CA2186998A1 - Use of protein-coding nucleic acid fragments as labels in immunoassays and nucleic acid hybridization assays - Google Patents

Abstract

An assay is carried out in which an analyte reacts with a reagent that is labelled by a nucleic acid fragment, preferably a DNA fragment, that expresses a protein. Preferably the expressed protein is an enzyme.
The analyte is detected or determined quantitatively by expressing the nucleic acid fragment and monitoring of the expressed protein.

Description

2 186~98 The present lnventlon relates to a novel method of lmmunoassay or nuclelc acld hybrldlzation assay of hlgh sensltlvlty.
Backqround of the Inventlon Immunoassay 1~ a powerful analytlcal technl~ue used wldely both ln the lnvestlgatlon of the flne structure and functlon of blologlcal systems and ln the cllnlcal laboratory for dlagnosls and monltorlng of varlous dlseases. In recent years, slgnlflcant advances have emerged ln lmmunoas~ay reagents lantlbodles) and detectlon systems. Progress ln antlbody englneerlng has allowed the productlon of antlbodle~ endowed wlth novel propertles, such as blspeclflc and catalytlc antlbodles. In parallel, conslderable efforts have been focused on the lmprovement of lmmunoassay sensltlvlty. Hlghly sensltlve lmmunoassays are expected to provlde valuable lnformatlon on antlgens found at levels that are close to or below the current assay detectlon llmlt ~e.g., antlgens lnvolved ln early steps of slgnal transductlon pathways), allow the monltorlng of tumor-speclflc products ln blood ~for early detectlon of relapse), and facllltate the sesrch for new dlagnostlc markers of dlsease. Sensltlvlty 18 determlned malnly by the detectablllty of the molecules used for antlbody labelllng. Radloactlve labels domlnated ln the lmmuno~ay fleld for at least 2 decades. However, the current trend 18 toward novel nonlsotoplc systems wlth superlor sensltlvltles. Nonlsotoplc lmmunoassays based on fluoroescent, chemllumlnescent, or enzyme labels have been developed and are commerclally avallsble. A problem sometlmes encountered when an enzyme 18 dlrectly llnked to an antlbody 18 that actlvlty of the antlbody or of the enzyme, or both, 18 destroyed. Thls happens, for example, wlth the enzyme luclferase.
Recently a hlghly sensltlve detectlon system, termed lmmuno-PCR (polymerase chaln re~ctlon), has been reported, see Sano, T., Smlth C.L., Cantor, C.R. Sclence 2186~98 1992 258 120-122. Immuno-PCR repllcates a DNA label, thus generatlng a large number of DNA coples whlch are then detected by electrophoresls.
8ummarY of the Inventlon In one aspect, the present lnventlon provldes a method for assaylng for an analyte, whlch method comprlses labelllng the analyte wlth a nuclelc acld fragment, prefer-ably a DNA fragment, that codes for a proteln, expresslng the nuclelc acld fragment that labels the analyte and detectlng the presence of the proteln that has been expressed by the nuclelc acld fragment.
In one embodlment of the lnventlon the analyte 18 an antlgen, the nuclelc acld fragment 18 attached to an antlbody and the analyte 18 labelled by means of the antlgen-antlbody reactlon. In another embodlment the analyte 18 a nuclelc acld sequence (target) and the nuclelc acld fragment 18 attached to a nuclelc acld probe that wlll hybrldlze wlth the target nuclelc acld sequence. In thls case the analyte 18 labelled by the hybrldlzatlon reactlon.
The amount of proteln expressed 18 dlrectly proportlonal to the amount of analyte detected by a reagent bearlng the nuclelc acld fragment, 80 the method permlts quantltatlve determlnatlon of the amount of analyte present. Thus lf the amount of expressed proteln 18 determlned there 18 provlded a dlrect quantltatlve measure of the amount of analyte.
Expresslon of a DNA fragment or template lnvolves transcrlptlon and translatlon. In transcrlptlon there wlll be formed from each DNA fragment several ldentlcal RNA
molecules. In translatlon there wlll be formed from each RNA molecule several ldentlcal proteln molecules. Hence there wlll occur an ampllflcatlon. Furthermore, lf the proteln 18 an enzyme and lf the enzyme 18 detected by means of lts catalytlc actlvlty on a substrate, there wlll occur further ampllflcatlon from the substrate turnover. Hence the lnventlon provldes an assay method of hlgh sensltivlty.

2 1 8~8 Detectlon of the expre~sed protein provldes a qualltatlve lndlcatlon of the presence of the analyte. Measurlng the quantlty of proteln expressed provldes a quantltatlve determlnatlon of the analyte.
Although lt 18 preferred to use a DNA fragment and thereby achleve ampllflcatlon ln both the transcrlptlon and translatlon stage~, lt 18 posslble to use a nuclelc acld fragment other than a DNA fragment, provlded that the fragment code~ for a proteln that wlll be expressed. Thus lt 18 posslble to use RNA as the nuclelc acld fragment.
It 18 posslble to carry out dual or multlanalyte assays lf, for example, two or more antlbodles, or two or more probes, speclflc for two or more dlfferent analytes are labelled wlth dlfferent nuclelc acld fragments that express two or more dlfferent protelns that can be detected separately. This conflguratlon permlts quantltatlve determlnatlon of more than one analyte ln the same sample.
Dlscusslon of the Preferred Embodlments An expresslble nuclelc acld fragment, preferably a DNA fragment, 18 used as a label of antlgens or antlbodles for the development of lmmunoassays, and as a label for nuclelc acld fragments ln a nuclelc acld hybrldlzatlon assay. The DNA fragment that serves as the template that expresses the detectable proteln sultably comprlses, (a~ an RNA polymerase promoter, ~b) a sequence that allows blndlng of rlbo~omes and lnltlatlon of translatlon of the produced RNA, and (c) a sequence that encodes an enzyme or other proteln whlch 18 detectable wlth hlgh sensltlvlty (e.g., a photoproteln). The DNA fragment can also lncorporate other approprlate ~equence~, for example promoter, enhancer or termlnatlon sequences, to enhance yleld durlng expresslon. The DNA label can be quantltated wlth hlgh sensltlvity, for example by measurlng enzymatlc actlvlty produced after expres~lon (transcrlptlon and translatlon). Transcrlptlon entalls synthesl~ of several RNA molecules for each DNA label. Translatlon, ln turn, produces more than one proteln molecule for each RNA.
Thus, many enzyme molecules can be syntheslzed from a slngle DNA label. Enzymatlc actlvlty i8 measured by uslng hlghly sensltlve (e.g., chemllumlnescent or fluorescent) reactlons. The hlgh sensltlvlty of the system 18 a result of the comblned ampllflcatlon due to transcrlptlon/
translatlon and the substrate turnover.
The mlnlmum length of the nuclelc acld fragment that serves as a template codlng for the expressed proteln 18 the mlnlmum length that contalns ~ll the sequences requlred to achleve the requlred expresslon. In most cases thls means that the fragment wlll be a lOOmer or hlgher, although a shorter sequence can be u~ed provlded that lt carrles out lts requlred functlon. There 18 no upper llmlt, although as the fragment lncreases ln length lt wlll become cumbersome. It 18 llkely that ln most cases the nuclelc acld fragment wlll not contaln greater than about 20 kllobases (kb), and often lt wlll contaln 10 kb or less.
The expresslon of the nuclelc acld fragment can be cell-free (ln vltro~. Op~lmum condltlons for transcrlptlon may dlffer from optlmum condltlons for translatlon. It 18 posslble to carry out transcrlptlon and translatlon ln two separate steps, each under optlmum condltlons and posslbly wlth a purlflcatlon step after transcrlptlon and before translatlon. There are commer-clally avallable ~lts for carrylng out cell-free transcrlp-tlon and translatlon sep~rately or together ln one step.
All these posslbllltles are wlthln the scope of the lnventlon. In some preferred embodlments, and to enhance the practlcallty of the system, expresslon of the DNA 18 accompllshed ln a slngle ~tep (coupled transcrlptlon/
translatlon).
An RNA label sultably comprlses (a) a sequence that allows blndlng of rlbosomes and lnltlatlon of translatlon and (b) ~ sequence that encodes an enzyme or other proteln whlch 1~ detectable wlth hlgh sensltlvlty.

2 1 86 ~9~

The RNA can al80 lncorporate other approprlate sequences, for example, enhancer or termlnatlon sequences.
An lmmunoassay for antlgens that 18 typlcal of one embodlment of the lnventlon 18 deslgned as follows.
The analyte, whlch 1~ an ~ntlgen from a sample, 1~ captured by two speclflc antlbodles, one belng lmmoblllzed on a solld phase (e.g., mlcrotlter wells or bead~) and the other (detectlon antlbody) con~ugated to an enzyme-coding DNA
fragment that serve~ as a label. After removal of the excess of reagents, the DNA bound to the lmmunocomplex 18 sub~ected to transcrlptlon and translatlon and the enzymatlc actlvlty 18 monltored.
Alternatlvely, the detectlon antlbody 18 labelled, for example wlth blotln, and, after completlon of the lmmunoreactlon, a streptavldln DNA con~ugate or an avldln DNA con~ugate ((strept)avldln DNA con~ugate) 18 added, complexes wlth the blotln-labelled antlbody and the con~ugated DNA fragment 18 sub~ected to transcrlptlon and translatlon as above.
A hybrldlzatlon assay can be deslgned as follows~
The nuclelc acld target (DNA or RNA) 18 hybrldlzed wlth two speclflc probes, one (the capture probe) belng lmmoblllzed on a solld phase and the other (the detectlon probe) labelled wlth blotln or a hapten. The solld phase can typlcally be a membrane of, for example, nltrocellulose or nylon, or a mlcrotlter well or beads of poly~tyrene. Then, the hybrlds are reacted wlth a (~trept~avldln-DNA con~ugate or an antl-hapten antlbody-DNA con~ugate. The DNA label 18 sub~ected to transcrlptlon~translatlon and the enzymatlc actlvlty produced 18 monltored.
Important appllcatlons of the lnventlon lnclude the followlng.
~ 1~ Analysls of antlgens or nuclelc acld~ found at levels that are close to or below the current assay detectlon llmlts, e.g., tumor-~peclflc products ln blologlcal fluld~ for monltorlng of cancer patlent~.

- 21~6~98 ~ 11) Analysls of mutatlons of nuclelc aclds, assoclated wlth genetlc dlsease.
~ 111) Analysls of speclflc antlgens and~or nuclelc aclds of pathogens for dlagnosls and monltorlng of 5 lnfectlous dlsease~.
(lv) As an analytlcal tool lt can be used ln the search for new dlagnostlc marker8 of dlsease.
It 18 preferred that the nuclelc acld fragment that expresses a proteln 18 a DNA fragment that expresses 10 an enzyme. It 18 pOBBlble to use a proteln that 18 not sn enzyme, however, provlded that the proteln 18 readlly detectsble ln small quantltles. ~xamples of such non-enzymatlc protelns lnclude aequorln (Wltkowskl et al, Analytlcal Cheml~try, Vol. 66, No. ll, June 1, 1994, p.
1837-1~40) and green fluorescent protelns (Kaln et al, ~lotechnlques, Vol. 19, No. 4, 1995 pp. 650-655). These protelns can be determlned accurately and ln small quantltles by means of thelr photo propertle~. The dlsclosures of these two papers are lncorporated hereln by 20 reference.
Enzymes to be expressed by the nuclelc acld fragment preferably have one or more of the followlng characterlst lc8 1 (a) They dlsplay full enzymatlc actlvlty wlthout any 25 requlrement for post-translatlonal modlflcatlonl (b) They can be readlly monltored ln the translatlon mlxture~
(c) The enzyme conslsts of a slngle polypeptlde chaln or 18 an ollgomer of ldentlcal BUbUnlt 8 ~ and ~0 (d) They are expressed by a short nuclelc acld fragment. An enzyme that dlsplays these characterlstlcs, and 1B therefore partlcularly preferred, 1~ luclferase.
Luclferase has a ~lngle polypeptlde chaln (550 amlno acld~) and cataly~e~ reactlon of the substrate luclferln wlth ~2 ~5 and ATP to glve off llght whlch can be measured quantlta-tlvely wlth, for example, a slngle photon counter or a - 218~9~

lumlnometer. Other enzymes that can be used lnclude, for example, alkallne phosphata~e, whlch can be detected quantltatlvely, for example, wlth a chemllumlnescent 8ub8trate 8uch as AMPPD (4-methoxy-4-(3-phosphonophenyl)-splro[l,2-dloxetane-3,2'-adamantane])~ see Ishll et al, Blocon~ugate Chem. 1993, 4, 3g-41, and ,B-galactosldase, whlch can be detected by lts reactlon wlth, for example, a chemllumlnescent substrate such a8 AMPGD ~3-~4-met~oxy-splroll,2-dloxetane-3,2'-tricyclo[3.3.1.1]decan]-4-yl)-phenyl-~-D-galactopyranoslde)~ see Jaln et al, Analytlcal Blochemlstry, 199, 119-12g (1991). The dlsclosures of these papers are lncorporated hereln by reference.
In one embodlment the nuclelc acld fragment codes for a peptlde or proteln that 18 ltself lnactlve but 18 actlvated when lt 18 ln comblnatlon wlth a complementary peptlde or proteln. The complementary peptlde or proteln 1Q also lnactlve by ltself. When the complementary pair meet they form a fully actlve enzyme and can be detected quantltatlvely by means of that enzymatlc actlvlty. An example 18 the nuclelc fragment encodlng for the a-peptlde of ,B-galactosldase and the complementary proteln 1B 13-galactosldase that lacks the ~-peptlde~ see for example Langley et al, Proc. Nat. Acad. Scl. U.S.A. Vol. 72, No. g pp. 1254-1257, Aprll 1975 and see also Langley et al, Blochemlstry, Vol. 15, No. 22, pp. 4866-4875, 1976, the dlsclosures of whlch are lncorporated hereln by reference.
By thls means lt 18 posslble to use a relatlvely small fragment of nuclelc acid to express a relatlvely small proteln that 18 then detected by the enzymatlc actlvlty of a much larger molecule. A nuclelc acld fragment codlng for the complete molecule would be very large and cumber~ome.
Nuclelc acld fragments (~NA or RNA) can be llnked to antlbodles or to other nuclelc acld sequences (probes) covalently or non-covalently. One preferred non-covalent method of llnklng an antlbody or a nuclelc acld probe to a nuclelc acld fragment lnvolves use of a blotln-(6trept)-2~ 86~98 avldln con~ugate. Avldln 18 a proteln of about 60 kllo-Dalton~ molecular welght that 18 available from raw egg whlte of blrds and amphlbla. Streptavldln 18 a slmllar proteln avallable from a streptococcus. The proteln 18 a glycoproteln that has four ldentlcal subunlts, each of whlch wlll blnd strongly but non-covalently wlth a molecule of blotln. In a preferred embodlment of the assay, there-fore, an antlbody or a nuclelc acld probe ls reacted flrst Wlth blot in . Thereafter there 1~ added to the blotlnylated antlbody or blotlnylated probe a nuclelc acld fragment that has been reacted wlth blotln and then wlth avldln or streptavldln ~sometimes referred to hereln as ~strept)-avldln). The nuclelc acld fragment 18 thereby llnked Wlth the antlbody or probe ln an antlbody- (or probe-) blotln-(strept)avldln-blotln-nuclelc acld con~ugate. The antlbody or the nuclelc acld probe 1~ blotlnylated before lt 18 reacted wlth the analyte. Provlded that the antlbody or the probe ha~ been ~lotlnylated, the further reactlon wlth (strept)avldln llnked to the nuclelc acld fragment can take place before or ~fter the antl~ody or the probe ha~ re~cted wlth the anslyte. Other non-covalent llnklng systems that can be used are sugar-lectln con~ugates and proteln A.
Nuclelc acld fragments (DNA or RNA templates) can be llnked covalently to antlbodles or to other nuclelc acld sequence~ (probes). An amlno group 18 lntroduced to the nucleic ~cld template, preferably at the end of the template. Thls can be accompllshed, for example, by uslng termlnal deoxynucleotldyl transfera~e ln the presence of amlnohexyl dATP. The amlno group 18 then thlolated u~lng N-succlnlmldyl-S-acetylthloacetate. Malelmldyl groups are added to the antlbody or to an amlno-modlfled nuclelc acld probe uslng sulfo-SMCC, avallable from Plerce, (Rockford, Illlnols). The con~ugatlon reactlon between the thlolated amlno-nuclelc acld template and the malelmldyl-antlbody or the malelmldyl-nuclelc acld probe 1B lnltlated by addlng hydroxylamlne to uncover the -SH groups on the nuclelc acld : ' .

~1 86~98 label. Purlflcatlon and characterlzatlon of t~e con~ugate can be done, for example, by chromatogr~phlc methods.
In the case of a nuclelc acld hybrldlzatlon assay the expresslble nuclelc acld fragment can be part of the nuclelc acld sequence that also lncludes the nuclelc acld probe that hybrldlze~ wlth the target DNA or RNA. Alterna-tlvely, two nuclelc acld sequences, one the detectlon probe and the other the expre~slble template, can ~e llnked ln any sultable manner, lncludlng the covalent and non-covalent llnklng me~ns dlscussed above wlth reference tothe lmmunoassay.
Brlef DescrlPtlon of the Drawlnqs Flgure 1 18 a schematlc lllustratlon of one embodlment of the lnventlon, lllustratlng by way of example an lmmunoassay of the lnventlon~
Flgure 2 18 a schematlc lllustratlon of one embodlment of the lnventlon, lllustratlng by way of example a nuclelc acld hybrldizatlon assay of the lnventlon~
Flgure 3(a) shows a plasmld contalnlng the T7 promoter and the codlng ~equence for the enzyme luclfer~se Flgure 3(b) shows the structure of the DNA
template that ls used as a label~
Flgure 3(c) shows the analysls of the dlge~ted plasmld by 0.7~ agarose gel electrophoresls and ethldlum bromlde stalnlng~
Flgure 4 ~hows quantltatlon of luclferase codlng DNA by transcrlptlon/translatlon. It 18 a graph of lumlnescence versus DNA template molecul'es~
Flgure 5 18 a graph of lumlnescence and tlme, lllustratlng the tlme dependence of the expresslon of an lmmoblllzed luclfera~e codlng DNA fragment~
Flgure 6 18 a graph of lumlnescence and number of antlgen molecule~, lndlcatlng a llnear relatlon~hlp~
Flgure 7 18 a graph showlng the relatlonshlp between lumlnescence and hybrldlzatlon tlme ln a nuclelc -~1 86~8 acld hybrldlz~tlon assay uslng DNA th~t expre~ses the enzyme luclferase as label~ and Flgure 8 18 a graph showlng the relatlonshlp between lumlne~cence and the concentratlon of streptavldln-DNA complex ln the nuclelc acld hybridlzatlon assay uslngDNA that expresses the enzyme luclfer~e as label.

Detslled Dlscusslon of the Inyentlon.

In the followlng detalled dlscusslon the lnven-tlon 18 descrlbed partlcularly wlth reference to an lmmunoassay and to a hybrldlzatlon assay, both carrled out uslng a DNA fragment that 1~ attached to an antlbody or probe by means of blotln and streptavldln and that expresses the en~yme luclferase. It should be appreclated, however, that the lnventlon 18 not conflned to the u~e of the~e materlals.

One embodlment of an lmmunoassay ln accordance wlth the lnventlon 18 lllustr~ted ln Flgure 1. Antlgen lmmoblllzed ln, for example, mlcrotlter wells 18 allowed to react wlth a blotlnyl~ted ~peclflc antlbody. The lmmunocomplex 18 then re~cted wlth streptavldln-DNA

template. The solld phase-bound DNA 18 sub~ected to a coupled (one-step) transcrlptlon/tr~nslatlon th~t produces enzyme molecules, whlch are subsequently detected by addlng the approprlate substrate.

One sultable con~ugatlon complex for att~chlng the DNA fragment to ~he lmmoblllzed antlbody 18 the blotln-(strept)avldln complex. As lllustrated below, blotlnylated DNA can be con~ugated wlth streptavldln and thereafter llnked vla the streptavldln to a blotlnylated antlbody for the requlred antlgen. Alternatlvely, elther blotln or blotln-(strept)avldln ls ~ttached to the antlbody and, correspondlngly, blotln-(~trept)avldln or blotln 18 attached to the DNA fragment. Formatlon of a complex between blotln and (strept)~vldln then crestes the requlred llnkage between the antlbody and the DNA fragment. The (~treptl~vldln-blotln complex can be formed, l.e., the DNA

21 86~g~ ' fragment can be llnked to the antlbody, elther before or after the antlbody complexes wlth the antlgen analyte.
One embodlment of a nuclelc acld hybrldlzatlon assay 18 lllustrated ln Flgure 2. An antlbody 18 lmmobll-lzed on a surface, for example ln a mlcrotlter well.Attached to the antlbody vla a hapten H, for example dlqoxlgenln, 18 a nuclelc acld probe. The probe 18 labelled wlth the hapten by means of an enzymatlcally applled tall st the 3' end of the probe. The tsrget nuclelc acld hybrldlzes wlth the lmmoblllzed probe, thereby becomlng lmmoblllzed ltself. After washlng there 18 sdded a further nuclelc acld probe, whlch probe a1BO hYbr1d1Ze8 wlth a portlon of the target nuclelc acld snd whlch probe 18 attached, vla a blotln-streptavldln-blotln con~ugate to a DNA template, whlch DNA template wlll express an enzyme that 18 detectable quantltatlvely when reacted wlth an approprlate substrate.
A probe, ln the chemlcal and blologlcal ~ense, 18 a molecule havlng a strong lnteractlon wlth a speclflc target and havlng a means of belng detected followlng the lnteractlon. The probes used ln the present lnventlon are nuclelc acld~ that hybrldlze wlth other nuclelc aclds. If the probes are double stranded DNA then they are readlly denatured by heat 80 that they can hybrldlze. ~ssentlal characterlstlcs of the probe are speclflclty and afflnlty.
The probe therefore has to contaln sufflclent nucleotldes to dlsplay the requlred speclflclty and afflnlty. In most cases a ~equence ln the range of about 20 nucleotldes to several kllobases wlll be requlred.
In an assay of the lnventlon, the label can be a DNA fragment that encodes an enzyme. The flrefly luclfera~e-codlng DNA 18 chosen as a model.
In one embodlment of the.lnventlon, an lmmuno-reactlon 18 carrled out wlth blotlnylated antlbodle~ whlch, after the lmmunoreactlon 1~ completed, are attached to the blotlnylated DNA template uslng streptavldln as a ~'brldge"

2 1 869~8 molecule. The DNA 18 then ~ub~ected to coupled cell-free transcrlptlon/translatlon, whlch generate~ several luclferase molecules. Luclferase catalyzes the reactlon of luclferln, ATP, and ~2 to produce oxyluclferln, AMP, pyrophosphate, C02, and llght. The lumlne~cence 18 proportlonal to the number of antlgen molecules present.
The assays can be conflgured ln a varlety of dlfferent ways, both competltlve and non-competltlve. An analyte can be lmmoblllzed on a surface ln a varlety of ways. One way, for lnstance~ 18 the sandwlch assay conflguratlon, ln whlch ~n antlbody (a capture antlbody) 18 attached to a surface, all posslble attachment sltes on the surface are thereafter blocked, a ~ample that may contaln an antlgen to the lmmoblllzed antlbody 18 then brought lnto contact wlth the surface bearlng the lmmoblllzed antlbody and, lf the ~ample contalns the antlgen there 18 formed an antlbody-antlgen complex. The ~olld phase 18 washed and thereafter there 1~ applled a detectlon solutlon contalnlng a further antlbody (a detectlon antlbody) for the antlgen, whlch detectlon antlbody 18 labelled wlth the nuclelc acld fragment or template that expresses the proteln.
In another conflguratlon a ~ample that may contaln an antlbody 1B applled to a surface under condl-tlons such that the antlbody, lf present, wlll blnd to the ~urface. After wa~hlng, a ~olutlon contalnlng labelled antlgen 18 applled and lf antlbody 18 present there 18 formed and can be detected a con~ugate of antlbody and labelled antlgen.
The same conflguratlons can be used wlth an lmmoblllzed nuclelc acld sequence (capture probe) and free nuclelc acld sequence (detectlon probe). The capture probe can be lmmoblllzed ln a varlety of way~. One way, lllustrated ln detall below, 18 to use an lmmoblllzed antlbody and a nuclelc acld probe to who~e 3' end there 1~
llnked a hapten, by means of an enzymatlcally applled tall.
The hapten lnteracts wlth the lmmoblllzed antlbody, thereby ~18~9~

lmmoblllzlng the nuclelc acld capture probe. Alternstlvely the probe can be covalently attached to a solld pha~e wlthout lnterventlon of a hapten. For example 5'-amlnohexyl labelled probe~ can be attached covalently to carboxylated poly~tyrene, e.g., polystyrene beads, uslng 1-ethyl-3-13-(dlmethylamlno)-propyl]dllmlde.HCl as coupllng reagent. Also, see Rasmussen et al, Analytlcal Blochemlstry 198, 138-142 (lg91), the dlsclosure of whlch 18 lncorporated hereln by re~erence. Thls descrlbes covalent bondlng of DNA onto Covallnk NH, a type o~
mlcrowell plate produced by A/S Nunc (Denmark). A "handle"
(M, 200) wlth a spacer arm endlng ln a secondary amlno group 18 grafted covalently onto a polystyrene ~urface as a solld phase for covalent lmmoblllzatlon and hybrldlzatlon of DNA. Furthermore, the target nuclelc acld can be lmmoblllzed dlrectly on the surface (e.g. as descrlbed ln the above reference). A probe carrylng the expresslble nuclelc acld label hybrldlzes to the target. In thls case only one probe 18 requlred for the detection and quantlta-tlon of the target nuclelc acld sequence.
In another conflguratlon a detectlon antlbody that 18 labelled wlth a nuclelc acld fragment and has been ralsed agalnst an antlgen expressed from cells can be used to ldentlfy the lmmoblllzed cells.
The lnventlon can be used ln competltlve a~says, used to detect the presence of an analyte ln small quantlty. A llmlted number of, say, antlbody sltes are provlded on a surface. A known sample of blotlnylated antlgen 18 applled ln excess to saturate all antlbody sltes, the sltes washed to remove excess antlgen. Thelmmunocomplex 18 then reacted wlth a streptavldln-nuclelc acld template con~ugate. The slgnal prov~ded by expresslon of the template 18 observed, thereby, monltorlng the generated proteln. In a sub~equent experlment wlth, agaln, llmlted antlbody sltes, blotlnylated antlgen ln admlxture wlth a sample that may contaln the unlabelled antlgen 18 2 1 86~

applled, washed and the slgnal provlded by proteln expres~lon 18 observed. Unlabelled antlgen ln the ssmple will compete wlth labelled antigen for the llmlted number of antlbody sltes and the antlbody sltes wlll be occupled by labelled or unlabelled sntlgen ln proportlon to the concentratlon of labelled and unlabelled antlgen. As only labelled antlgen wlll glve a ~lgnal, the dlmlnutlon of the slgnal, as compared wlth the case where no unla~elled antlgen i8 present, provldes a measure of the concentratlon of tne unlabelled antlgen ln the sample.
The lnventlon 18 further lllustrsted ln the followlng examples.
BXP~RIM~NTAL 8~CTION
In~trumentatlon. Lumlnescence measurements were carrled out uslng a llquld sclntlllatlon counter (Model LS-6500, ~eckman Instruments Inc., Fullerton, CA) ln the slngle-photon monltorlng mode. An lmaglng den~itometer (Model G~-670, Blo-Rad Laboratorles Ltd., Mlsslssauga, Canada), along wlth the Molecular Analyst verslon 1.0 software, was used for quantltatlon of DNA fragments after agarose gel electrophoresls. The mlnlature horlzontal gel system MLB-06 from Tyler Research Corp. (Edmonton, Canada) W~8 used for electrophore~ls. Tlme-resolved fluorescence was measured wlth the CFl 615 Immunoanalyzer from CyberFluor Dlvlslon, Nordlon Internatlonal lToronto, Canada). Bxcltatlon and emlsslon wavelengths were set at 337 and 615 nm, respectlvely. Hlgh-performance llquld chromatography (HPLC) was performed using the Shlmadzu sy~tem ~Shlmadzu Corp., Kyoto, Japan~ wlth absorbance monltorlng. ~ G24 envlronmental lncubator shaker from New Brunswlck 8clentlflc (Edlson~ NJ) wa~ employed for culturlng bacterla.
Materlal~. For prepar~tlon of a DNA template that encodes luclferase, there was used a plasmld contalned, a~
a control DNA, ln the TNT T7 wheat germ extract, whlch 1 transcrlptlon/translatlon system commerclally avallable -21 86~9~

from Promega Corp. (Madlson~ WI). A schematlc dlagram of the plssmld 18 shown ln Flgure 3~a), whlch shows the restrlctlon sltes of the enzymes Alw44 and PvuII, the T7 promoter and the luciferase codlng sequence Luc. Wlzard maxlprep~ DNA purlflcatlon system and beetle luclferln were also from Promega. Restrlctlon enzymes Alw44 I snd PvuII, ss well as the Geneclean DNA purlflcatlon system, were purchased from BloCan 8clentlflc (Mlsslssauga, Canada).
Ultrapure 2'-deoxyrlbonucleoslde 5'-trlphosphates (dNTPs), the Klenow fragment of the ~scherlchl~ coll DNA polymerase I, and coenzyme A (CoA) were from Pharmacla ~lotech (Montreal, Canada). Llnear DNA markers (A-DNA dlgested wlth EcoRI and HlndIII) contalnlng fragments from 0.12 to 21.2 kbp, supercoiled DNA markers (slzes 2.07-16.2 kbp), streptavldln, magneslum carbonate pentahydrate, and trlclne were from 81gma (~t. Louis, M0). Flrefly luclferase (from Photlnu~ pyrd~ alkallne phosphatase-labelled streptavldln, adenoslne trlphosphate (ATP), bovlne serum albumln, and the "blocking reagent" (Catalog No. 1096 176) were obtalned from Boehrlnger (~aval, Canada). Blotln-14-dCTP (blotln attached at the Ng posltlon of cytldlne by a 14-atom llnker) and "U"-bottom polystyrene mlcrotlter wells (Nunc, Maxlsorp) were obtalned from Llfe Technologles (~urllngton, Canada). Whlte, flat-bottom polystyrene wells, Mlcrollte 2, were from Dynatech Laboratorles Inc.
(Chantllly, VA). Monoclonal antl-thyrotropln antlbody was from Medlx ~lochemlca (Flnland). ~lotlnylated goat antl-mou~e antlbody was from Jackson Immunoresearch Laboratorles, Inc. (dlstrlbuted by BloCan). The phosphate ester of fluorosallcyllc acld (FSAP) was from CyberFluor.
Mlcrocon-30 mlcroconcentrators were purchased from Amlcon Inc. (~everly, MA).
The blocklng solutlon contalned 1% blocklng reagent ln 0.1 mol/L maleate and 0.15 mol/L NaCl, pH 7.5.
The wash solutlon conslsted of 50 mmol/L Trls, pH 7.4, 0.15 mol/L NaCl, and 0.1~ (v~v Tween-20). The phosphate-21 ~6q9~

buffered ~allne ~P~S) contalned 10 mmol/L sodlum phosphate, 1.8 mmol/L potasslum phosphate, 0.14 mol~L NaCl, and 2.7 mmol/L KCl, pH 7.4. The Trls-EDTA (TB) buffer conslsted of 10 mmol/L Trl~ and 1 mmol/L EDTA, pH ~Ø A wheat germ-based tran~crlptlon/translatlon mlxture (wheat germ TNTsy~tem from Promega) was prepared ~ccordlng to the manu-facturer's lnstructlons. The complete mlxture conslsted of wheat germ extract (contalnlng rlbosomes, tRNA, and other tran~lation factors), T7 RNA polymera~e, and amlno aclds ln the approprlate buffer.
Preparatlon and Purlflcatlon of tho DNA Template. For growlng 0. col l JM 109 cells, preparatlon of competent cells, and transformatlon wlth the plasmld DNA, ~tandard procedures were followed~ ~ee Sambrook J.~ Frltsch, E.F.
Manlatls, T. Molecular Clonlng. A Laboratory Manual, 2nd ed.~ Cold 6prlng Harbour Laboratory Press~ Cold Sprlng Harbour, N.Y., 1989, the dlsclosure of whlch 18 lncorporated hereln by reference. Transformed bacterla were grown overnlght ln LB broth (10 g/L tryptone, 5 g/L
yesst extract, 0.17 mol/~ NaCl, and 2 mmol/L NaOH) contalnlng 0.1 g/L amplclllln. The plasmld DNA was purlfled from a 1 L bacterlal culture wlth the Wlzard maxlpreps DNA purlflcatlon system accordlng to the manufacturer's lnstructlons. The slze of the plasmld was conflrmed by agarose (0.7~) gel electrophore~ls and ethldlum bromlde Rtalnlng, u~lng the ~upercolled DNA
markers. The plasmld concentratlon was determlned from the absorbance at 260 nm. For preparatlon of the DNA template, 40 ~g of plasmld DNA was flrst dlgested for 90 mln at 370C
wlth 240 unlt~ of Alw44I ln 150 ~L of dlgestlon buffer (lO
mM Trls-HCl, pH 7.9, 50 mM NaCl, 10 mM MgC12, and 1 mM
dlthiothreltol). Subsequently, a flll-ln reactlon was lnltlated by addlng 150 ~ of a ~olutlon contalning 80 ~mol/L of each of dATP, dGTP, dTTP, and blotln-14-dCTP a~
well as 40 unlts of the Klenow fragment of the DNA
polymera~e I. After lncubatlon for 10 mln at room 2~ 86~98 temperature, the reactlon wa8 termlnated by heatlng the mlxture at 70~C for 5 mln. 8ub~equently, 2gO unlt~ of PvuII were added, followed by a 90 mln lncubatlon at 37~C.
After dlgestlon, the DNA fr~gment~ were separated by agaro~e gel (0.7~) electrophore~l~ and stalned wlth ethldlum bromlde. Flgure 3(c) shows lane 1, llnear DNA
marker~ (1.4 ~g)~ lane 2, pla~mld ~1 ~g) dlgested wlth Alw44 (fragment slzes, 2.59, 1.25 and 0.50 kbp)l lane 3, plasmld (1 ~g) ~fter fllllng-ln reactlon and dlgestlon wlth PvuII ~fragment slzes. 2.10, 1.25 and 0.50 kbp).
The band corre~pondlng td 2.1 kbp was excl~ed, and the DNA was purlfled u~lng the Geneclean purlflcatlon system and recovered ln water. To quantltate the purlfled DNA template, there were performed another electrophoresls and stalnlng, as above. A lane contalnlng the llnear DNA
markers was also lncluded for constructlon of a callbratlon curve. The gel wa~ photographed under UV excltatlon uslng a Polarold 665 fllm, and the negatives were scanned by the densltometer. The structure of the blotlnylated DNA
template 1~ shown ln Flgure 3(b). A slngle blotln molecule has been added down~tream of the luclferase-codlng sequence .-Prepar~tlon ~nd Purlflc~tlon of 8treptavldln-DNA
Templ~te Comple~. The streptavldln-DNA templdte complex was prepared ln a flnal volume of 80 ~L contalnlng 3.6 ~g (2.6 pmol) of ~blotlnylated) DNA templ~te and 5 ~g (84 pmol) of streptavldln ln TE buffer. After lncubatlng for 30 mln at room temperature, the complex ~50 ~L) was purlfled by HPLC u~lng a ~lze excluslon column (the 810-Sll Sec 400-5, 300 mm x 7.8 mm from ~lo-Rad Laboratorle~) lsocratlcally. The moblle pha~e wa~ 50 mM NaH2-P04, 50 mM
Na2HP04, and 150 mM NaCl, pH 6.8. The flow rate was 0.5 mL/mln. Ab~orbance was monltored at 260 nm. A 1 mL
fractlon, corre~pondlng to the vold volume peak, was collected. Next, 100 ~L o~ blocklng solutlon wa~ added (a~
a carrler), and the mlxture wa~ concentrated down to about 21 86~98 60 ~L by u~lng Mlcrocon-30 mlcroconcentrators. A 3 ~L
allquot of thls prep~ratlon was electrophoresed, and the DNA concentratlon was determlned by scannlng densltometry as above ~ee Preparatlon ~nd Purlflcatlon of th~ DNA
Template).
Luclferaee A~say. The substrate solutlon for luclferase contalned 20 mmol/L trlclne, 1.1 mmol/L
magneslum carbon~te pent~hydrate, 2.7 mmol/~ Mg804, 0.1 mmol/L EDTA, 33 mmol/L dlthlothreltol, 270 ~mol/L CoA, 530 ~mol~L ATP, and 470 ~mol/L luclfer~n, pH 7.8. For the luciferase assay, 10 ~L of the sample was added to 50 ~L of ~ubstr~te solutlon ln a mlcrocentrlfuge tube. The tube was placed ln a glass sclntlllatlon vlal, and the lumlnescence was measured for 1 mln uslng the llquld sclntlllatlon counter ln the slngle-photon monltorlng mode.
Quantltatlon of Immoblllzed Antl~en by Bxpresslon Immuno~s~ay. Solutlons wlth varlous analyte concentratlons were prepared by dllutlng a monoclonal antl-thyrotropln antlbody ln coatlng buffer ~0.1 mol~L carbonate buffer, pH
9.6). The an~lyte was lmmoblllzed by plpettlng 25 ~L of solutlon lnto "U" bottom polystyrene mlcrotlter well~ and lncubatlng overnight at 4~C. The wells were then washed once wlth wash solutlon, and the remalnlng blndlng sltes were blocked for 90 mln at room temperature wlth blocklng solu~lon. Afterward, the wells were washed once as above, snd to e~ch well wa~ added 25 ~L of 5 ~g/mL blotlnylated ~goat antl-mouse antlbody, dlluted ln blocklng solutlon.
The lmmunoreactlon was allowed to proceed for 30 mln, and the wells were washed four tlmes to remove the excess blotlnylated antlbody. Next, 25 ~L~well of the streptavldln-DNA template complex ~0.65 ~g/mL wlth respect to DNA), dlluted ln blocklng solutlon, wa~ added. The wells were lncubated for 10 mln to allow for blndlng of the complex to blotlnylated antlbody, ~nd the excess complex was removed by washlng flve tlmes wlth wash solutlon and three tlmes wlth T~ buffer. Subsequently, 25 ~L of the ~ 1 8~8 tran~crlptlon/tran~latlon mlxture wa~ added lnto each well and lncubated at 30~C for 90 mln, to allow expresslon of the DNA template bound to the lmmunocomplexes. At the end of thls perlod, the syntheslzed luclferase wa~ measured by addlng 10 ~L of the reactlon mlxture to 50 ~L of ~ubstrate solution (as above).
~ nzyme-Ampllfled, Time-Resolved Fluorescence Immunos~s~y of I~moblllzed Antlgen. (Com4ar~tlve) The reactlons lnvolved ln enzyme-ampllfled, tlme-resolved fluorescence lmmunoassay, up to the addltlon of blotlnylated antlbody, were as descrlbed above (see Quantltatlon of Immoblllzed Antlgen by Expresslon Immunoas~ay). Next, 25 ~L of a ~olutlon contalnlng 400 unlts/L streptavldln-alkallne phosphataqe, 6~ bovlne ~erum albumln, 50 mmol/L Trls, and 0.5 g/L NaN3, pH 8.0 were added and lncubated for 15 mln. The wells were washed four tlmes wlth wash solutlon, ~nd 25 ~/well of substrate solutlon (1 mmol~L fluorosallcyl phosphate, 0.1 mol/L Trls-HCl, 0.1 mol/L NaCl, 1 mmol/L MgC12, pH 9.1) was added.
After a 30 mln lncubqtlon at room temperature, the solutlon was transferred into whlte, flat-bottom mlcrotlter wells, and 75 ~L/well of a 0.4 mol/L NaOH, 2 mmol/L T~3+, 3 mmol/L
EDTA, and 1 mol/L Trls, pH 12.5, solutlon was added. The wells were ~haken for 1 mln, and the fluorescence was measured wlth a tlme-re~olved fluorometer.
The DNA template that was used as label was a llnear DNA fragment prepared from a plasmld ~4.3 kbp) contalnlng the luclferase-codlng sequence downstream of a T7 RNA polymerase promoter (Flgure 3(a)). The plasmld was flrst dlgested wlth Alw4gI, a reactlon that produced three fragments. The recessed 3' ends created by Alw44I were fllled-ln wlth the Klenow fragment of DNA polymerase I ln the presence of dATP, dGTP, dTTP, and blotln-dCTP. At the end of thls step, both termlnl of each DNA fragment were blotlnylated. Subsequent dlgestlon wlth F~mII removed a 0.49 kbp part from the blgge~t fragment, ~ust upstredm from 2 1 ~q8 the T7 promoter, thus leavlng a 2.1 kbp fragment labelled wlth blotln only ~t the one termlnus ~Flgure 3(b), (c)).
The fragment~ were ~eparated by agaro~e gel electrophore~l~
and stalned wlth ethldlum bromlde. The 2.1 kbp band was excl~ed, and the DNA was purlfled and u~ed a~ a label ~DNA
template). The concentratlon of the DNA template was determlned by 8cannlng den~ltometry.
To test lf the DNA template was blotlnylated, 0.5 ~g of the template were mlxed Wlth a 30-fold molar excess of streptavldln (dlluted ln T8 buffer) and lncubated for 30 mln. ~olutlons of the DNA wlth and wlthout streptavldln were then electrophoresed and stalned. In the pre~ence of streptavldln, the DNA moblllty decreased, due to the formatlon of strept~vldln-blotln-DNA complexes. In parallel, a non-blotlnylated DNA template was prepared exactly as above but uslng dCTP lnstead of blotln-dCTP.
The electrophoretlc moblllty of thls fragment was (as expected) not affected by the presence of streptavldln.
~lotlnylated and non-blotlnylated DNA templateR showed ldentlc~l mobllltles ln the absence of ~treptavldln.
To asses~ the performance of transcrlptlon/
translatlon as an analytlcal sy~tem, varlous amounts of DNA
template were sub~ected to a coupled transcrlptlon/
translatlon reactlon for 90 mln at 30~C, ln a total volume of 25 ~L. After completlon of the reactlon, 10 ~L allquots of the mlxture~ were added to 50 ~L of luclferase substrate solutlon, and the lumlnescence was measured for 1 mln.
Flgure 4 shows that the lumlnescence 18 llnearly related to the number of DNA template molecules ln the range of 3 x 103-8 x 106. The curvsture observed at hlgher numbers of molecules 18 due to s~turatlon of the llquld ~clntll-latlon counter from the llght produced. From the slgnals obtalned at varlous DNA levels and ~ luclferase callbratlon curve, prepared by dllutlng commerclally avalldble purlfled luclferase ln the transcrlptlon~translatlon reactlon mlx-~1 86998 ture, lt wa~ estlmated that 12-14 molecule~ of luclferase are synthe~lzed from each DNA template molecule.
The tlme dependence of luclferase synthesls from lmmoblllzed DNA template was studled by blndlng the blotlnylated template on streptavldln-coated wells, ~ollowed by coupled tran8crlptlon~tr~n~1~tlon. Well~ were coated overnlght wlth 25 ~L of 1.4 ~g/mL streptavldln ln PBS and then washed three tlmes wlth wash solutlon and blocked for 30 mln wlth blocklng solutlon. The wells were lncubated for 30 mln wlth 25 ~L of a 4.6 ng/ml (Figure 5~1)) or a 0.9 ng/ml (Flgure 5~2)) solutlon of blotlnylated DNA template dlluted ln blocklng solutlon. The wells were then washed three tlmes as above and once wlth T6 buffer.
Next 25 ~L of transcrlptlonitranslatlon mlxture wa~ added.
Durlng lncubatlon at 30~C, 1 ~L allquots were removed, and the luclfera~e was measured. The results ~Flgure 5~ show that luclferase synthesls reaches a plateau after 90 min.
The plateau has also been observed ln llquld phase transcrlptlon~translatlon (DNA template ln solutlon) and 18 probably due to lnactlvatlon of translatlon factors wlth prolonged lncubatlon.
The DNA template was attached to blotlnylated antlbodles uslng ~treptavldln as a llnker. There was flrst prepared a 1-1 complex of the blotlnylated DNA wlth streptavldln by reàctlng the template wlth a 30-fold molar exces~ of streptavldln. The complex was separated from free streptavldln by slze-exclu~lon HPLC. The streptavldln-DNA template complex was eluted at the vold volume ~9.5 mln), whereas free streptavldln came off at 20.3 mln.
To a~sess the performance of the lmmunoassay of the lnventlon, varlou~ amount~ of antlgen were lmmoblllzed (an antl-thyrotropln monoclonsl antlbody) on mlcrotlter wells and a blotlnylated goat antl-mouse antlbody was used for detectlon. After completlon of the lmmunoreactlon, the ~treptavldln-DNA was bound to the lmmunocomplexes~ the 2 1 ~6~8 excess reagent wa~ washed out and the transcrlptlon/
translatlon mlxture added dlrectly to the solld phase.
Durlng the 90 mln lncubatlon perlod, DNA template molecules bound to the lmmunocomplexes were expressed, and luclferase was syntheslzed. Allquots of the reactlon mlxtures were then mlxed wlth substrate solutlon, and the lumlnescence was measured. The results are presented ln Flgure 6.
There 18 a llne~r relatlonshlp between lumlnescence and the number of antlgen molecules pre~ent ln the well, ln the range of 5 x 104-1 x 108 molecules. After antlgen lmmoblllzatlon and blocklng, the tlme requlred for detectlon wss about 130 mln. The Coefflclent of varlatlon (cv) obtalned at the level of lo6 molecules was 7.5~.
Immuno~ssay ln ~ccordance wlth the lnventlon was comp~red dlrectly wlth enzyme-amplifled! tlme-resolved fluorometrlc lmmunoassay, one of the most sensltlve lmmunoassay sy~tems currently avallable. In thls assay, after completlon of the lmmunoreactlon as descrlbed above, alkallne phosphatase-labelled streptavldln wafl added lnto the wells. Subsequent hydrolysis of the substrate (FSAP) produced fluoro~allcylate, whlch forms fluorescent complexes wlth Tb3+-EDTA. The fluorescence was llnearly related to the number of antlgen molecules ln the range of 5 x 106-1 x 109. Flgure 6 shows that the lmmunoassay ln accordance wlth the lnventlon 18 more sensltlve than the tlme-resolved fluorometrlc lmmunoassay.
In lts preferred embodlments, the assay of the lnventlon does not requlre posttranslatlonal modlflcatlon of the syntheslzed proteln for full enzymatlc actlvlty.
Thls 18 deslrable because the current ln vltro transcrlptlon/translatlon systems do readlly not allow for speclflc modlflcatlons after expresslon, although such modlflcatlons are not outslde the scope of the lnventlon.
Prevlous attempts at dlrect chemlcal con~ugatlon of luclferase to antlbodles have shown that the enzyme 18 lnactlvated durlng coupllng reactlons. In contrast, by uslng sn enzyme-codlng DNA fragment as l~bel, lnstead of the enzyme lt~elf a~ ln the prevlou~ attempts, problems of lnsctlvatlon due to coupllng ~re avolded, a slgnlflcant ampllflcatlon 18 achleved, and the generated enzyme molecule~ are free ln the solutlon. Thl~ 18 s dlstlnct advantsge of the lnventlon.
DNAs are generally more stsble than protelns (enzyme~), and because of thelr negatlve charges are not prone to non-speclflc blndlng. Nucles~es (lf present ln the ssmple) wlll not normally affect sn as~sy, becau~e all the sample constltuents that could degrade the DNA label or lnterfere wlth the transcrlptlon~translatlon reactlons are efflclently removed by several washlng steps prlor to addltlon of the nuclelc acld-labelled reagent snd the expresslon of the nuclelc acld template.

OPtlmlzatlon of the hybrldlzatlon tlme Labelllna of 3'-dlaoxlqenln-tsiled csPturlnq DNA Probe The followlng resgent~ were mlxed.
20 2~ ~L dlstllled water 12 ~L reactlon buffer (lM potas~lum cacodylate, 125 mM Trls.Cl, 1.25 mg~mL B~A, pH 6.6) 12 ~L 25 mM cobslt chlorlde 1 ~L dNTP's 25 1 ~L 1 mM dlgoxlgenln-dUTP
3 ~L 100 pmol~L capturlng probe 150 U termlnal transfersse The reagents were lncubated at 370C for 45 mln. At the end of the lncubatlon, the labelled probe WdB purlfled twlce by 8eph~dex gel chromatography, snd wss eluted ln 10 mM sodlum phosphate buffer pH 6.8.

~ 1 869 98 Labellln~ of 3'-blotln-talled detectlon DNA Probe The followlng reagents were mlxed~
6.5 ~L dlstllled water 4 ~L resctlon buffer (lM potasslum cacodylate, 125 mM Trl~.Cl, 1.25 mg/mL ~8A, pH 6.6) 4 ~L 25 mM cobalt chlorlde 1 ~L 10 mM dATP
2.5 ~L 0.4 mM blotln-dATP
1 ~L 100 pmol/~L detectlon probe 10 24 U termlnsl trsnsferase The resgents were lncubated at 37~C for 40 mln. At the end of the lncubatlon, the labelled probe was purlfled twlce by ~ephadex gel chromatography, and wa~ eluted ln 10 mM sodlum phosphate buffer pH 6. a .

Method 25 ~L of 5 ~g/mh polyclon~l antl-dlgoxlgenln antlbody (from ~oehrlnger Mannhelm) ln 0.1 M ~odlum hydrogen carbonate pH 9.6 wa~ added lnto each one of a number of U-bottomed polystyrene mlcrotlter well~, and waq lncubated at 4~C overnlght. The wells were wa~hed twlce wlth a wa~hlng buffer ~50 mM Trls, 0.15 M sodlum chlorlde, and 0.1~ (v/v) Tween 20, pH 7.4). The wellQ were blocked wlth 100 ~L of ~ blocklng solutlon (1% blocklng reagent ln 0.1 M maleate and 0.15 M 60dlum chlorlde, pH 7.5) for 60 mln, then wa~hed twlce wlth the washlng buffer.
25 ~L of 2.4 fmol/~L 3'-dlgoxlgenln-talled capturlng DNA probe ln the blocklng ~olutlon was added to each well, and wa~ lncubsted for 60 mln. wlth shaklng. The well~ were washed three tlme~ wlth the washlng buf~er. 10 ~L of 10 fmol/~lL 3'-blotln-talled detectlon DNA probe ln 1%
blocklng reagent, 0.06 M ~odlum cltrate and 0.6 M sodlum chlorlde, pH 7 wa8 added, and wa~ lncubated at 42~C for 5 mln. 15 ~L of 0.03 fmol/~L denatured DNA target wa~ added, and was lncu~ated at 42~C for varlous tlme~ wlth ~haklng.

/
71 86~9~
(DNA target ln 1~ blocklng reagent, was denatured at 95~C
for 7 mln, and W~B then kept on lce). The wells were washed three tlmes wlth the w~shlng buffer. 25 ~L of 0.86 ng/~L streptavidln-DNA template complex, prepared as descrlbed above, ln 1~ blocklng reegent, 0.06 M sodlum cltrate, and 0.6 M sodium chlorlde, pH 7 was added, and wa~
lncubated for 15 mln wlth shaklng. The well~ were wa~hed ~our tlmes wlth w~shlng buffer, followed by washlng one tlme wlth TB buffer ~10 mM Trls.Cl and 1 mM EDTA, pH 7.6) at 4~C. 25 ~L of the abovementloned wheat germ transcrlptlon-translatlon mlxture (T7 ~NA polymerase, and wheat germ extract) was added, and was lncubated at 30~C
for 90 mln wlth shaklng. 10 ~L of the transcrlptlon-translatlon mlxture was mlxed wlth 50 IIL Or substrate solutlon (20 mM trlclne, 1.1 mM magneslum carbonate pentahydrate, 2.7 mM magneslum ~ulphate, 0.1 mM ~DTA, 33 mM
dlthlothreltol, 270 ~M CoA, 530 ~M ATP, and 470 ~M
luclferln, pH 7.~) ln a mlcrocentrlfuge tube. The tube was placed ln a glass sclntlllatlon vlal, and the lumlnescence W~8 m~asured for 1 mln uslng a llquld ~clntlllatlon counter ln a ~lngle photon monltorlng mode. The result~ are glven ln Table 1 and al~o pre~ented ln Flgure 7, a graph of slgnal and hybrldlzatlon tlme.

Table 1 25Hybrldlzatlon ~8ackground Slgnal wlth Slgnal -Tlme 0. 5 fmol ~ackground Target (min) (CPM) (CPM) (CPH) 21 8~8 s The nuclelc acld t~rget was a 200 bp sequence from a leukaemlc cell. It was obtalned by ampllflcatlon of BCR-A~L mRNA from the cell llne K562. The tsrget had the followlng ~equence, 41 ACTGTCCACA GCATTCCGCT GACCATCAAT AAGGAAGATG ~0 Bl ATGAGTCTCC GGGGCTCTAT GGGTTTCTGA ATGTCATCGT 120 The capturlng DNA probe was B 26mer thst hybrldlzed wlth po~ltlon 139-164, havlng the sequence 5~-GCTGAAGGG~ GAACTCTGCTTA-3'. The detectlon DNA probe wa~ a 22mer that hybrldized wlth posltlon 179-200, havlng the sequence, 5'-TCAGACCCTGAGGCTCAAAGTC-3'.

OPtlmlzatlon of the concentratlon of strePtavldln-DNA
temPlate comPlex Method The DNA probe~, DNA target and procedures were the same as ln the optlmlzatlon of the hybrldlzatlon tlme, except the followlng.
1. Both capturlng and detectlon DNA probes were dlluted ln the blocklng solutlon.
2. The denatured DNA target was dlluted ln phosph~te-buffered ssllne (0.1~ M sodlum chlorlde, 10 mM sodlum phosphate, snd 1.7 mM potasslum phosphste, pH 7.4) snd 0.14 (v/v) Tween 20.
3. The streptavldln-DNA template complex was dlluted ln the blocklng ~olutlon.
4. After the lncubatlon of the streptsvldln-DNA template complex, the wells were washed flve tlmes wlth the washlng Puffer, followed by wa~hlng three tlmes wlth TE buffer at room temperature.
Th~ results are glven ln T~ble 2 218~99~

Table 2 Streptavldln-DNA 8ackground Slgnal wlth Slgnal -Complex (ng~L) 0.5 fmol ~ackground Target (CPM) (CPM) (CPM) 0.11 30064 55591 25527 0.22 39872 485344 445472 0.43 53832 2144616 2090784 0.86 614g3 3702220 3640777 1.72 65154 6240275 6175121 3.44 101843 569ag6l 5597118 1~ The re~ult~ are al~o pre~ented ln Flgure ~, a graph of lumlne~cence and concentratlon of ~treptavldln-DNA
complex.

Claims (12)

1. CLAIMS, l. A method for assaying for an analyte, which method comprises labelling the analyte with a nucleic acid fragment that codes for a protein, expressing the nucleic acid fragment that labels the analyte and detecting the presence of the protein that has been expressed by the nucleic acid fragment.
2. 2. A method according to claim 1 wherein the nucleic acid fragment is a DNA fragment.
3. 3. A method according to claim 1 which includes the step of measuring the amount of protein expressed, thereby determining quantitatively the amount of analyte.
4. 4. A method according to claim 1 wherein the analyte is an antigen that binds to an immobilized antibody.
5. 5. A method according to claim 4 wherein a sample that may contain antigen is reacted with an immobilized antibody for the said antigen to bind the antigen, another antibody is added to react with bound antigen, thereby binding the said other antibody which said other antibody is labelled with the said nucleic acid fragment.
6. 6. A method according to claim 5 wherein the nucleic acid fragment is a DNA fragment that is bound to the said other antibody by means of biotin and streptavidin.
7. 7. A method according to claim 6 wherein the said other antibody is conjugated with the DNA fragment before the said other antibody is reacted with the bound antigen.
8. 8. A method according to claim 6 wherein the said other antibody is reacted with the bound antigen and is thereafter conjugated with the said DNA fragment.
9. 9. A method according to claim 1 wherein the analyte is a nucleic acid that hybridizes with an immobilized nucleic acid probe.
10. 10. A method according to claim 9 wherein a sample that may contain a target nucleic acid is hybridized with an immobilized nucleic acid probe to bind the target nucleic acid, another nucleic acid probe is added to hybridize with the bound target nucleic acid, which said other nucleic acid probe is labelled with the said expressible nucleic acid fragment.
11. 11. A method according to claim l wherein the expressed protein is an enzyme.
12. 12. A method according to claim 11 wherein the enzyme is luciferase and its presence is detected by means of its catalytic effect in the luminescent reaction of luciferin with O2 and adenosine triphosphate (ATP).