeled virus peak (retention times of 13-17 minutes) wa6 again identified in the same manner a5 above, by looking for the f irst RLU peak through counting samples of the collected fractions. The fractions onl-S7lnin~ the first RLU peak were pooled and L~JL~_C_.li ~' the purified labeled virus tracer .
B) PL~aLation of Anti-human IgG solid phase Nouse anti-human IgGFc ~ 77A1 antibody was covalently linked to PNP with glutaraldellyde (in 20 mM sodium pho..~..a~e, pH 7). This is a modification of the method described in BioTerhni~ 3, 156 ~1985).
C) p ~ r~lLiOn of - --lrnAl anti-Human IgGFc (clone 157 . 8D6 . 5D3 ) Fusion PL~C~`]~ Method uses polyethylene glycol to fuse isolated mouse spleen cells from i ; ~d donors with SP2/0 Ag4-1 mouse myeloma cells (Galfrie and Milstein, (1981) ,Methods in Enzymolog,v 73:3) .
Scr~n i n- PL .~ e: Secretion of specif ic antibody was tested for by the ability of the cell ~u~L.. at~.,L to link acridinium ester labeled human IgG to human IgG
immobilized on P~L~I -JI~-I ;C particles. The monoclonal antibody was shown to be specif ic f or human IgGFc and nonreactive to human IgG Fab and Fab2 rL _ , human IgA, IgM, IgE, and IgD when tested by western blot (as per Proc. Nat. Acad. Sci. U.S.A., 76: 4350 (1979) ) .
~n~ i ht-~'V IsotvL~e The antibody isotype is IgGl, identified by using the Zymed MonoAb ID EIA kit (Zymed Lab Inc., San Francisco, CA).

2 l 86463 Wo 95/27702 r D) PL.~aL~Lion of -lnn~l Anti-Rubella ~ntibody (clone MI.109 . lOE7 . 4A3 ) F~la;cn PL~ r~ 8ame as above.
Screenina PL VCedUL ~: Secretion of specif ic antibody S was tested for by the ability of the cell n~ L.~ L to bind rubella virus. The lnnAl binding to antigen was ~tect~l by ELISA (as per J.E. Coligan, Current Protocol~
in T ~logy Vol. 1 (1991) section 2.1.3). Clones were hown to be speciric by fi.~ cLivity to uninfected culture controls.
E) Rubella IgG Capture Ch~-m;ll~m;n~c~ e ~ y Performance of the different rubella virus tracers was evaluated in a manual Magic Lite~ (Ciba Corning Diagnostics, Medfield, MA) assay format in triplicate. All 13erum samples were diluted five-fold with the diluent (1 x DP8S, BioWhittaker, Walkersville, MD) containing 50%
calf serum, 1% Triton X-100, 1% normal mouse serum, and 0.1% sodium azide. Diluted sample (50 ~IL), Anti-human IgG
solid pha~e (80 I-g in 250 ~LL), and diluted rubella virus tracer (100 ,uL) were incubated to~e~h~t for 7.5 minutes at 37 C in a Sarstedt poly ,LyL~ I-e tube (Newton, NC) . The reaction mix wa5 s=~ar~ted, a8pirated and washed twice with 1 mL 1 x DPBS (BioWhittaker, Walkersville, MD). The final pellet was reconstituted with 100 ,uL of water and its nh-,mill-m;~ ed on M~-II 1 ; Ler (Ciba Corning Diagnostics, Medfield, MA). Since the two rubella virus tracers (prepared as de~cribed above) varied in their spe~ifi~ ~h~m;l ;n~c^~ e activity, the titering ~tudy was ~ cl over different ranges of input counts, using nine rubella IgG samples as shown in Table 3.

wos5/277o2 2 ~ 8 6 4 6 3 ~ ' -RllhPllA IqG t`Antl7re Assav Re~llts of NSP--nMAR ~lhPllA
Titerinq S~ 1Y
S~mple NSp--nMAR RllhPllA tl2T3J, %~V. P/N~
l~Q65 . 95X10611 . lX10613 . 3Xl06 24 . 3Y1 0646 . lxl06 Diluent 1233 1793 2473 2757 4253 9360 ~CV1.9 2.6 9.4 3.3 5.5 17.2 P/N1.0 0.9 1.0 0.8 1.1 1.6 Neg~tive RAB ~7 1233 2037 2447 3367 4023 5987 %CV 3.3 20.1 3.9 23 8.5 7.5 P/N 1.0 1.0 1.0 1.0 1.0 1.0 1531aB 36 1383 1897 2587 2880 6357 8357 %CV 13.3 13.2 4.7 0.9 16.3 21.7 P/N 1.1 0.9 1.1 0.9 1.6 1.4 Low +

%CV 2.7 6.1 2.~ 6.4 9.1 7.6 P/N 1.1 1.4 2.2 2.1 2.6 3.3 caP lS 1473 3170 5283 7003 10263 19737 %CV 2.7 3 3.2 2.7 6.3 7.9 25P/N 1.2 1.6 2.2 2.1 2.6 3.3 Mid-High+

%CV 3.8 3.3 4 3.2 1.3 30P/N 2.9 7.1 13.1 12.0 16.6 23.8 R~67 10970 52003 120987 151000 262317 536050 %CV 2.7 6.4 5 2.4 1.06 2.3 P/N 8.9 25.5 49.4 44.8 65.2 89.5 35%CV 1.9 1.9 8 3.4 4.6 3.5 P/N 6.0 17.3 30.7 28.1 41.7 57.2 %CV 2.2 3.3 4.9 1.7 2.2 10.6 P/N 9 .1 27 . 7 45 . 5 42 . 3 62 . 0 85 . 9 %CV1.4 6.5 12.3 3.2 3.2 3.4 P/N9.3 27.0 46.5 43.5 65.4 90.6 The DNAE labeled rubella tracer was titered over a wider range due to a 4-5 fold greater degree of in.u,yu,~Lion of DMAE into the labeled virus tracer _ , d to NSP-DMAE.
In order to compare assay conditions using a comparable W0 95/27702 2 ~ 8 6 4 6 3 P ~
mass of rubella tracers, the DMAE rubella tracer total input range must be several times higher than that of NSP-DMAE rubella condition. The titering study results ted; v vc:d P/N ratios as the level of NSP-DMAE
rubella tracer invL~ sed~ while the P/N ratios generated by DMAE rubella tracer plateaued at lO x 106 RLU/test.
The selection of 24 x 106 RLU/test as the optimal input level for the NSP-DMAE rubella tracer was based on the results of higher P/N ratios, while maintaining low n~n~p^~ ic binding with the control diluent. The comparable assay conditions using DNAE rubella tracer is 125 x 106 RLU/test. When these two conditions are -red, it i8 clear that NSP-D15AE rubella tracer is the superior reagent with P/N ratios ranging from 2 to 4 fold greater than those generated by DMAE rubella tracer.
The testing of these two rubella tracers was oY}~AnA~A
to a population of twenty rubella IgG samples (ranging from 0.3-500 IU/mL) and 4 potential ~L~D-L~aCtiVe samples (all identified as rubella IgG negative by Rubazyme assay (Abbott Labs, Chicago, IL) (Table 4A).
The study was c~nA~tecl using the following total inputs for each reagent; NSP-DMAE rubella tracer (24 x 106 RLU/test) and DMAE rubella tracer (125 x 106 RLU/test).
The population data suggested that the sensitivity of the rubella IgG capture assay is affected markly by the class of the PAAE utilized to directly label the virus. The P/N
(positive to negative result) ratios generated from employing NSP-DMAE rubella tracer are considerably greater than those generated by the DNAE rubella tracer (FIG. 6).
These results reflect the ability of the assay performed with hydrophilic NSP-D~E rubella tracer to more clearly dis~;n~;~h positive samples from negative samples than conventional DMAE tracer. This is particularly important in the diagnostically critical low positive population.

wo 9512770~ 2 1 8 6 4 6 3 P~ 0. M
The impact of hydrophilic NSP-DMAE rubella tracer on the specificity of the potentially uLo~.~L~active samples w~s evaluated. Using a P/N ratio cutoff UVLL~ ;n~ to the CAP10 sample, the results indicate no false positive ;~Dn~ rntionS resulting from the use of the rubella tracers with these types of samples (Table 4B).
The results o~ both of these studies clearly suggest NSP-DMAE-NHS is a superior reagent for the direct 1 AhDl l; n~ of rubella virus . This reagent results in a rubella ~, ep~L~Lion which specifically binds to a wlde range of rubella IgG levels to produce specific RLU Va1Ue8 which are clearly dis~;n~;~hAhl~ from those of a rubella IgG negative population. FUL t ~:, these conditions exist without incurring false positive results with potDntiAlly uL~ ,L-active samples.
TABLD
p~-n~ tion gtudY ~ NSp--nMAR RllhDl l A ;~n~ nMAR ~llhDl l :~
Tracers Rubella IgG Capture Assay (P/N ratio) Pl~ NSP--nM~ Rubella nMATC ~-lhDl 1 A
RAB17 1.0 1.1 AP05874--4 1. 0 1. 0 AS04912--9 1.1 1.1 RAB 36 1.1 1.2 AS14837-8 1. 3 1. 0 CAP 10 1.8 1.2 AS01721-3 2 .1 1. 0 AP27771-3 2 . 4 1. 0 AR99860--4 2.5 0.8 CAP 15 2.5 1.0 TSI 3164 5.8 1.8 AS06137-4 8 . 0 1. 3 GPl-27 23.9 2.8 AP41719-0 24.9 3.5 GP2--9 2 6 . 2 3 .1 GP2-18 27 . 7 3 . 4 AR86731-7 51.1 5.6 AR96863-2 58.7 5.9 AS15758-2 68 . 5 6 . 9 AR74744-4 88 . 9 9 . 8 .

WO95/2~702 2186463 P~ . s q~ABLB ~.B
Intererence Samples Rubella Assay (P/N ratio) (All ~ella IaG -~ NSP-DNAE 1. l 0 725 5 VZV+E02606-135 1. 4 1.1 0 .183 ANAfl2615N-910513 1.7 1.1 0.793 F) Comparison with Ciba Corning Magic Lite~ control Assay and Abbott INx Assay The evaluation of NSP-DMAE rubella tracer was extended even further, to include an investigation of how this Magic Lite~ rubella IgG assay using NSP-DMAE rubella tracer _ ~s with (1) Ciba Corning Diagnostics ~agic Lite0 assay twhich uses a complex of llnl ;Ihc~ rubella and NSP-DMAE labeled anti-rubella ~ ~cl-~n~l antibody lOE7) and (2) IMx Rubella IgG (Abbott, Chicago, Il). The sample population tested by both Nagic LiteG assays ~nrll-A~A thirty-nine rubella IgG samples identified as positive or negative for rubella specific IgG by IMx (Table 5). It is interesting to note that the Positive result/Negative result (P/N) ratios generated by NSP-DMAE
rubella tracer are signif icantly greater than observed with the Magic Lite0 control assay format. This is particularly evident in the rubella IgG high positive samples and may ref lect competition between the rubella specif ic IgG and the NSP-DMAE-MAblOE7 tracer f or binding ~ites on the llnl ~h~ A rubella virus . Based on a P/N
ratio cutoff ~Ll-~L~ lin~J to the CAP10 sample, the Nagic Lite~ rubella IgG assay util;~;n~ NSP-DMAE rubella tracer ' LL~ted 97.4% a~ ~ with the Magic Lite~D rubella control assay and 92 . 3% &~L ~ with IMx rubella~ IgG.

. .

~ W095127~02 21~6463 9 s F:~n~....... l ~opul~tion gtudv ~ n-- NSP--DMAF~ R--h~ll~ Tracer NSP-DNAE Rubella +
Rubella NSP-nMAFl OE7 I~
5 R~B 17 1. 0 1. 0 I~L~re AP05874-4 1. 0 O. 9 n~ptve A504912-9 1.1 O. 9 ~at~e R~B 36 1.1 1. 0 I~L~e A514837-8 1. 3 0 . 9 ~atve 10 CAP 10 1. 8 1.1 p~;it~e A501721-3 2.1 1.05 rP~e AP27771-3 2 . 4 1.1 n~t~e AR99860--4 2.5 1.1 ~e CAP 15 2 . 5 1. 2 p~ t~e 15 A500541-9 2 . 8 1. 2 p~ t~ie AR97400-3 3 . 2 1. 3 p E~t~e AP41598-1 4 . O 1. 5 p~t~e TSI 3164 5.8 2.0 p~t~e A506137-4 8 . 0 1. 7 pX t~,e 20 A500407-3 9.9 2.4 p~t~e TSI 3156 13.6 3.0 p~t~e GP2--3 2 3 . 4 3 . 7 p~8; t~e GPl-27 23.9 3.6 p~tve AP41719-0 24 . 9 2 . 6 p~ t~e 25 GP2-9 26 . 2 3 . 9 p~ t~e GPl--1 26 . 3 3 . 7 p~ t~e GP2-11 26.9 4.1 p~ t~re GP2--18 27 . 7 4 .1 ~t~e GP2--4 28 .1 4 . 4 ~t~e 30 TSI 3167 33 . 4 6. 2 ~tt~e GP2--22 39 . 7 5. 4 ~t ~e GP2-14 42 . 0 4 .1 x~tve GPl--17 43 . 3 7 . 4 ~t ~e GP2--35 44 . 6 7 . 8 p~ t~e 35 TSI 3163 48.7 13.1 p~t~e A515788--7 49 . 3 5 . 3 p~t~e DLl--3510--388 51. 0 11. 6 p~8 t~e AR86731-7 51.1 9 . 6 p~ t~e AR96863-2 58 . 7 10 . 6 p~ tve 40 AP35925-6 62 . 0 7 . 9 p~t~e HG68085-5 63.8 16.5 p~ t~le A515758-2 68 . 5 11. 3 p E;at~e AR74744-4 88.9 27.9 p~ t~e 45 ESlUlPLlS 11.
D~lalS L ~ of prot- l n Thi~ example shows that a lar~er quantity of NSP-DNAE
can be covalently attached to a protein, such as BSA
(bovine serum albumin) because of the hydrophilic nature of NsP-D~AE. A solu.ion of ~ (10 ~ 50 lm~ol~ i~ O.1 ~ car~onate buf~er, p~ 9, ~2.7 mL~ wa5 treated wit~ a 50~ tiOr~ 0~ ~0 e~ui~alents o~ either D ~~N~S ester or NSP~D~F~ IS ester ~4.~ mg eaoht 7.~ ol) in D~F ~30~
,uL~. lrhe reactiorl utilizing 3}~-NHS was sli~htly cloudy whereas the reaction ~-ith NSP-D~aE-NRS re3~ained completel~
clear thraughout the rezction. ~ne re~ nq were stirr~d at room t~ Cli.ULe for 24 hours and the labele~ proteins wer~ isola~ed by size-exclusion cllromatography on a S~-rl~ r G-~S col~n ~3.7 x ~12 cl3) eluted with lQ r phosphate ~fi~e~ p~I 8. ~he protein ~raction was collecte~
in each case, concentrated and ~epurif ied on ~he sa~e Sephadex G-25 column~ After the second concentration, the qu~tity of the labeled protein recove~ed frs~ e~ch reaction and the extent Ole Acridinium Ester taE) labeling was esti~hated by performing a protei~ analysis ~8io-Ra~
protein assay~ ~nd by 31easuring the chemilllTn;neFc~nt activity cf the la~eled protein, respecti~ely. From these nea~ t~, the protein was found to be ;~ P~ with 2~ appr~xinLately 14 D~; ~olecules and Z~ ~SP-D~ ~olecules.
rle 12 Conjugatio~ ~f 1~8P--D~ lab~lcd ~A ~B~vi~e SesU~ a~ i to 5~-32P-labc~led 508.c~-3'-~lei~ide:
This exa3lple cho~s how tbe protein carr:Ler pre-labeled ~ith pluratity of NSP-~ can bQ covalently attached with a 2~ er oligonucleotifle which we c lled 508.CF-N~2, q'he c)ligonucleotide was firs~ ~adio-la3~eled with 32-P at the 5' end phosphi~t~ ~lsing the ~onven~icnal radio-1~1 in~ technique commonly lcno;m to those slcil~ed in the arts. q~he purpose of 32P-l ~h~-l i ng~ of t~e ol; gor-~lcleotid~ was ~o provide a ha}~ale to ~ua~ti~y t~e n~ cles~ide once conjugated with the prc~tein carrier.
The 3'-end of the oligohucleotide carries an ~mint-~7k~1 group to allow t~ ~eac:tion with a bi-~uhc:tional cross-_ _ _ _ _ _ _ _ . . _ _ . . _ _ _ _ _ _ .

,. .
linker zs desc~ibed ~elow so that a sul~hyd~yl-reactive im;~n group can be zttached. Methods o~ prep~ring oligonucleodie wi~h Aminna3lcyl group at the 3'--end is well 3cno~n (See ~ser's Manual on D~A Mo~i~ication ~ea~ents for Use in huto~ated DNA Syn~ hresis , Doc. No. PBC22783-1, CLC~ ECH La~oratories, Inc., Palo Alto, CA, 19~9 ) .
PreDar~s~;n~ of 32p - l2lh~led S013.CF-tl~aleimide:
A solution of 32P-508.C~-NH2 ~ 4~ nmoles~ in 50 m~
carbonate (600 ,llL, p~ 8. 4~ was treated with sulfoc~ i n; m; dyl --(N l~;m;~ ot yl)cy~ ohpy~ne-l--carbclx~rlate (sulfo-SMCC, lo ~g, 22.~nol~. ~he reaction was stirred ~t roo~ t~mpera' ~r~ ~Qr 30 minutes ~'ollowing which the product was p~ri~ied direGtly by preparative ~IPLC on a c8 col~ 0 . 7 x 25 cm) using a gradient of 85 to 20~ acetonitrile ~'n o.l ~ triethylammoniu~ ~cetate ~uffer, p~ 7.0, over 20 minutes at a ~low of 2.3 IDL/mi~
~nd I~V detection at 260 n~ (t3~ s 14.~ ~in. product; tR =
12~7 ~in~ s~arting ~terial~. The eluent off the ~PLC.
containing the product ~as lyophili2ed to ~rynes~: yield 25 n~o~es ~60~.
~o ~ ti~n of the NsP~ rAr labeled ~SA to 32P-5~8 CF--m~ m; de:
~SP-D~aE) 21-BSA was prepared by rezLctmg BSA with 30 ~uivalents o~ NSP-DXhE-N~S ~s aescribed a~ove. T~e labe~d protein (6~g, 9~ nnole~ in 100 mM rhosrh~te ~er containin~ S rM E~A, p~ 8, w~s treated with 2-~inothiolane ~50 e~uivalents, o. 6 mg, Pierce, 3o Roc}~ford, Il~ .
The reaction was stirred under nitrogen at room t~ ~ for 1 hour. ~he thiolated protein was then isolated ~ size-exclu-zion chro~natc.~ Ly on a col~n af S.~rh~Y G-z5 ~3.~ x ~2 cm) u~iing 20 ~I~N rhr~rh~te~ 1 EDTA, p~ 6.8, as eluent. The protein. frac~ion o~ the W095/27~02 21 86463 r~ ss column was ~ ted to a volume of approximately 2 mL in a ~,~eed ~- c. The protein solution was then mixed with 5'-32P-labeled 508.CF-3' lPim;rlo (8.9 nmole) and the resulting sol~lti~ln wa5 stirred at room ~ LUL~:
under nitrogen for 16 hours. The reaction was then y~ d by the addition of 600 nmole o~ }JL~ ~tic acid. After stirring for an additional 2 hours at room t a~ULe~ the reaction mixture was ~ ted to a volume of about 0.5 mL by centrifugal ultrafiltration.
A mixture of the ~ .,.. juy~te and u~ Juy~ted protein was s~p~L~ted from unreacted 508.CF leimitl~ by native Polyacrylamide Gel ElecL~v~l.~,le~is ~PAGE) on a 7.5%
denaity gel (3~ cross-linked) in TBE buffer, pH 8, run at aor.:.Ld~.L current of 10 mA at 4 deg C. The mixture of the av.. JuyaLe and the u-~c~ juyclted protein was eluted from the gel matrix by a crush and soak method in PBS
buffer (138 _M NaCl, 8.1 mM Na2HP04, 2.7 mN KCl, 1.2 mM
RH2P04, pH 7.4).
To further separate the cc...; uy~te which inauL~,r~-tes the ol ig~mlc leotide, from the u~w~juyc-Led protein, an affinity purification is called for. A
typical example of separating the conjugate with the inc~LLJ~L~Ited rll ignn~ otide ~rom the cont~m;n~ting ullaulli ~yated protein is the utilization of a solid phase (e.g. fl-nrtion~li70~l polyaLyr~.-e or controlled pore glass) ; ~i l i 70-l with the _ ~ y oli7r~n lrlootide. By performing a hybridization, separation, and washing steps, one can capture the desired cu..juy~ll e onto the solid phas, wash away the u~ juy~Jted protein, and continue on with a r~ clnq step to regain the purifed ~ ~..juy~,te from the solid phase. Such purification/enrichment steps can be easily done by those skilled in the arts of nucleic acid hybridization .

~ wossl277o2 2 ~ ~ 64 63 r~
IS~mpl~ 13.
Or nt~body-D~A ~anti~ 5~8.CF) J, te This example rurther illustrates the range of uses for the ~ _ ' of the instant invention. In this example antibody-DNA c;~ juy~te suitable for carrying a large number of ~h~m;lllmin~c~nt tags is generated. The juy~Le can be ~,.~a~ed in three steps using an alternative thiol - lPimlAr- CO~'r~ in~ chemistry.
Preparation of ~hi~ ted ~nti-TSll AnA col~iu~ ion to r~ ;m;AQ-508.CF: A 50ll~t;nrl of anti-TSH (20 mg, 133 nmoles) in 2 mL 0.2 M phoD~l,ate buffer, pH 8, c~ntA;n;
150 mM NaCl and 5 mM EDTA was treated with 2-iminothiolane (Traut's reagellt, 0.37 mg, 20 equivalents) under nitrogen. Th~ reaction was stirred at room t ~ u.a for 1 hour under nitrogen and the thiolated protein was isolated by size-F-Yrll~ n t~y~ y on a column of Serh~AoY G-25 (3.7 x 42 cm) using 10 mM phG:,~h~Le, pH 6.8, containing 0.5 mM
EDTA as eluent. The protein fraction eluting orf the column (yield 17 mg, 113 nmoles) was cv..~.. -.L,.lted under reduced ~ in a D~ed ~,-c to a volume of approximately 2 mL. This solution was then miYed with ;m;A---sog.cF (12 nmoles). The reaction was stirred at room t~ u,~ for 24 hours under nitrogen by which time the solution had turned slightly cloudy.
The reaction mixture was then loaded on a column of DEAE-c~lllllose (1 x 15 cm) equilibrated with 20 ~M
phosphate, pH 7. The column was eluted with a gradient of 0 to 200 mM NaCl in 20 mM phosphate, pH 7, followed by 200 mM NaCl in 20 mM phosph~te, pH 7, to elute excess ullL-:~.Led protein. Elution with 500 mM NaCl in the same bu~er, eluted a mixture Or the c~,..j~.y~.te and unreacted DNA. Following 0~,...;6.lL,c.Lion of the high salt fraction orf the ion-~Y~h~n~e column by ultrafiltration, wo ss/27702 2 1 ~ 6 4 6 3 r~
~eparation of the ~;ullJuyate from unreacted DNA was achieved by size-~Yrlllaion chromatography on a column of S~rhA-l~Y G-75 tl.5 x 40 cm) using water as eluent at 0.5 mL/min. The solution of the cv--juyaLe isolated by this pLu `édUL~ was 1yorh~ cl to dryness.
Characterization of the ~;u..; uyGte and calculation 0~ ~c~l.juy~lte yield was performed by W
..~e.iLLù~.ut ~Ly. The W D~e~;LLu~ of the ~ol~3uyaLe isolated by the p~ùCeduLe described above was identical in appearance to that of a 1:1 mixture of 508 . CF and anti-TSH. This W ~e~.~L~u was characterized by an absorption maximum at -270 nm and a 1:1 CuL~
between the A~60 and Azso values- The value of A26c and A280 in the W ~ LL u u of the ~u~j uyate was increased by a ~actor of 1. 5 when ~ ~~ with DNA alone and protein alone respectively. From this result, the yield of the 1:1 c;vl~3uyate of 508.CF and anti-TSH was calculated to be 1.6 nmoles (13~ overall from r~ mide 508.CF).
The 6u..juy~lLes thus ~Lud,~,ed can be covalently linked to NHS-DMAE rh~-m; ll-mlne-cc--nt label by the previously deDcribed ~Lvc~-luL~c, among others.

.

Claims (35)

What is claimed is:

1. An acridinium ester of the following formula:

wherein R1 is alkyl, alkenyl, alkynyl, aryl or aralkyl, having up to 24 carbons and up to 20 heteroatoms selected from the group consisting of nitrogen, oxygen, phosphorous and sulfur; and R2, R3, R5, and R7 are hydrogen, amino, hydroxyl, halide, nitro, -CN, -SO3H, -SCN, -R, -OR, -NHCOR, -COR, -COOR, or -CONHR, wherein R is alkyl, alkenyl, alkynyl, aryl, or aralkyl, having up to 24 carbons and up to 20 heteroatoms selected from the group consisting of nitrogen, oxygen, phosphorous, and sulfur; and R4 and R8 are alkyl, alkenyl, alkynyl, aralkyl or alkoxyl having up to 8 carbons, with no branching wherein the side chain groups have more than 2 carbons;
and R6 represents the following substitutions: R6 = R9 -where R9 is not required but optionally can be an alkyl, or aralkyl group having up to 5 heteroatoms which can be P, S, N, or O, and R10 is an electrophile, a leaving group, a group with these two combined natures, or selected from the following structures;

-N3, a halide, -COOH

Where Y is a halide and R is alkyl, aryl, aralkyl group; and where one position selected from Rx, Ry and Rz is R6 while the other two positions are R5 and R7.

2. An acridinium ester of claim 1 where R1 is a sulfopropyl or sulfoethyl group; R2 is a hydrogen, methoxy, ethoxy, nitro or halogen; R3, R5, and R7 are hydrogen; R4 and R? are methyl, ethyl or isopropyl groups; and R6 is N-succinimidyloxycarbonyl, N-succinimidyloxycarbonylalkyl, or carboxylate.

3. An acridinium ester conjugate of claim 1 where the acridinium ester is conjugated directly or indirectly to a compound or macromolecule.

4. An acridinium ester conjugate of claim 3 where the conjugation.

occurs by bifunctional cross-linker.

5. An acridinium ester conjugate of claim 3 where the conjugation occurs by hexyl-1,6-diamine, ethylene diamine, or aminocaproic acid.

6. An acridinium ester conjugate of claim 3 where the macromolecule is selected from the group consisting of protein, peptide, inactivated protein, DNA, RNA, oligonucleotide, polysaccharide, neurotransmitter, hormone, steroid hormone, virus, bacterium, toxin and cytokine.

7. An acridinium ester conjugate of claim 6 where the protein is selected from the group consisting of antibody, antibody fragments, avidin, streptavidin, allergen, receptor protein, DNA binding protein, inactivated protein, neurotransmitter, hormone, viral antigen, bacterial antigen, toxin and cytokine.

8. An acridinium ester conjugate of claim 3 where the compound is a hapten or small biologically active molecule.

9. An acridinium ester conjugate of claim 8 where the hapten is a steroid hormone.

10. An acridinium ester conjugate of Claim 9 where the steroid hormone is testosterone, and the bridge and linker arm are connected by a C-19-C linkage, olefinic C-19 linkage or C19-O linkage.

11. An acridinium ester conjugate of Claim 9 where the steroid hormone is testosterone, and where the conjugation occurs by hexyl-1,6-diamine, ethylene diamine, or aminocaproic acid.

12. An acridinium ester conjugate of claim 9 where said steroid is a testosterone derivative selected from the group consisting of StructuresI through VII

wherein A is a functionalized group selected from the group consisting of -OH, -N(R1)2, -NHR1, nitro, halide, -OR1, -OCOR1, -Tosylate, -mesylate, -azide, -isocyanate, -thioisocyanate, -SR1, nitro, -SH, -SSR1, -phosphonate, -sulfonate, -NHCOOR1, -NHCOR1, -NHCONH2, hydrazines, cyanide, and -R1; where R1 is an alkyl, alkenyl, alkynyl, aralkyl containing up to 10 carbons and up to 5 heteroatoms; where B is -(CH2)n where n = 1-4 or -C=;
when B is -C=, A is preferably omitted; where X is a carboxylate, -COOR2, -CONHR2, (where R2 is an alkyl, alkenyl, alkynyl, aryl, aralkyl containing up to 15 carbons and up to 5 heteroatoms), or a carbonyl attached with proper leaving groups, including but not limited to halide, N-hydroxysuccinimide, and imidazole.

13. An acridinium ester conjugate of claim 3 wherein said macromolecule is an infectious agent or pathogen.

14. A method of synthesis of the precursor of the acridinium ester of claim 1 where is reacted with a compound of the formula where R2,R3,R5, R7 are hydrogen, halide, nitro, -R, -OR, -CN,-NHCOR, -COR, -COOR, or -CONHR, where R is alkyl, alkenyl , alkynyl, or aralkyl, and where R4 & R8 are R and R is as defined above.

15. A method of claim 14 for the synthesis of DMAeE-NHS, wherein R4 and R8 are methyl and R2, R3, R5 and R7 are hydrogen.

16. A method of measuring the amount of analyte in a sample comprising a. detecting an analyte specifically and in proportion to its concentration in a sample, and b. measuring a signal which is generated by the chemiluminescent, hydrophilic acridinium ester of claim 1 that is directly or indirectly proportional to the concentration of an analyte in a sample.

17. A method of claim 16 for detecting an analyte in a sample comprising a. contacting a sample with a hydrophilic acridinium ester labeled detector molecule, said detector molecule optionally being a complex of two or more molecular entities, b. sequestering bound detector and analyte, c. washing away excess detector, and d. measuring a signal from an analyte bound detector.

18. A method of claim 16 for detecting an analyte in a sample comprising a. contacting the sample with a hydrophilic acridinium ester labeled competitive tracer, and a specific binder for an analyte, b. retrieving the specific binder, and c. determining a signal generated by the bound tracer.

19. A method of claim 16 for detecting an analyte in a sample comprising a. contacting a sample with a hydrophilic acridinium ester labeled competitive tracer, and a specific binder for an analyte, b. retrieving the specific binder, and c. determining a signal generated by the unbound tracer.

20. A method of claim 16 for detecting an analyte in a sample comprising a. contacting a sample with a first specific binder, and a second hydrophilic acridinium ester labeled specific binder, and b. detecting a signal.

21. A method of claim 16 for detecting an analyte in a sample comprising a. contacting a sample with hydrophilic acridinium ester labelled detector and a competitive binder to the detector, b. sequestering the detector bound to the competitive binder, c. washing away excess detector and the detector bound analyte, and d. measuring a signal from the detector bound to the competitive binder.

22. A method as in claim 21 where a releasing agent is used.

23. A method as in claim 21 where no releasing agent is used.

24. An acridinium ester conjugate of claim 3 wherein an oligonucleotide is conjugated to a macromolecule, said macromolecule being further conjugated to multiple acridinium esters and optionally multiple hydrophilic polymers.

25. An acridinium ester conjugate of claim 24, wherein said oligonucleotide is a gene probe.

26 The acridinium ester of claim 1 which contains a hydrophilic moiety, said hydrophilic moiety comprising a highly ionizable group.

27. The acridinium ester of claim 26 wherein said ionizable group is sulfate, sulfonate, phosphate, or phosphonate.

28. The acridinium ester of claim 1 wherein said R1 group is sulfopropyl or sulfoethyl.

29. The acridinium ester of claim 1 wherein said R2 or R3 is a substituted or unsubstituted aryl fused to said acridinium moiety.

30. The acridinium ester of claim 1 wherein said aryl in R2 or R3 can be a benzene ring fused to the acridinium nucleus to form a benz[a]-acridinium ester, benz[b]-acridinium ester, or benz[c]-acridinium ester.

31. The acridinium ester conjugate of claim 8 wherein said hapten is an aminoglycoside.

32. The acridinium ester conjugate of claim 8 wherein said hapten is a therapeutic drug, steroid, or a controlled chemical substance.

33. The acridinium ester conjugate of claim 13 wherein said infectious agent is selected from the group consisting of Rubella, Hepatitis of various classes and subtypes, Toxoplasma, Cytomegalovirus, HIV, and Chlamidya.

34. The acridinium ester of claim 24 wherein said hydrophilic polymers are polyethylene glycol.

35. A testosterone derivative from the group consisting of Structures I
through VII, said derivative being an intermediate in the synthesis of the compound of claim 12, wherein A is a functionalized group selected from the group consisting of -OH, -N(R1)2, -NHR1, nitro, halide, -OR1, -OCOR1, -Tosylate, -mesylate, -azide, -isocyanate, -thioisocyanate, -SR1, nitro, -SH, -SSR1, -phosphonate, -sulfonate, -NHCOOR1, -NHCOR1, -NHCONH2, hydrazines, cyanide, and -R1; where R1 is an alkyl, alkenyl, alkynyl, aralkyl containing up to 10 carbons and up to 5 heteroatoms; where B is -(CH2)n where n = 1-4 or -C=;
when B is -C=, A is preferably omitted; where X is a carboxylate, -COOR2, -CONHR2, (where R2 is an alkyl, alkenyl, alkynyl, aryl, aralkyl containing up to 15 carbons and up to 5 heteroatoms), or a carbonyl attached with proper leaving groups, including but not limited to halide, N-hydroxysuccinimide, and imidazole.