CA1170179A - Assay method and reagent therefore - Google Patents

Abstract

Abstract of the Disclosure A method is disclosed for determining a ligand or receptor, which comprises carrying out an assay for the ligand or receptor, the assay requiring a labelled component, wherein the labelled component is a conjugate between (i) a ligand or a receptor and (ii) a primary enzyme that is itself capable of producing or removing a modulator for a secondary system or that is the first enzyme in an enzyme system that is capable of producing or removing a modulator for a secondary system, and determining that portion of labelled component to be determined by allowing the primary enzyme and any other enzymes in the enzyme system, to produce or remove the modulator for the secondary system, allowing the secondary system to function in the presence of absence as appropriate of the modulator, and determining a product of the secondary system.

Description

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The present lnve~tion relateA to as9ay~ w~l ch u~e rea~ent that ~ enzyme-lin~ed~
. Various methods are available for the detection and/or iden~ificatlon of substancea in~ for e~ample~
sample6 of body materiale7 Immunoae3ays make use o~ the specificity of the reaction o~ an ant~gen with its anti-body for the detectlon o~ ~ubatance~ which are antigenic or can be made antlgenic or, conver~ely~ are ant~bodies or derivatives thereof. Immunoassay~ may be used for the detection o~ an~ substance o~ any origi~ provided it fall~ within one of the above categor~es, and are parti-cularly u~eful for testing sample~ of body materials ~or the detection of various types o~ sub~tances, especially naturally occurring substance~, for example, hormones, the content of which may change under certain circumstance~, for example, pregnancy; substance~ which may be present in the body under certain circumstances but whlch ar~ ~t normally pre~ent, for e~ample, particular tumour antigena .
speciflcall~ a~eociated with mali~nant states; and non-naturall~ occurring ~ub~tances, Yor examplep certain dru~.
~ s indicated above, immunoassay~ may al~o be used forthe detection of a~tibodles rather than antigen69 ~or example, in autolmmune disea~e~ and certain cancer~, and al80 to detect certain infectious dlsea~es which gi~e rise to altered specific antibody titres in a~fected indivld-ualc. I~ the latter ca~e, the diaease may be detected -and, if desired, it~ cour~e ~ollowed while pre~qent in the iDd1v1dual to mon1tor, fur eIample, respDnse to treatmeut, ~ ' ~" ' , .

or previou~ iniection may be detected, for example, in the testing for rubella~
Immunoa~ay~ may be used for qualitative or quanti-tative determination~. Colour reactiens and precipita-tlon reactions, ~or example, using ~ate~ particlz~ *orvisualisation, are o*$en used in qualitative methods to indicate the pre~ence cr ab~ence o~ the sub~tance under inve~tigation.
In quantitative as~ays, one of the components iæ
usually labellea in ~ome way, for e~ample, with a radio-i~otope or with a fluorescent group. Radioactive labels have a number o~ di~ad~antages~ however, including the C08t and complexity o~ measuring equlpment (when compar-ed with colourimetric assay~), health hazQrds as~ociated with radioisotope~, the real limit to ~eneitivity cause~d by the degree to which radioi~otope~ may be incorporatsd in anti~en~ and antibodie~ and the-inevitsble decay of the label on ~torage.
S~milarly, fluore~cent làbel~ also require ~pensive -20 equipment for their determination, and ha~e the further dl~advantage that immunofluore~cent P~6ay~ are particu-larly d~fficult to ~tandardi~e and to quantitate. The asses~ment of result~ i~ ~ery subjectiYe and can re~ult in an unacceptable degree o~ variation amon~ worker~.
Other physical and physico-chemlcal method~ are al80 available for the detection o~ further type~ o~
labels for antibodie~ and antigens, but theae ofte~
have limited applicability and again require ~pecialised : ,. : . . .

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and e~pen~i~e apparatus.
Attempts have been made to get round the problems a~sociated with radioisotope Rnd fluorescent label~ b~ the use of enzyme~ as labels. The enzymes previou~ly pro-5 posed have been cho~en ~or their abillty to c~talysereactions which.are relati~ely eR~y to mea~ure and which also proceed ~t a high rate (cf. ~ngrall, ~. ~Md Perlm~nn~
P~, ~1972). The Journsl of I~munology ~Q~, 129~o The enzymes propo~ed are, for e~ple, those which generate 10 a coloured end product or which produce ~ substrste for a second enzyme, which ~ubstr~te iB u~ed up ~n the genera-tion of a coloured end product 7 There iB, however, the problem of ~ntroduoing the enzyme label ln a high enough concentrati~n and al~o the 15 problem that ~imply attaching an enzyme to an ant~body or an antlgen may re6ult in a certain 10B8 of catalytic ac-tivit~. The number oi labelled molecule~ to be detected may be relati~ely small, æo it iB important that the label can be detected easll~.
As indicated abo~e, lmmunoa~8ay8 utili8e the ~peciiicity o~ the interaction bet~een ~n~ibodlea and antigens t~ datect and/or determine these sub~tancee, There are, moreo~er~
other ~nalo~ous pairs of 8ub~anceB th~t haYe an analogou3 specificit~ for each other; these are the receptor~ that 25 occur in the bod~, often in a~oclation with cells, and their complementary partner~. ~here iB considerablo over lap between a~tibodies and ~ntigens and other li~ands aad receptor~, e~g. a sub~t~nce that iB a~ antigen ma~ al~o be the partner ~or a ~on-antibody receptor~ ~oreover, lt ha~
30 proved dl~ficult to ~Bt~gui8h ~ome lYmph~id cell receptors from ant~bod;y molecules.

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O ~ 3 a~ples of partner~ ~or non-antibody receptor~ ~re sub-. stances produced by the body it~elI, for example, hormone~,opiates, alld chemlcal lntermediates in the nervou~ ystem~ and materials originatin~ e:cternally, for e~ample" viru0e~ and 5 toxlns . These receptore and their partners rec~æniee each other and b~d ~peclYically w:ith one ~nother in the eame manner a8 do antibodie~ and antigen~ and they can be u~ed in ~say~ that are directly e.~ OgOU8 to immUnoa~Bay~ ior the detectlon E~nd~or dete.m{natlon of either partner.
The present lnvention i~ ba~d on the obaerv~tion thE~t the enzyme used a~ the label in an immunoassay or analo~ous a89ay may be ~ en~yme that pr~duces, directl~r or indirectly, ~ substance that i8 capable o~ uencing a catalytic e~ent without ltselr be~g consumed during the catalytic event.
The lnvention accordingly provides a method ior de-termlning a llgand or receptor~ ~hlch c~mprises carry~ng out an a~say ~or the llgand or receptor9 the as~ay requir-~ne a labelled component, whereln the labelled compo~ent i~ a co~u~ate between (1) a li~and or a receptor and (il) a primary enzyme th~t iB ltee~f capable of producin~
or rem~in~ a modulator (as hereinafter defined) for 2 ~econdary ~ystem or that 1B the M ret enz~me ln an enzyme system that ~8 capable of producing or removi~e ~ modulator (as h.erelna~ter de~ined) ~or ~ secondary system, ~nd ~5 determ~nin~ that portlon of labelled component to be de-termlned b~ Pllowin~ the primaIy a~zyme and any other enzymes in the en~yme system, to produce ~r rem~ve the modulator for th~ ~ec~ndary ~ystem~ allo~ng the secondary sy~tem to ~u~ction in the presence or absenc~ (as appro-priate) of the modulator; and determining a product ~f the .

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_ - 6 secondar~ stem. B~ producing or removing a~ appropriat~
the modulator, ~Imp]ification is acheived by the production of substantially more than one molecule of product of the secondary system per molecule of modulator.
The terms ~ligand" and "receptor~ are u~ed in the present Specification to denote ~ complementary ~air o~
substances that are capable of recoenisin~ the ~peciflc spatial an~ charge configuration of each other and of b~nding specifically with each other.
~iands are~ for e~amp~e, antl~en~, haptens, and the partner6 of cell- ~nd non-cell associated, non-antibody receptors, and receptor~ are, for e~ample, ~ntibodie~ and non-cell and cell-aseociated non-antibody reoeptors. The *erm ~non-antibody" receptors as used herein include~ non-antibody receptor6 obtained from natural souuces and those produced ~ynthetically or semi-syntheticslly9 and 1 BO
includes analogues thereof that are capable ~f binding to the appropriate partner. S~m~larly, the~r respective partners may be obtaIned ~rom natural s~urces~ or msy be synthetic or semi-~ynthetic, or an~lvgue~ of natural part-ners pro~ided that they are cap~ble of binding to the appropriate receptor.
The antibod~ component of an antib~dy-enzyme c~n~u-gate o~ the inven$ion may be ~ny immunoglobulin obt~ine~
~rom any souroe~ provided that it is suitable for tak~ng part in the desirea assay. In ~ome case~, lt may be preferable to u6e a heterogene~us ~ntibody pDpulatlon~
for e~ample, as obtained from ~ ~hole blo~d ~ample, where-aB in other ca~e~ it m~y be preferable to u~e ~onDclonal 3~ antib~die~. ~urthermore,:there ma~ ~e ue~d mi~ed antibodies, that ia t~ ~ay~ ~ntibodles h~vin~ ht and hesvy chalns - - ~ .

O l ~ g . . -- 7 origin~tlng in di~ferent molecule~, the mixed ~ntlbod~eK
being produced b~ hybr~dleation.
I$ w~ll be appreciated that~ inatead of b~ing bOuna to a complete immu~oglobul~n m~lecul~ the enzyme msy be b~und to ~ ~ultable immunDglobL~in fragment. ~ccording-ly, the term "antibody~ ~hen u~ed herein denote~ any lmmunoglobulin molecule or any ~ragment o~ a~ immu~oglo-bulin molecule cDnts~nin~ arl intPct anti~en binding site ~nd being capable of being b~und to the enzyme without eubstantlally inter~ering ~ith the antigen binding.
E~mple~ of sult~ble ~mmunoglobulln fr~gments are Fab and (~ab~)2 ~ragment~. . .
The antigen component of an antlgen-enzyme con~u-gate of the invention 1B any antigen that i9 capable o~
being bound to the enzyme without ~ub~tantially lnter~
fering ~ith itB antibody binding capacity. The term ~antig~nn ~hen used in ths pre~ent speciflcatio~ includea h~ptens, and "antigen-enzyme c~nJu~te" include~ hapten-e~zyme con~ugate, u~e~ otherwise ind~cated.
The term "mod~lator" i8 u~ea herei~ to denote a BUb-~tance thst giVeB riBe to a catalytlc event but of wh~ch there iB no net con~umption during the ~atalytic event.
The term "enzyme~ ~B used herein to denote ~ par-ticular ~nzyme nctivity~ (An enzyme m~ h~ve the form Or a diacrete molecule or an enzyme c~mple~ which may display more than one enzyme acti~it~.~
~ he term ~pr~m~ry en3yme B~Btem~ iB used i~ the prese~t speci~icatlon to denote a s~stem that compri~es .

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an enzyme conjugate of the invention and that iH capable of producing or removi~ a m~dulator for the secondary ~ y6tem. The primary enzyme ~y~tem may ¢omprise the primary enzyme as the only enzyme, or it may com-prise a 6erie~ of enzymes of which the primary enzymei~ $he fir~t~
The term "~eGondary ~yfite~" is used hereln to denote a reaction or reactions ~odulated by the product o~
- the primary enzyme eyatem, that i~ to say, the primary 'enzyme system produce~ or re~move~ a sub~ance that, in the presence of the,aecondary system, ~ve~ ri~e ~o a catalytic event without being consumed (in net terms) durine the catalytic event. An example of a type of modulator that may be produced by the primary enzyme sDstem is an enzyme activator) and enzyme inhibitor~ are an example of another group of modulator~. In the latter case, the primary enzyme ~ystem mu~t be capable of remoYing an inhibltor to !'switch on" the second~ry enzyme sy~tem.
' In aome case~, the primsry enzyme ~yctem may be capable of simultaneou~ly producin~ nn'acti~ator for a secondary enzyme ay~tem and of remo~inp~ an inhibitor therefor. .
An e~ample of a further $ype oI modula~or is a 6ub-atrate or co~ctor ~or a ~econdary s~-stem t~lat , 25 i8 capable of regenerating the ~ubstrate or . cofactor, Such a ~econdary ay~tem involve~ a cycle. l`he modulator "~witche~ on" the cycle, ~hich can tXen continue to turn almo8t indefinitely, provided there ~s a aufficier~t .
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supply of the apnropriate sub~trate~. The cycle compri~estwo or more reactions, at lea~t one o~ which may be en~yme cataly~ed. ~he other reaction(s~ in the cycle may each be enzyme cataly~ed or not.
The modulator may be physically separated fro~ the ~econdary system, for example, it may be pre~ent in a cell or vesicle. In th~ case, the primary enzyme system produces the modulator by causing all or ~ome of the modulator to become available to the secondary ~ystem, for example9 by cau~i:n~ the cell or ve~icle to rupture or become permeable The use in as~ays o~' the enzyme conju~ate o~
the invention circumvents many of the problems and di~-'advantages encountered w'ith radioactive and fluorescent ~5 'labels, and has advantage6 over previou~ly proposed uses o~ enzyme~ as lab'els, ~or example, tlle sensitivity of the assay is improved and the product of the primar~ en~yme eystem i~ not con~umed in the reaction for the determination of the label~
By producine or removin~ a modulator ~or the secvn-dary 9y~tem7 eg. by producing an activator and/or removin~ an inhibitor for the secondary enzyme ~ystem, or by producin~ ~ regeneratable substrate or coenzyme for the 6econdary enzyme sy6tem, or ~ re~eneratable substrllte or ~yGtem9 25 co~actor ~or a. nbn-en~yrric ,l nmpliiication i8 aChleVed.
~ach molecule of modulator re~ults in the produc$ion of substantially more than one molecule of product of the econdary . ~ystem. T~e modulat~r produced or re- -'. ', ' '' ., ~ . . .

O 1 ~ 9 moved by the primary enzyme system can be reg~rded a~ a catalyst ~or the ~econdary fiy~tem, e~, the pre~ence of an activator or removal of inhibitor "~witche~ on" an enzyme, and a re~eneratable ~ubstrate or co~actor "switches on" a ~eco~dnry c;ycle Y~l~ch can then continu~
to turn with determinable product being produced at each turn of the cycle. Thi8 i8 in direct contrast to those previous propo~ed enzyme labels for lmmunoa9says where the enzyme bound in an enzyme conjugate either produoes a determinable product directly or produce~ a ~ubstrate for a further enzyme reaction in a simple li~ear, usually l:l, r~tio. The amplification serves to increase .the sensitivity o~ the assay directly by cau~ing ~he pro-duction of larger numbers of determinable molecule~ than would be produced directly by lig~r.d or rec.eptor bound enzyme, and thus help~ to overcome one of the di6-ad~antages of the previously proposed use of enzymes, ~hat is to ~ay, the tendency to inactivation of certain enzymes on conjugati~n, A further advantaee i8 that the reactions involvine the primary enzyme 8y9tem and the ~econdary ~y6tcm mar be carried out ~eparately. Thi~ ~ive~ ~,reater flexi-bility with regard to the time and place at which the reactions are carried out. It i9 also ~enerally ea3ier to quanti.tate the secondary '6y6t~m i~ the reactions are carried out ~eparately ~rom those of the primary ~y~tem~ Moreover9 there i~ greater freedom in the choice of en~yme~ ~or the ~econda~y s~tem a8 tilere can be used in ', .

thi~ ~ystem enzyme~ that are not suitable ~or con~uea-tion to a ligand or receptor, ~or example, a BeCOn-dary enzyme 8y9tem ma~ comprise ~n in~oluble enzyme or an unstable enzyme.
As indicated above, the primary enzyme ~ystem u~ed in the method of the invention may comprise the primary enzyme, that le to say, the enzyme present in the con~u-gate, as the only enzym~, nr it may comprise more than one enzyme, only the primary enzyme bein~ bound to a li~and or r~ceptor with each other enzyme generally producing $he sub~trate for the ne~t enzyme. It may be preferable to use a reaction chain a~ short a~ possible, ~or e~mple, using the primary enzyme ~nly to produce or remove the modulator for the secondary enzyme system.
A ~econdury enz~e sys-tem, too~ may comprise one or more enzyme~. In the ca~e of activation and/or in-hibition, there may be only one enzyme in the ~econdary enzyme ~ystem, or the modulated enzyme may be part of a chain or cycle compri~ing other enzymes and/or non-en~yme catalysed reactions. When the modulator i9 a regeneratable ~ubstrate or cofactor~ a cycle i9 lnvolv~.
As indicated above, a cycle m~y comprise at least one enzyme cataly~ed reaction, or any one or more of the reaction~ in the cycle may be not catalysed by an enzyme ie a cycle m~y be ~Jholly chemicnl; n~loll~r enzymic; or part ~llemical, part enzymic9 There may be used a secondary enzyme sy~tem in which one ~econdary enzyme pr~duce~ a ~ub~tance that i8 . . ', . , , : . , ;
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modulatory for a further enzyme, so an extra multiple amplification step is incorporated in the system. Further modulated enzymes and/or modulator-producing enzymes may be used in series.
A secondary enzyme system may be a mixed system comprising enzymes subject to different types of modulation, eg. one or more enzymes subject to activation and/or inhibition, and a cycle capable of regenerating a substrate or coenzyme. This, too, may result in multiple amplification.
The choice of primary enzyme system and secondary system are, of course, linked, as the primary enzyme system must be capable of modulating the secondary system.
Dealing first with the secondary system, this may comprise an enzyme system that is subject to regulation, either by --~
activation or by inhibition. The enzyme system may be natural-ly subject to regulation or may have been modified to become so. Alternatively, a secondary enzyme system may be capable of regenerating a substrate or cofactor (coenzyme~ that is produced by the primary enzyme system and also causing con-commitantly the build-up of a substance that is detectable either directly or indirectly. The use of secondary system that generates modulator in this manner is highly advantageous as it may be employed to give rise to a particularly rapid build up in the .

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determinable substance ~ modulator for ~ ~ecorldary ~r6tem may be n~tur~l ~r synthetic, and a "p~ -moal~ator" may be converted by the primary system ~nto a modulator by removal of the protecting moiety e~. protectil~ groups or protecting peptides. A natural modulator may ~ave been modified such ~hat ~t is inactive until acted up~n by the primary en~yme system. P~etal ions are modulator~ for ~ome enzymes~ A compartmentation ey~tem as described belo~
may be used with metal ions.
~ he eecondary system ~r~er~bly prcdl~ces a determinable aub9tance or uses a ~ubstrate that can be readily determined directly9 ~or example, spectrophoto metrically, b~ colour, by ~taining, manometrically, b~ llght 15 production e~ usin~ ATP on ~ir~ly ~xtr~ct9 or n~icrobio-logically eg by using bacteria with ~pecific nutritional requirements , or by mea~uring physicochemical changes~
eg. conductance changes, The determinable substance producea may, however, be determined indirectly, by act-ing as the substrate for one or more further reaction(s~
- producing a readily determinable end product or may be a regulator for a further reaction. An e~lzyme system m~y9 ~or e~ample, catalyse a reaction in which carbon dioxide i8 produced eg. u8ing py~uvate decarbo~ylase; in which the oxygen ten~ion i~ changed, eg~ using glucose ~idase with measurement by an o~ygen electrode; or in which D~P-hydraz~ ne can be used to produce a coloured end product~
It is partic~iarly convenient to utilise an enzyme 8y9tem .

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capable of producing NAD or a compound that can partake in a reac-tion in which NAD/NADH interconversion is involved. (~bbreviations used in this specification are set out before the Examples.) Some examples of primary enzyme systems comprising one enzyme only with associated secondary enzyme systems also compris-ing one enzyme only are given in Table 1 below, by way of example only:
Table I
Primar~ enzyme Secondary enzyme 1. Enzyme that produces Enzyme subject to activa-tion by cyclic ~MP eg. adenylate cyclic AMP eg. phosphorylase B
cyclase (4.6.1.1) kinase (2.7.1.38), pyruvate carboxylase (6.4.1.1) phosphoenol pyruvate kinase (2.7.1.40)

2. Glyoxylate reductase (1.1.1.26) Isocitrate dehydrogenase (lL142) (reduces glyoxylate) (inhibited by mixture of glyoxy-late and oxaloacetate)

3. Enzyme that removes ATP, Enzyme inhibited by ATP, espec~
especially that converts ATP ially when also activated by to ADP eg. ATPase (3.6.1.3) ADP eg. isocitrate dehydrogenase e.g. apyrase (3.6.1.5) (1.1.1.42)

4. Glutathione reductase~.6.4.2) Glyoxylase (3.1.2.6 or 4.4.1.5) (produces glutathione) (activated by glutathione)

5. Fumarase (4.2.1.2) or fumaryl Mitochondrial NAD-linked malic acetoacetate lyase (3.7.1.2) enzyme (l.1.1.38 or 1.1.1.39) ~produce fumarate) (activated by fumarate) In the examples given above, the primary enzyme system comprises one enzyme only. As indicated previously, however, the primary enzyme system may comprise several enzymes, only the first being bound in a conjugate. An example of such a system , with a single enzyme in the second system, is given below:

~ l~V :~7~3 ¦Primary enzyme system¦

*pyruvate kinase* ~
Pyridoxal~ TP

pyridoxal ~ (2.7.1.35) Pyridoxal ~
phosphate ADP

recondary enzyme¦ ~ activator for system I amino acid decarboxylase A further example of such a system is the following:
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Primary enzyme system *glucokinase* f Glucose t2.7.1.2) - ~Glucose-6-phospate phospho-glucokinase (2.7.1.10) Glucose-1,6-di-phosp-~phate rSecondary enzyme r , activator for system ¦Glucose-l-phos-phate ~
~ phosphogluco-. mutase (2.7.5.1) Glucose 6-phosphate~
(For this method, the phosphoglucomutase must be in the de-phosphorylated condition. The enzyme can be de-phosphorylated by exposing it to fluoride ions.) In a~ diagrams, *.... * denotes the primary ie. conjugated enzyme, and there are given only those components of the various reactions that are necessary for the understanding of the reaction schemes.
As indicated previously, a secondary enzyme system may comprise only one enzyme, or it may comprise several enzymes, more than one ~ B

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-of which may be subject to re~ulation by a modulator, ïf desired.
An example of a system comprising a chain of reactions in -the secondary system is the following:
Primary enzyme *glutathione* f system reductase (1.6.4.2) Glutathione Secondary enzyme lactivator for system 1 4-Maleylaceto-acetate \ maleylacetoacetate . isomerase (5.2.1.2) 4-Fumarylaceto acetate \ 4-fumarylacetoacetate J lyase (3.7.1.2) Fumaric acid~
activator for ~
mitochondrial N.~D-linked malic enzyme (1.1.1.38 or 1.1.1.39) In this case, the product of the primary enzyme system is the modulator for the first enzyme in the secondary enzyme system, and extra multiple amplification is achieved by the inclusion of a further modulated enzyme in the secondary system.
It may be preferable to use pximary and secondary enzyme systems as simple as possible. When more enzymes are incorporated, however, particularly if they are themselves subject to modulation, different advantages may be achieved, for example, with regard to the sensitivity of the assay.
An example of a simple system that can be extended with the addition of extra enzymes is that ..~. .
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utilising E.coli Type I pyruvate kinase (PK) (2.7.1.40) as the secondary enzyme system with phosphofructokinase (2.7.1.11) as the primary enzyme. Phosphofructokinase (2.7.1.11) produces fruc-tose-1,6-diphosphate (FDP), which is a very potent activator for E.coli Type I pyruvate kinase (2.7.1.40) (in the presence of certain amounts of phosphoenolpyruvate, cf. M. Malcovati, G.
Valentini, H.L. Xornberg, Acta vitamin. enzymol. (Milano) 1973, 27,96.
The system described above can be extended by providing ATP, which is required by phosphofructokinase (2.7.1.11), indirect-ly by means of an enzyme capable of converting ATP to ADP, for example, mammalian pyruvate kinase or E.coli Type II pyruvate kinase (2.7.1.40). Such a system has the advantage that it gener-ates more ATP (the modulator) during the reaction to drive the activator-producing enzyme faster.

¦Primary enzyme system¦
(2.7.1.40) ~ ADP
*pyruvate kinase*
(non-regulated) ~ATP~
F6P ~ ~ I
t2.7.1.11) FDP ~ ~ ADP

, , actlvator for Secondary enzyme . I
system _ ~ I

PEP pyruvate ,--ADP
) kinase type I
pyruvate ~y (2.7.1.40) ~ ATP~
As soon as the primary enzyme (non-regulated pyruvate kinase) produces some ATP, phosphofructokinase .

i6 able to produce fructo~e-1,6-diphosphate (FDP), which in turn activates the ~DP-sensitive pyruvate kinase (which is preferably present i~ relatively high con-centrations). This pyruvate kinase, too, convert~ ADP
to ATP which, if both primary and secondary enzyme systems are allowed to react toeether in one ve~el~
further increases the production of ~DP by phosphofruc-tokinase. This leads to augmented stimulation of the ~DP-sensitive p~ruvate kina9e, and a substantially ex-plosive increase in the production of pyruvate fromphosphoenolpyruvate~ ~he pyruvate can be converted to lactate by lactic dehydrogenase (1.1.1.27), with concommitant oxidation of NADH which can be followed photometrically, Alternatively, the pyruvate c~n be reacted with D~P-~5 hydrazine to give a coloured end product.
An e~ample of a ~ystem in which the modulator,once produced by the prim~ry enzyme syste~, is ~elf-producin~, is that involving the conversion of comple-ment factor C3 to C3b ~ rOll O~S:

~rimary enzyme system 1 C3 C3-converta~e (3.4.21.43)(for example of the classical pathway) ~econdary enzyme , __ __ ~ 3 - 25 ~y~tem _ ' C3 ¦ activates C3-convertase(3.4.21.47) of the alter-native pathway L -~ ~3b D

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Complement component C3 i8 clea~ed proteolytically to form the frA~ments C3b and C3a. There are two distinct C~-convertases which ca~ do this: that of the ~o-called class~cal pathway of com~lement activation and that of the alternative pathway. ~ignificantly, the latter i~
unlike the former in that lt requires C~b itsel~ to function.
~: In the reaction ~cheme shown above, the C~b formed by the independent C3~convertase, in acti~ating the de-pendent convertase3 results in an increase in the forma-tion of C3b. ~his pos~tive feed-baok system would operate until maximal activation wa~ achieved and very much more C3b was being produced than by the primary system alone9 ~ither C3j ~b, C3a or the biologlcal effect~ of these mediator~ ln, for example, cell lysi~
or provocation of anaphylactoid reaction0 can be followed to indicate presence of the primary enzyme sy~tem~
A~ indicated ubo~e~ the primar~ en~yme ~ystem muy prod~cc a regeneratable 6ub6tr~t~ ~or ~ s~cond.lry sy6tem. In this-case, a seconunry en-zyme ~y~tem m~y com-prise two or more enzyme~. Preferably one of the enzymes produces or use~ a ~ubstance that is readily determinable.
A two-enzyme system is, for exampleg as set out sche-matically below:
~ ubstance X A
25Enzyme 2 ~nzyme 1 C ~ubstance Y ~
Subst~nce X is the product sf the primary enzyme system.
~he ~econdary enzyme system ~hould be selected ~o that .
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there is build-up of at least one of B and D and/or a decrease in at least one of A and C (insofar as they exist), as X recycles via Y. Provided suitable amounts of A and C are present, substance X
will be continually recycled, using one molecule of each of A and C per cycle and producing one molecule of each of B and D. At least one of A, B, C and D is preferably readily determinable it~
self or may participate in one or more further reactions to give a determinable product or to use a determinable substrate. Moxe than two enzymes may be combined in larger cycles or in interconnecting cycles. Alternatively, substance Y may be the product of the primary enzyme system. (In either case, the cycle may be reversed, if desired.) An example of a system described in general terms above is that in which the secondary enzyme system comprises fructose diphosphatase and phosphofructokinase.

Pi ~ ~ Fructose-6-phosphate ~~~` ATP
Fructose-di ~ ~ ¦ Phospho-phosphatase I ~ fructo-(3.1.3.11) ~ Fructose-1,6-diphosphate~ ~kinase (2.7.1.11) `-~ ADP
It is possible to determine the production or consumption of any of ATP, ADP and inorganic phosphate (Pi).
The primary enzyme system for the above secondary enzyme system should be one that is capable of producing either fructose-

6-phosphate or fructose-1,6-diphosphate. Fructose-~-phosphate may be produced by a primary enzyme system in which phosphoglucomutase (2.7.5.1) is the primary enzyme,converting ylucose-l-phosphate to glucose-6-phosphate, which is then converted by phosphoglucoisomer-ase (5.3.1.9) to , . . ' ' ' .

l1 7(~1 ~9 fructose-6-phosphate. Fructose-6-phosphate may also be produced from glucosamine-6-phosphate by glucosamine-6-phosphate deaminase (~3.1.19 or 5.3.1.10). Fructose-1,6-diphosphate may be produced from glyceraldehyde-3-phosphate and dihydroxy-acetone phosphate by the action of aldolase (4.1,2.13), to enter the cycle at the bottom.
A further example of a substrate cycle used in a secondary enzyme system is the following:

.
Primary enzyme APS
system \ *Adenosine-5'-triphosphate ~ sulfurylase * (2.7.7.4) - ATP \ ' - --~
Secondary t enzyme pyruvate system pyruvate \ ATP phosphohydrolase (3.6.1.3) kinase \ or other ATPase (3.6.1.3) PEP (2.7.1.40) ~ADP
(Adenosine-5'-triphosphate sulfurylase converts adenosine-3'-phosphate-5'-phosphosulphate to ATP and SO~.) Alternative enzymes for the prod~ction of ATP in the primary system are, for example, 20 pyruVate phosphate dikinase (2.7.9.1), which catalyses the fol-lowing reaction: PEP + ~MP + pyrophosphate > pyruvate + ATP +
Pi, and ATP:D-ribose-5-phosphate pyrophosphotransferase (2.7.1.15) which converts AMP and 5-phosphoribose-1-pyrophosphate to ATP and D-ribose-5-phosphate.

This system may be modified and extended, providing an example of a system involving both a substrate cycle and a modul~t-ed enzyme:

~~, '' ~ ' .

i :

7 '~
- ~2 -Primary enzyme system ) *Enzym~ l*
f ~ - ~ ~' ~ ATpJ~
/ ~ phosphofructo- ~F6 Secondary Enzyme 2 ~ kinase (2.7.1.11)( system _ ADP~ ~FDP
activator jfor ADP~
E.coli pyruvate kinase ~-PEP
ATP Typ~e I (2.7.1.40) Enzyme 1: see above pyruvate Enzyme 2: phosphoglycerate kinase (1.1.1.95) or non-regulatory pyruvate kinase (2.7.1.40) The third reaction is preferably carried out separately from the second reaction.
A system involving regeneratable coenzyme is directly analogous to a system involving a regeneratable substrate as described in general terms above.
An example of such a system is the following, in which NADP
is the regeneratable coenzyme:
Primary enzyme system NAD\ ~ ATP

*NAD-kinase*
(2.7.1.23) P ~u~n~ itrate -NADPH dehydrogenase ~
(1.1.1.42) ~DNP-hydrazine ~Secondary enzyme ¦ coloured product Lsystem As mentloned above, a modulator, for example, a substrate or coenzyme, may take part in a secondary enzyme cycle in which not all the reactions are enzyme-catalysed. A simple example of such a system is as follows:

B

-.

O l'i' ~23~

Vncataly~ed D ~ ~ub~tance X A
- chemical ~ ~ Enzyme reaction C ~ ~ubstance ~

~ ither substance X or substance Y may be the' product of the primary enzyme system. An advanta~e of ~uch a ~y~tem i~ that it may be possible to u~e a total of only two enzymes: one in the primary enzyme sy~tem - and one in the secondary system.
An example of a group of reactions that may par~
ticipate in such ~ cycle are oxidation-reduction reac-tions, for e~ample~ involving NAD/NADH or NADP/NADP~I
interconver~ion~ A~ e~ample of such a eycle i9 givenbelow:

Uncatalysed D ~ ~ NAD(~) A
chemical y ~ ~nz~me reaction C J~ NAD(P)~ B

Substance6 capable of reduction with concommitant oxida-tion o~ Nl~(~H are well known, for example, (4,5-dimethylthiazolyl-2)-2,5-di~henyltetrazolium (~ITT tetrazolium~ i8 particularly useful, because on reduction it gives a blue coloured product and, more--over, the result~ re linear with reeard to the NAD(P).
A secondary enzyme cycle invo~vine an NAD(~)/NAD(P)H
interco~version may therefore be determined directlyO
In the case of secondary enzyme cycleY involving NAD(P)/NAD(P)H interconversions, it i~ preferable to use a-primary enzyme ~ystem capable of produclng NAD or NADP, for example, NADP may be'~roduced from NAD by . N~D-I~inase, and NAD may be produ~ed ~rom NAD-dihydroxy-.

.
;' ' ' ' " . ' ' , ' .

0 1'~ ~
- 2~

acetone ~ nicotinamide by NADase (DPNase)-Examples of such cycles are given below:

Primary NAD
enzyme *NAD-kinase*
system ~ ~(2.7.1.23) ADP~

blue ~ \ Secondary coloured ~ NADP-linked enzyme product ~ isocitrate system MTT ~ ~NADPH dehydrogenase tetrazolium (1.1.1.42) ¦Primary ¦ NAD~dihydroxyacetone enzyme + nicotinamide system r * NADase* (DPNase) (3.2.2.5) .-~
.-- \ - \ ~ NAD --~

blue ~ / Secondary coloured enzyme product alcohol system MTT___~' ~ dehydrogenase tetrazolium ~NADH (1.1.1.1) A modulator may morevover, take part in a secondary system that does not comprise any enzymes. In such a case, the modulator is a substrate or cofactor for a cycle in which the modulator is regenerated, so there is no net consumption thereof (of the wholly and partly catalysed cycles mentioned above).
A possible example of such a cycle is that in which the primary enzyme is peroxidase (1.11.1.7), which removes iodine (in the form of iodide ions) from thyroxine. The iodide ions may then 3 0 be recycled in the secondary system via iodine, eg. as follows:

' ~3 , 1 1 7 () 1. ~ ~ 1 Thyroxine *peroxidase*
Primary (1.11.1.7) Enzyme system Secondary reactant Enzyme which gives a quinone system oxidation product ~ I2 Alternatively, use may be made of a system using an iodide-iodine cyclic reaction (see Clinical Chemistry principles and Techniques, R.J. Henry, Harper & Row, New York 1964, p. 7) as follows:

As ~ ~ ~ I2 ~ Ce As indicated above, the modulator may be physically separated from the secondary system, the primary enzyme system causing the modulator to become available. The modulator may be an activator or inhibitor or a regeneratable substrate or GO-factor. Metal ions are examples of modulators that may becompartmentalised. Metal ions are modulators for a number of enzymes, for example Mg and Mn are modulators for pyruvate kinase (2.7.1.40) and isocitrate dehydrogenase (1.1.1.42).
The modulator may be present in relatively high concentra-tions in, for example, a synthetic or semi-synthetic vesicle or an organelle or cell, or may B
.
.. ~
, , .. , . ~ ~, ;

. , .

~imply be prese~ t in a normal oell. The primary en~yme system may rupture or lyee the vesicle or cell~ or may simply make it more p~rmeable, to the modulator at least, so that there i8 leakage of sufficient amount~ o~
m~dulator to affect the secondary sy~tam.
C3, 'l~c~
The use of lysczyme~and trypsin may be particular-ly ~uitable for the di~ruption oi celle, organelles, and ~esicle~. Either lysozyme or tryp~in may be bound in the de~ired conjugate with the other being present in the reaction mediwm for di6ruption of the ætructure.
, In general, the tryp~in i8 preferably bound in the con-!~ ~ugate, with hi~her concentrations of lysozyme in the reaction medium.
1ysozyme itself is capable of ly~ing the micro-organism Micrococcus l,Ysodeil~tu~. This microoreani~m ' , may there~or~ be used as ~uch or may have its internal - concentration of a modulator increa~ed, to be ru~tured a~ desired by a primary enzyme 6y~tem compri~ing ly~o-'zyme a~ the primary enzyme.
The ~ethod of the invention may be used ~or detect-ing ligands and receptor~ of n~tural or rlon-, natura~ ori~in. Immunoas~ays have wide application, in both clinical and non~clinical field~; they are parti-cularly useful in any circumstance where it is nece~3ary to detect ~nd/or determine small or very small amounts of sub~tances. Clinical u~es include~ *or example, the detection and/or determination of blood group ~ubstance~, ' of Au~tralia antigen, of di~ea~e~ of various microbial .
- ' . .

g _ - 27 -~rlgln~ eg. di~eases cau~ed by vlru~, bacteria or fungi, or para~itic disea~e~, of hormones~ o~ ~ub~tanoes that ~ay be present under certaln condltlon~) for e~ample, durl~g pregnancy, ior e~ample~ pregn~ncy-~peclf~c proteln~ ~nd foetal protein~ ip foetal materlal and even in maternal material eg. blood, or ~n as~ocistion ~ith certain m~l~g-nant states, ~f antibodie~ ~a~ociated with a~ oimmune disea~e~ ~nd certain ¢~ncer~ and drug8. Immunoas~aye are , ~ .
part~cularly use~ul in ~orensio ~nve~tigatlons, as the 1~; ~mounts o~ ~ubstanceR to be detected ana~or determ~nea i~
often very Bmall~ Dru~ detestion~ bath in ~oren~ic investl-gatlon~ and in lnvestigation~ associated ~i~h human ~nd animal ~porting events may b~ advsntageously carried out by immunoassQys. The methcd o~ the inven~ion may be used . 15 for detecting.any of the aboY~ sub~t~nce~ and slso aDy other substunce th~t can .be dstect~d and/or dctermined b~
an lmmunoaB~a;sr~
~ BBay8 analOgOUB to immunoR8say~ ~or the detec~lon and~or determinatian o~ li ~ ds and receptor~ other th~n . 20 antlgen/antibod~ pa~r~ are also use~ul ~$1cally and other-~ise. Such ligands are, ~or ~Eample~ ho~mones, ~or ~ample~
in8ulln~ glucagon, piultsry hormo~e~ egO vasopre~in~ o~y-tocin and trophic hormonea, ~teroid hormones and ga~tric hormones; chemlcal intermediates ~nd 8i~nal~ ln the nervouB
~ystem9 for e~ample9 acetylcholine, opiates and ~hetr analo-gues, naloxones and encephalin6;, chalo~es, ~e~elopmental 8ignal8 and cell interaction ~gnRls, other ~turally . occurrlng chemicala egO histamine; .and~substance~ orig~n~t-ing outside the bod~ eg. viruse~ and to~ina.-:
- . .

.

, 1 :L 7V 1 7 ~

An a~ay for a ~ub~tance that can be o~nsldered to be both an antlgen and the partner ~or a ~on-antibod~ receptor may be carTied out UBi~ elther the corresponding antibody or the other receptor a~ partner~ An immunoa~a~ ma~ have the advantage that the antibod~ csn be obtained more readily and c~eaply th~n the other receptor. Conver~aly~ ~t may be difficult to raise an antibody to a particular antl~en, ln : which case the use o~ the non-antibody receptor 1B pa~ef'er~
able, ~n a~say using a non-antibody receptor m~ be even ,~0 more ~pecif.~c than an aeeay u~ing an ~nt1body ~g~inst the '' ' corre~ponding l~gand because a ~peei~ic receptor generally bind~ at the active part of the llgand molecule, wher,eas the corre~ponding antibody~usually,bind~,at ~nother part oi the molecule, giving greater po~sibillty of cro~s-reac-15 tions.
Antibodies and antlgens have a'~peciric aff~nity foreach other; when~ howe~er, an a~tlbody-antlgen complex has been ~ormed, thls comple~ and complement factDr Olq can,form "' a ligand-receptor ~air~ Factor alq ~ay therefor be u~ed i~
2~ ~n assay of the inventlon to detect ant~body-~ntigen com-ple~es o~ any t~pe.
The assay technique to be used in the meth~d o~ the in~ention i~ an~r immunoas~y or analogou~ teChIliqU~ in which a labelled llgand or recep~or 18 u~ed a~ one of the 25 components. Th~ ~ssay may be quslitative, quantitativs or semlquantit~t~ve. ~uch as~ay tech~lques are well k~own, and ~nclude, for exRmple~ competltlve bi~d~ng techniques, ~_ called ~sand~ich~ te~h~iques9 and any ~odiflcation~ ther~or, ~or e:~ample, technlques ~hlch uss sompetitiYe binding and 30 "sandwlch" techni~ues together in one IB88ay., ~The term "eand-~hich" technlques as u~ed herei~ include~ ~3o~called "~ntl-globulin~ as~
I~ 8 ~omp~tltl~e blnding a~sa;y there i~, ~or . .

example~ competition between the unknown amount of one component and a ~tandard amount of the same component for a standard amount of it~ complementary component.
One of the known components :i9 generally bound to a solid matri~; whereby it c n be readily isolated and one of the known component~ iB ~enerally labelled in some way.
In one method uf carrying out a competitive bind--- ing assay, a calibration curve may first be ~et up as .....
]O ,~ollows: An antigen is bound to a solid matri~ and' then allowed to come into contact with a 601ution ron-taining its ~pecific antibody. The antibody i9 then taken out of ~olution onto the matrix, and if the anti-body or anti~en ie labelled, measurement of label of the material on the matri~ eive~ a mea~ure of the, amount of specific antibody-antigen combination that - has taken place. Thi6 combination may be interfered , with i~ a modification o~ thi~ method, by fir~t mixing the antibody ~Tith thè s~ne, but 601uble, antigen, If a laree exces~ of soluble antieen is used'the antibody bin~s this and thus re~ains in 901ution with it~
, ~pecific anti~en-bindin~ ~ites ~turated ~nd therefore unable to bind with matrix-linked anti,~en. Sllbst~n-tially no labelling will then ~p~e~r in ~ecific a590-clation with the ~olid m~trix. ~e~6 than 3aturatine ~nount6 of ~oluble antieen will result in more free antibody available for combination with matrix-linked antl~en~ The ~y~tem can there~ore be calibrated in .
..
: . , , , ' .
.- ' ''' ' ' .... ''' ~ ' -.

.

l'i' 9 .. -- 30 ~
terms of ~oluble ~ntigen and then u~ed 1to determine quan-~itatlvely ~mounts of antigen pre~3ent ln ~un~own~ ~3Qmp'l e~.
Alternativel~ the 8s8ay may be organieed oc~ver~el;y euch that tha amount o~ antib~dy remaining in t3olution aiter 5 the Elddltion of matriJt bound ~Itigen 113 that ~hlch i~ quan-titated .
As~asrs u~ng a ligand snd a non-antibody receptor. are - carried out in a dlrectly.analogous mar~er, u~ing the li~nd .
---a~ the antigen and the receptor a~ the ~tibod;y.
General~y, "8andwich~ techr~que~ are ba~ed on the f`ollow-ing: one component of an l$g~nd~receptor couple, (generally bonded to a ~olid matri~)J iB cont~cted with the sample con-ta~ning ~n unknown amount of the compo~ent to be deter ~ ed.
Thi~ bound to the fir6t component (~nd ~e~rally matrl~-l~n~ed ~ia the iirst component), i~ determ~Qd by the ~se o~
a further ~ample o~ the ~rst component tWhich hRB bee~ la-belled in some ~a~., and i~ not matri~ bound), or a third com-ponent thRt haa speci~ic af~inity ~or the component t~ be determined and that iB itself labelled. (The ch~i~ ma~ be longer $han thiB.~ .
In a "s~ndwich~ a~say uaing a ligand and no~-~nt.ibody receptor, either the ligand or the receptor may be matrix-bound~ I~ the receptor i8 m~tri~-bound~ then ~ further ~ample of that reoeptor may be used ~ter the ligand hae been b~und 2~ thereto,:~r ~ mi3ed ~s~sy maD be carr~ed out~ ~e~ the re~
cept~r ~ matri~-bound~ the li~a~d 1~ bound thereto~ a~d then labelled antibcdy agai~st the lig~d, i8 u~ed to detect th~
b~und ligandD
' . : .

.. . . . .

O 1 '~ ~

"Sandwich'l techniques have certain advantages over competitive binding techniques, for example, ~urther amplifica-tion may be achieved because there will oten be more than one receptor site for the conjugated ~enzyme to bind, so more than one molecule can bind per molecule of substance to be deter-mined, thus increasing the sensitivity of the assay.
A further advantage of sandwich techniques is that it may be possible to use a standard enzyme conjugate, which may simplify the determination and is often more convenient when carrying out assays for different substances. An example of a system using a standard conjugate is that in which a sheep antibody against the antigen to be determined is bound to a solid matrix, the antigen to be determined is then contacted with the sheep antibody, free antigen is removed, a goat ---antibody against the antigen is contacted with the antigen, free antibody is removed, and finally a standard labelled antibody with specificity for the antigenic determinants on goat antibodies is contacted with the goat antibody.
After an assay has been carried ou~ according to the chosen technique or mixture of techniques, it is usually necessary to separate the portion of enzyme conjugate that is to be assayed from the portions of enzyme conjugate that are present in the assay reaction mixture but are not to be assayed. This is facilitated ~y the conjugate being bound, during the assay, to an insoluble matrix.

~ he matrix may be~ for e~ample~ Bephadex~a pla~tics materi~l eg. nylon, cellulose or ~ derivatlve ther~of7 ~or example, brom~cetylcellulose ~cf~ Sel~ et ~19 loc Cit) ~
~ome receptors, both an~ibodiea and other receptor~5 may be cell-as~ociated ~n ivo; some antibodle~ are present ~n cell surface~ some receptors are ~180 a~oci~ted ~ith cell surfaces, and some receptors are preaent inside cellQ. Cell-hssoci~ted receptors of any typ~ may be used a~ter l~olation and puri~lcation, or the9 may be used in ~soci2tlon ~ith 10 ali or part oi ~he -ell ia . ~lready matr~--bound .
&enerallg7 the m~ bound oon~ugate iB as~ayedl, but that portio~ of the c~n~u~ le~t in suspension may be ass~yed .
The enzyme bound in the con~u~3ate is then generally 15 ailowed to cataly~e the a~propriate r~ctiGn. II two or more reac*ion6 ~re required to modulate the secondary B~tem~
thes~ may be oarried out simulta~eously or ~uccessively.as separste reacticns, in each case in 6~1 tU or ~n di~ferent reaction vss~els. T~e reaction~) catE~lysed b~ the secondary 20 ~ystem may alGo be carried out simultaneousl~ with those o~
the primary enzyme 6ystem ~ie. on modulation by the pri~ary enzyme ~ystem) or ~ eeparats reactio~ nd ma;y be oarrled out in eitu or in another reactio~ ve~e7.
It iB often preferable to allo~ the pr~na~r e~z;srme 25 - ey~tem to react for a predetermined tlme, and then t~
allo~ the modula*or tQ contact the seconda2~ system ~or a . predetermined time. ~e mentio~ed above, ~eparati~g the primary and ~lecondsry sy~tem~ make~ it f3 D ~:

.
. ', - ~ . ' ' . .
, more ~a~y to obtain qu~ntlt~.ve results und U~ve8 groater fr~edom in t~e choice Or enzymes.
It is ~so possible, in some cases, to release the primary enzyme from the en~yme con~u~ate after the portion of en%yme con~ugate to be as~ayed ha~ been i90-lated~ for example, if the enzvme is bound in the con-~u~ate by disulphide bonds, it may be released by the action of dithiothreitolO If this is done, the free enzyme i~ reacted sub~equently as described above for .10 enzyme conjugate~. .
In general it i9 preferred to have the ~econd~ry ~ ys~em prlmed, th~t i~ to ~ay, to h~ve all the components pre~ent in optimal amounts and under optimal reaction conditions 80 that the presence or remov~.l ol 1~ the a~propriate modulator will result in an immediate and optimal reactiGn. In ~o~e cases, however, it may be des.irable to ~elay the action of the secondary system;
This may be done by not combinin~ the product of the pri~l~ry ~ystem (the modul~tor) with the second~ry ~ys-tem, or by omittin~ one or mole of the component3 ofthe second~ry system. Addition of the missing component ._.... . will then initiate the reaction.

. . , , ' ,' , ' .

~, , . ' . : ~ ' ' ' -- . .

.

7 0 ~

o~ the lnventlon ' A, conjugatelmay be bound to a micro-titre plateJ
a test strip or dip ~tlek. In the ~ormer case, the whole reaction ~equence may be carried out on the platen Nicrotitre plates are well k~own ~n the art, and ones ~uitable for application in previous~y proposed enzyme immunoassays (~lisa, or ~nzyme-linked Immun~-sorbent assa~s 3 are available commercially. Test str1ps ~n~ dip sticks are al~o known.
In the method of the present invention, the enzyme conju~ate may be determined in situ ie. each of the reactions, startin~ with that catalysed by the primary enzyme, may be carried out on the micro titre plate.
Te~t strips and di~-sticks may be u~ed analoeously , to micro-titre plate~.
~ kit compri~ing components suitable lor carrying out an immunoa~say of the invention i5 al~o part of the present invention.
A conjugate of th~ invention m~y be ~repared by a~y method suitable for bindinf, li~ands and cn7;,~mes or rcceprorA and enzyme~. Such ; methods are well known and often utili~e bifunctional ,,- agents. The two component~ are preferably bonded ~.uch that the ligand-rece~)tor binding~ 9iteB on .lig~nds and receptor~ are not ~ub tantially . .

.
', ' . ' ~ ~ ~ . ' ~ :1 7 ~

impaired, an'd also that the active site of the enzyme is not inactivated~

As has been expiained herei.nbefore, the-modulator for the secondary system may serve to activate the secondary system by its production or serve t~ ac-tivate tbe secondary system by its removal if it is an inhibitor.
Of $hese two alternative methods it is generally most desirable that the modulator activates the secondary system when it is produced by the primary system. Thus a~favoured lo method of this invention comprises carrying out an assay for the li~and or receptor, the assay,requiring a conjugate .¦
between the ligand or receptor and a primary enzyme that .
is itself capable of producing a modulator for a secondary system and determining that portion of labelled component ~5 to be determined by allowing the primary enzyme to function so that sai~d modulator is produced, whereby the secondary system is caused to function, and determining a product ~f the secondary system, As previously indicated herein the method of this invention is particularly suitable when adapted for the determination of an antige~. Also as previously indicated herein the method of thls invention is particularly suitable when adapted for the determination of.an antibody.

, .

', ' ' . ' .

'.

01~

As previously indicated the amplification achieved in the method of this invention occurs because the secondary system produces substantially more molecules of detectable substance than are produced by the primary system once it is "switched on" by the primary system. Particularly rapid rises in the presence of the detectable product of the secondary system can occur when the secondary system is capable of regenerating a substrate or co-factor (i.e. a modulator) for the secondary system that is produced by the primary enzyme system This aspect of the invention in which the modulator is a substrate or co-factor for a secondar~ system that is capable of gener-ating the substrate or co-factor is particularly favoured.
Yet more favourably in-this form of the invention the secondary system comprises a cycle capable of generating the substrate or co-factor. Systems which can be adapted to favourable purposes include (a) a primary system phos,~1~ f ruC~ t~qse ~ 1.1,f/) comprising-~e~ofr~ ~nc and a secondary system (~7~ o) comprising pyruvate kinase~type I and (b) a primary system D comprising phosphofructokinase and non-regulated pyruvate kinase and the secondary system comprises pyruvate kinase type I.

As hereinbefore indicated the modulator may be generated in this invention in (a) a secondary system comprising one en~yme catalyzed reaction and one non-enzyme catalyzed chemical reaction (b) a secondary syætem ., ,, .. - ' . ., . ' ' ', , , '. , , ' .

'' ` ~

:1~'701~9 that does not comprise any enzyme catalyzed reactions or (c) a secondary system comprising two enzyme catalyzed reactions.

As previously indicated particularly apt systems for use in this invention comprises oxidation/reduction systems, of which those employin~r NAD/NADH interconversions and NADP/NADPH interconversions are most suitable.

As made apparent héreinbefore, the method of this invention is most aptly performed using systems in which the secondary systems has all components present except the modulator in optimal amounts before the modulator is allowed to control the secondary system. ~In this aspe~t the modulator will not be an inhibitor).

. Desirably the oxidation/reduction system employed is one interconverting NAD and NADH. A preferred modulator in such a system is NAD which most aptly is produced from NADP by a conjugate between a ligand or réceptor and a phosphatase In one aspect this invention provides a method for determining a ligand or receptor which method , . . .
. .
., ' ,. ' , ~' ' ' :11701'''~

comprises carryin~ Ollt an assay for the ligand or receptor, the assay requiring a labelled component wherein the labelled component is a conjugate bet~een a ligand or a receptor and a phosphatase capable of producing NA3 ~h'ich is a modulator for a secondary system which in-terconverts NAD and ~ADH and determining that portion of labelled component to be determined by a,llowing the conjug~te between the ligand or receptor and phosphatase to produce NAD, allowing the secondary system ' 10 to function in the presence of the NAD, and determining a product of the secondary system, Since this ~orm of my invention relies upon the pro-duction of NAD (the modulator for the secondary system) by the action of a conjugate of a phosphatase) the skilled worker will appreciate that the assay will be carried out in the presence o~ N~DP. Schematically therefore the latter part of the assay may be represented thus: .
' NADP
- ~ phosphQtase conjugate reduced pr~duct ~ NAD ~ oxidizable reagent reducible product A NADI~ ~ oxidized pr~duct ~ither the oxidized product or the reduced product .
..

.

~ ~ .

''``` ~1~01'~'~ "

may be determined during this assay. I believe that one of -the considerable advantages of/this amplif~ed~assay is that it allows for the detection o~ a product b~ e~e or by the use of simple optical measuring de~isesO One-partîcularly suitable method of producing an opticallydetectable colour change is to employ MT~ tetrazolium (also known as thioæolyl blue or 3-(~,4-dimethylthiazolyl-2)-2,5-diphenyltetra~olium) which on reduction can change ~rom a yellow colour to a dark colour (~or example a blueish/ greyish/ blackish colour). ~hus a favoured reduc~ble reagent ~or use is M~T tetrazo~ium.
~his reagent often produces a more marked colour change in the presence of an electron transfer reagent such as P~S (also known as phenazine ethosul~hate), ' ' . ' ' ' ' : ' '' ,':
A favoured oxidizible reagent for use in the NAD/NADH cycle is an alcohol such as ethanjol )hich can be oxidized by alcohol dehydrogenase~into an oxidized product such as acetaldehyde~ ..
~: ' . ~rom the foregoing the skilled worker will 20- appreciate that a more detailed representation of the preceeding schematic representation may be written thuso - , ., . - .. ` ., .. ~

.... .

.. .. ', '. ,' ' , ' ' - '' ' , ' -; . -. '. ' - ' ' ' ,' ' ' ' ', - - - ` ` , ' . ' ' t ~0 179 ~o NADP

~ phosphatase conjugate dark coloured NAD ethanol alcohol reduced product ~ ~ ~ dehydrogenasef//./~l~

MTT NAD~ acetaldehyde '.
Once the preceding primed cycle is switched OD by the production of NAD by the primary cycle, a colour change (by eye or machine) can be detected.
Clearly the system Will utilize NAD-free reagents since the modulator should not be introduced except by the action of the phospho:ase conjugate on the NADP.

~he phosphatase conjugate may be from any convenient source~ The phosphatase may be of the alkaline type or the acid type. ~he phosphatase may be bound to a ligand or receptor using any convenien-t method SUCh as by reaction with a bifunctional conjugating reagent such as SPDP (N-succinlmidyl-2-(2-pyridyldithio )propionate or other like agent.

.
The skilled worker will a~preciate that SUCh methods Of conjugating enzymes are well known in the art.

,, . ,' ': ' '', ~
- ; ~ . ,, .. -. . .
.- . , ~ :, .
.
.

, rjl ~

Most desirably the phosphatase is conjugated to an antibody or an antigen. Preferably the phosphatase is conjugated to an antibody. The antibody may be an antibody against any antigen including those ins,tances where the antigen is another antibody.

~ rom another aspect, this invention provides a method of determining a conjugate between a ligand or receptor and a phosphatase which method comprises contacting said conjugate with NADP and the components l~ of a NAD/NADH cycle other than NAD or NADH and deter-mining a product of that cycle~
.
~- Most ~uitably the ligand and receptor are an antibody or antigen although other moieties are en-visaged,,for example a drug and its receptor.

, ~rom another view, this invention provides a method of determining'a conjugate between an antibody or antigen and a phosph~tase which method comprises contacting said conjugate with NADP and the components of a NAD/NADH cycle other than NAD and NADH and determining a product of that cycle.
.
.

- . .

.
.. . . . .
- . . - .

Naturally the ~etllod will be carried out under conditions such that when NAD is produced by the conjugate the cycle will operate. Since sufficient amounts of the reagents required to drive the cycle will be presen~ an amplification is achieved that greatly increases the sensitivity of the detection method. Normally the reagents employed are in sufficient excess so that their concentrations do not limit the amplification achievable.

Most aptly the conjugate employed is a conjugate of alkaline phosphatase (3.1.3.1) since it has been discovered that the optimal pH for the cyclic reactions is very suitable for the operation of alkaline phosphatase (pH 8-10-5, more aptly 9-9.5 for example 9.3). This allows one to cause the primary reaction and the cycle to proceed simultaneously if desired. If a conjugate of acid phosphatase (3.1.3.2) is used the initial reaction is generally carried out at pH 4-6, for example 5.6. At this pH the cycle does not work efficient-ly so that when the pH is raised to allow the cycle to operate the acid phosphotase is effectively switched off and produces no further NAD. This has the advantage that simpler kinetics and easier quantification can be achieved.

;.~r '` .1 :l~Ol~9 -~3-Alkaline pH ~alues as outlined above may be achieved using conventional buffers such as an ethanolamine/HCi buffer~ Acid pH values as outlined abo~e may also be achieved using conventional buffers such as a citrate buffer (buffers of both types may be obtained ~rom com~ercial supplies such as Sygma or the like).

The assays of this invention may be carried out at any non-extreme temperature such as 5-45C
but generally it is preferred to carry out the assay at ambient temperaturesO
.. . .

, - Most desirably this invention provides a method ;5 of amplifying an enzyme linked immunoabsorbent assay ~ ISA) system which utilizes a phosphatase linked l 15 to a ligand or-receptor wherein the amplification is achieved by using the phosphatase linked to a ligand or receptor to produce NAD from NADP which NAD starts , a NAD/NADP cycle one product of which is determinable.
~ .

~he reagents and products of this amplified .system may be as hereinfore described.

. -- . .
~ ' .. - ' .
.
., : . . . .
.. . . . .
.,~ , :- . .
'.

:1 ~'70 1 7 9 ~ XS~ systems ~hich may be amplified in this manner include those adapted to the detection of: Malaria;
hmoebiasis; Schis~omosiasis; Onchocerciasis; ~oxoplasmosis;
Hydatidosis; Trlchinella; ~abesia; ~eishmaniàsis; Trypanosome;
cytomegaIovirus; Hepatitis ~ an-tigen; Measles; Rubella;
Pla~t Viruscs; ~rythrocyte ~ntigens; ~actor Viii-related antigens; antibodies against ru~ellaS cholera, ~.coli;
Salmonella O antigen; markers of oncological importance such as alpha-foeto-protein; hormones such as thyroid hormones and sex hormones etc; baculoviruses; ge~tamicin; etc.

~ISA systems are well known - ~ee for example':
Voller, A & ~idwelll D~. (1975) ~rit. J. Exp. Path. 56:338 - ~ngvall, E & Perlman, P~ (1971) Immunochem. 8:871 Voller, A., ~idwell, D , Huldt, G. & EngYall, ~. (1974 15 ~ull. Wld. Hlth. Org~ 51:209 ~arlsson, H.E., ~ina~erg, A.A. & Hammerstein, S. (1972) Infect~ I~nun.6.70~ ' VolIer,A, ~idwell, J.E. & ~artlett, A (1976) ~icroplate Enzyme Immunoassays of Virus Infections- from Manuals of Clinical Immunology, Chapt. 69 (~d. Rose~ N. & ~riedman, H), Am. Soc. Microbiol. p506.
Veldkamp9 J. & Vis~er, A.M. (1975) Brit. J. Vener. Diso ~:227 Vol~er, A., ~idwell, D.E. & ~artlett', A. (1976) Bull. Wld.
Hlth, Org. 53:55 ..
~, ' , ' :

-~5-Voller, A~, ~idwell, D.~, & Bartlett, A. (1976) ~he Application of Mic~o-Plate Enzyme-~inked Immunoso~bent Assays to Some Infectiou~ Diseases in ~he ~irst Internation-al Symposium on I~mun~enzymatic ~echniques INSERM Symposium No 2 (~d. Feldman et al3 North Holland Publishing Co.

An extremely effective ~ISA system is presently available as Rubelisa ~est Kit .~rom M~A. ~ioproducts, ~alkersville, Maryland 21793, USA. ~his Test Kit (their cataloque number 30-3000) is a sensitive method for the detexmination of Rubella virus IgG antibody in human serum.
However the method recommends a relatively long final incubation period and the use of a spectrophotometer.
Amplification of this test b~ the method of this invention allows for the reduction of the incubation period and allows visual determination (without the necessary use of a spec-trophotometer~. .
. . ' .

-~ 1~ 0 1~

- ~6 -The following abbreviations are used in the present specifi.cation:
GlP, Glucose~l~Phosphate;
G6P, Glucose-6-Phesphate;
F6P, Fructose-S-Phosphate;
FDP, Fructose-1,6-Diphosph~te;
ATP, Adenosine Triphosphate;
ADP, Adenosine Diphosphate;
AMP, Adenosine Monophosphate;
PEP, Phosphoenolpyruvate;

NAD, Nicotinamide Adenine Dinucleotide;
NADH, Reduced NAD;
¢a~
PGM, Phosphoglucomutase;
m~rase ~ 9 PGI`, Phosphoglucose ~sff~e~*e~;
~,~/ //) PFK, Phosphofructokinase~
(~.7 /
PK, Pyruvate Kinase;
LDH, Lactic Dehydrogenase;
FDPase, Fructose-1.6~Diphosphatase, DNP, Dinltrophenol;
. - N~DP, Nicotinamide Adenine Dinucleotide Phosphate;
. NADPH, Reduced~NADP;
APS,. Adenosine 3~-Phosphate 5'-Phosphosulphate.

..

.~ . , ', :
: .

1 1 70 1 ~ 9 -~7-The ~ollowing ~xamples illustrate the invention.
EXAI~PL~ 1 ~J a) Preparation of Pyru~ate Kinase~xtract Culture~ o~ E.col1 ~tra~n Kl-l were ~rown on a synthetic medium containing t!ssential nutrients and glycerol as carbon source, until well into their loga-rithmic pha~e of growth~ The cells were harve~ted, washed by centrifugation and resuspended in a sonication bll~fer of 5mM pho6phate, 1~1 ~DTA, 2mM mercaptoethanol pH 7.5 in an amount of approximately 20 ~g dry weight per ml. They were then disrupted with an M~E :ultra-~onic disintegrator for 4 minutesO The resulting crude extract was separated from cell debris by centrifugation.
/ 40~
As well as containing pyruvate kinase~ (~) thi~ e~tract also contalned an interfering i~oenzyme of PK with differ-ent propertie~,-and al90 a number of other -~nzyme 3ctivi-ties which interfered with the determination. It was found, however, that it was po~ible to remove all of these contaminating actiYit~ee simply by heating the - 20 e~tract to 55 ~ for 20 minutes. Thi~ heat-treated e~-tract wa~ uaed in this Example.
b) Preparation of the immunoabsorbent Bromoacetyl cellulose con~ugated human serum albu-min BAC-HSA wa~ prepared exactly a3' described in ~olid Phase A~oay of Radioactlve Antibody to Soluble Antieens by Self, C.H., Tew, J.G., Cook, R.G. and Stavitsky, Ao~
~l973) In _ _ ochemi~try, 11, ~27-2~5, ' ', ' . - .

, .

:

~01~9 c) Preparation of t~e Enzyme-Antibody ConiuFIate ~ hie was ba~ed on Pr~te~n thiolatio~ and rever~ible protein-protein conjueation. N-~uccinimidyl 3 (2-pyridyldithio) propionate a new heterobifunctional 5 ~eagent by aarl~son, J., Drevin, H., A~en~ R. ~1978) . -- in BiochemO J~ . 72~-7~7.
~Both the enz~me (pho~phofruGtokinase~"PFX"~ which .. .. had been prepared from ~tearothermophilis and purified :to crystalline purity by mean9 of ~IP and ATP a~iinity ~hromatogr~phy) and the antibo~y (IgG~ obtained ~rom ~les-Yeda ~td., Rehovot~ Isr~el), ~ere prepared for conjugation by ~eparate passage through a Sephade~
G-25 ~ine) column equilibrated with the D coupling buffer'1 of O.IM sodium phosphate pH 7.5 containing NaCl at O~lM. One mg of each protein was applied. One ml c~ tbe IgG eluant of optical density at 280 nm o~ 0~67 O.D. units was taken, ~haken gently while a ~ive-~old molar e~ce~s o~ the conjugating agent (N-succinim~dyl 2-(2-pyridyldithio) propionate, "~PDP") was added from 20 . a stock solution (5mM) in eth~nol. The mixture ~a~
allowed to stand for 30 minutes at 23~C with occasional shakIng. Then with rap~d etlrrlng, 1 ml of the PFK
eluant, also of optical density at 280 nm of 0~ 67 OoD~
units, was added. The mixture wa~ allowed to remain Ht room temperature ~or 24 hour~. During thi~ time the ~ewly introduoed labile di~ulphide group~ on the IgG
underwe~t e~change react1ons with the ~re-e~i3tlng reactlve thiol groups o~ the ~ to give ri6e to P~ IgG

7 ~ A /~

.

- . .
.. , ~ ... .

' 0 1 7 ~

con~ugate~.
d) Method of Assa.~
Into each of two ~mall test tubes were put 20 ~1 of the P~ G con~ugate~ ~uman ~erum albumin (0~1 ml .5 of a 10 m ~ml ~olution) was ~dded to one tube and ~ovlne ~erum album~n (0~1 ml of a 10 mg/ml solution) added to the other. BSA wa~ added to the second tube ~o that -: ` any non-specific.protein effect on the conju~ate would . ... be duplicated in both tube~. The volume~ of the tubes - 10 were then made up to a standard 1DO ml with phosphate-buf~ered ~aline (PBS). The content~ of the ~ubes were mi~ed and incubated at 37a for 15 minute~q -They were then a~ded ~eparately ~o two aliquots o~ BAC-HSA (each 0,1 ml of the standard suspension which had been wa6hed 4 time8 in PBS) in micro-centrifuge tubes. The capped tubes were then ~haken and incub~ted at ~7C for ~
further 15 minuteB. --They ~ere-then centrifuged ln a micro-centrifuge at ~ull ~peed for one minute, the supernatant ~olution discarded, the ~olid wa~hed by the addition of 1.~ ml of ~BS to each tune, the contents mixed on a vorte~ mixer and centrifuged ae abo~e~ ~he wa~hing procedure was repeated 4 time~ to en3ure removal of ~nbound contaminating PFE. To each tube was then ~ added 0~94 ml of the as~ay buffer (lOn~l dimethylgluta-- 25 rate p~ 6.8, 5 mM, ~IgC12~ and then 50 ~1 of a 40mM
solution of fructose 6-phosphate and 10 ~1 of a 40mM
solution of' ade~osine tripho~phate. ~he contents of the tubes were mi~ed by mea~s of the ~ortex mixer ~nd then ,. . . .
' .
-~701 ~9 incu~ated with.gentle ~haking at 37C for two and a half hour~0 ~fter this the tubes were once more ce~tri-- ~uged at full speed ~or.one minute and the supernatant solutions taken off and a~sayed for evidence of pre-vlou6 ~F~ activity ln the tube~3 during the two ~nd ahali ~our i~cubation. ~he particular ~y~tem enabled a direct comparison to be made between ths tri~gered amplifier method of the pre~ent invention and a conven-,........ tional 1:1 coupled enzymic detection method. For the : . -10 former3 the ~upernatant solution wa~ te~ted ~or its ability to activate.a primed P~ assay eet-up with~ut ~DP. For the latter the ADP produced concomitantly with the FDP was assayed by following the NhDH oxidation it o~
produced in a d~rectly coupled pyru~ate kina~e~- lactate (/,/.1 ~) ~-: 15 dehydrogenase~y~tem ~et-up to operate optimally (with . ,.~ added FDP) but without ADP. . The two a~ay 6yætems were~
.~,".,._ .
. therefore, ccmpo~Rd a8 shown in Table 1.
..~ .~
Table 1 A~say component~ for the ADP and ~DP aesay~
Primed ampli~ier Con~entional Component .(FDP determining) coupled (~DP
- determining~
NAD~ 0.1~1 O.lmM
~DH (Tnctato 14.4 UnitB 14.4 unit~
De~lydrogena se PK(I) extract 10 ~1 10 ~1 P~P ~ 2.0mM 2.0~;
ADP 2.0mM ~ NI~
~DP NIL . . 1.0~l ATP 0.4_1 0.4m~l '~ ' -51~
T'able 1 contd.0 supernatant ~olution 100 ~1 100 yl a~e~y bu~er - to 1 ml to 1 ml ~ The ma~i~al amount of P~'P wa~ used which could be added without causin~ unwanted background actlvation of the pyruvate kina~e in the ~DP-determining aesays.
; Results ~ . i~ Primed amplifier (FDP-dependent) assy.
.-.~ Thi~ waa set ~p such that ~ull activation of the 10. sy~tem produced a rate of o~idation of NADH
7.9 nmole~/min( ~ convenient.r~te to me~s~re).
The re~ults of adding 100 ~1 o~ the supernatant ~olut~on ~rom the immunoabsorbent~
- either previously e~posed to ~SA or BSA are shown ln Table 20 ~ . ~ ' ' .
Primed amplifier ~DP-dependent~ assay re9ult9 supernatant rate o~ NADH oxidation from BSA-exposed tube 4.8.nmoles /min (61 ~ maximal) ~rom H~A-e~posed tube nil ii) Conventional-coupled (ADP-dependent) as~ay~
The result~ of adding 100 ~11 of either supernatant eolu~ion to the ADP-dependent ~y~tem are 3hown in Table 3 0 2~ .

' ." ' .. . . .
-.

-` ~1'70;l7g -52~

?able ~
;Conventional-coupled ~ADP-dependent) a~aay resulta , supern~tant rate o~ NADH o~idation ~rom ~A~expoaed tube 0.12 nmole6/~i~
(1~6 ~ ma~lmal)- h~
:'j irom H~A-e~posed tube nil ; ~ ~hi~ figure on repeated iassays was clo~e to back-.: ground and di~`~icult to quantitate accurately.
~he reBult~ show clearly that:
.:
; a) the ~FE-IgG conjugate was inhibited in its binding to io BAC-HS~ by ~d much more than by B~A.
b) the aenaitivity o~ the primed-ampli~ier system was much higher than the conventional-coupled reaction-.

:

;

0 ~ 7 ~ -EXA~IPLE 2 1~PG;~
&~P [~
PGI
O
~second~ry~ p ATP
enzyn;e ~ ~ ,~
~yGtem FDPa~e ~ ) P~{

FDP4 ~ A:DP , ' P~P
~ ~ f ~ P~
`' ~ . ~ ' . . ~ ATP :Pyruvate NAl~H. ¦

N~4D ~
~actate ~ -.

'~

1.' , , ' ~ ~
.

. . '. , : . ; '' ' .:. . ' - . ' : . ' .
, ' ' .

~.7V1 ~3 The reaction sequence is outlined above. Phosphoglucomutase (2.7.5.1) (PGM) was chosen to be the enzyme to be llnked to an antibody. The presence of the antibody is determinable by the ability of the conjugate to catalyse the formation of G6P from GIP. This in the presence of phosphoglucose isomerase (5.3.1.9) (PGI) results in the sequential formation of F6P
which is then itself acted upon by phosphofructokinase (2.7.1.11) (PFK) to give FDP. This latter reaction generates one molecule of ADP from ATP for every molecule of F6P convert-ed to FDP. The final detection system shown in the diagram takes advantage of this, in that it is independent on ADP.
This system results in oxidation of NAD~ which is monitored spectrophotometrically by the concomitant decrease in optical density of the mixture to ultraviolet radiation of 340 nm. --~
The secondary enzyme cycle operates as follows. In the presence of fructose diphosphatase (3.1~3.11) (FDPase) the FDP generated by the system, and which otherwise would play no further part in the system, is reconverted into F6P
(without involvement of ADP/ATP) and is thus available again for a further turn of the cycle, to give rise to another molecule of ADP for each turn of the cycle.
The amplification of the activity of PGM achieved by the catalytic secondary enzyme cycle may be readily seen by comparing the NAD~ oxidation resulting from the corresponding 1:1 linked enzyme system which does not have FDPase (3.1.3.11) present ie. in which F6P is not recycled.

s .
~ 54 -.

:1~7017~

Method of enzyme assay All deter~inations were performed using lml quartz cuvettes with a path-length of lcm. N~DH oxidation was monitored by the decrease in absorbance at 34Onm. The reactions were carried out in a buffer comprising: 25mM
Tris~HCl, lOmM MgC12, lmM EDTA at pH8.00 and 30 C. The final reaction mixtures were as shown in Table 4. LDH and PK were obtained from Boehringer Mannheim and the other enzymes from the Sigma Chemical Company.

Results of enzyme assays These are shown in Figure 4 and represent the activities of the systems after they had been incubated for 12 minutes at 30C, after initiation.
The results indicate that: --(i) the basic 1:1 linked enzyme reaction sequence worked (compare first to third column), (ii) the catalytic substrate cycle formed by inclusion of FDPase into the system markedly increased the activity of the system for the standard amount of PGM ~column two).
(iii) the background activity of the total system, including the substrate cycle, in the absence of PGM is very low (column three).

. .

I :L7V 17~

Table 4 components~ 1:1 linked including no PGM
(no cycle) cycle (background) NADH O.lmM O.lmM O.lmM
ATP O.4mM O.4mM O.4mM
PEP 2.0mM 2.OmM 2.OmM
_ __ GlP 2.OmM 2.OmM 2.OmM
LDH 1.1 U 1.1 V 1.1 U
PK 0.04 U 0.04 U 0.04 U
PGI 0.2 U 0.2 U 0.2 U
PFK 0.02 U 0.02 U 0.02 U
FDPase NONE ADDED 0.1 U 0.1 U
PGM 0.06 U 0.06 U NONE ADDED

oxldation rate _ _ _ of NADH 0.475 4.72 0.064 tn mole/minute) 'U' stands for units of enzyme activity~ However as different conditions were used to measure each enzyme by their various manufactures the various activities do not necessarily correspond to each other in the final assay mixtures but simply denote the standard amounts of each preparation used.

Assay buffer of 25mM Tris-HCl, lOmM MgC12, lmM EDTA, at pH 8.00 added to each assay to a final volume of l.OOml.

,~, ' , ~ ' : ~

.~

1 :1 70 1 ~J 9 ~inkin~ the substrate cycle to anti'body-antigen reactions.
After demon~tr~ting the amplification afforded by the substrate cycle as above, .the~
usefulne~s o~ the ~y~tem in detecting antibody (or anti~en) was asses~ed~ Thi~ was done by conjugating the prlmary ~ e:nzyme, PG~7 to IgG
wi~h specifici~y again~t humian ~erum albumin_ .

Formation of the con~ugate A6 in Example 13 the hetero- -bifunctional linking reagent SPDP was used, how-ever in this instance both enzyme and antibody ~er~ treated before con~ugationO
One mg of the IgG (Miles Yeda ~td) wae passed through a ~ephadex G 25 column previously ~quilibrated with the Icoupling-buffer' (O.lM
sodi~n pho~phate pH 7.5 containing 0.1M NaCl)~
~he antibody eluted from the column was then shalcen gently wlth a ten-fold excess of SPDP which was added from a 5mM stoc]c solution in ethanol. ~he mixture was allowed to sta~d for ~0 minutes at 23C
with occaslonal shaking. It wa~ then passed through .
another SephadexG- 25 column, this time equiIibrated with 0.1M ~odium acetate at pH 4.5 containing O.lM
NaCl. ~he.proteln fraction of the elua~t was taken an equal volume of 100mM dithiothreitol was added wi~h rapi.d stirring. ~he mixture was left for 20 mlnute~ at 23C after which it ~Jas laEsed.th~ough - . . .

. .

.! - .1170179 another Sephadex G-25 column equilibrated with the original'coupling buffer'.
At the ~ame tlme~ 1mg of PGM wa~ pa~ed through a G-25 column equilibrated with coupling buffer. The ma~or protein ba11d of the eluant was then~expo~ed to a ten-fold excess-of SPDP as above c~nd left al~o for 30 m1nutes at 23C. It wa~ then mixed rapidly with the treated antibody and left to ~ta~d for 2 hour9 at 23 C to allow the con~ugate to .
. -lO form.
- Full Conjugate A~say :~. . Into each o~ two small test tubes were ~ put 0.2ml of the PGM-IgG con~ugate. Human serum album~n (0.1ml of a 10mg/ml solution) waB added to one tube and.the ~ame amount of bovine ~erum.albumin to the other; 0.7ml of phosphate~bu~ered ~aline ~ ; .
(P~S) wa~ then added to both tubes and the contents mixed,and incubated at 3?C for 15 minutes. ~hey were then added separately to two aliquots of BSA-HSA
(each 0,1ml of the ~tandar(l ~u~pension WhiCIl hnd been washed 4 times in ~S) in micro-centrifu~e tubesO
~he capped tube~ were-then shaken and incubated at ~7C for a further 15 minutes. ~hey were tl1en cent-.-~ .
rifuged in a micro-centrifuge at full speed for one minute9 the supernatant solution diæearded, the ~olid washed by the additibn of ~.5 ml of P~S to eaoh tube~
.
the content~ mixed on a vortex m~xer and centrifu~ed a~ above. ~he wash~ng procedure was re~eated 4 times .

..

: ' ~ 3 ~01 ~3 to en~ure removal of unbound contamlnating ~GM~ To _ ; each tube was then added 50~1 of a 40~ ~olution o~
~lucose-1-phoaphate and assay bu~fer (25mM ~ris-HCl., 10~1 MgC129 1mM EDTAJ pH 8.00~ to a final ~olume of lml. ~he ~ontents were mixed and then incubated wi~h gentlë shaking for two and a half hour3 at 37C. After incubation, the tube~ were cent~lfuged at full ~peed for one minute and the Yupernatant solution~ taken off witll rasteur pipette~ and the content~ a~sayed 1~ for evidence of PGM activity during the two and a half hour incubat~on, ~hey were assayed in two way~.
By the direct 1:1 linked enzyme sequence outlined above and also by the ~equence inc~uding the secondary enzyme cycle (FDPase included~ ~he assay3 were set up a~ previously de~cribed.but included 0.2ml of : the ~uperanatant ~olution~ and thus 0.2ml le~ of the separately added as~ay.bu~fer in each case~
Result~ `
~hese are shown in Table 5. They show two ~o feature~:
(i 3 that the ~ubstrate cycle inclusion into the as~ay provides for much greater activity o~
the syBtem ~or a given amount of conjugateq (i~ ) that the ByS~em is capable of showing the 25 . inhibition of uptake of the conjugate onto -- BACi-HSA by ~oluble HSA as a~ainst the control . of ~SA.
',' ' ' ' .

.
.,. ' . ' . '' : . i -' ' . ;
~:. - - - - - . . . ' .. ;
' 0~7 ~able 5 Activltie~ of ~AC-HSA bound PGM-IgG conju~ate6 i~
terms of final NADH oxidation ~ln n moles/minute~

wlthout sub~tratewith sub~trate supernatant cycle cy¢le HSA-exposed tube 0.19 - 2.21 SA-exposed tube 0.36 . ~-7 ..j., ~=~

, - ' ~ ' .
.

. . .' ' . ' ', ', ' ' , ., ': ' . ' ' -. . . . . .

, . . . .
- '~ ' ~ ' .. :
- ~ ~

.

1 1 70 L 7 ~

_xample 3 Detection of Pyruvate kinase (II) In order to demonstrate the enhancement in sensitivity brought about by the method of this invention an assay for pyruvate kinase (II) was carried out with and without a secondary system to produce amplification.
The two assays may,be represented as follows:

Assay without Ampli~ication Assay with amplification PEP ~ ADP PEP ADP
~ PK(II) ~ 2PK(II)~
Pyr ATP Pyr ATP~ E6P
~ PKF
NADH\ ADP. FDP
~L~H
NAD lactate ~,~

ADP PEP
~ PK(I)~
ATP Pyr NAD~
d L~H ~
1 act ate NAD
.

, .,. ' ' , '. . ,' . . ~ ' .
~ ' ' ~ ' ' ' :

In the' assay system without amplification PK(II) converts PEP and ADP into pyruvate and NAD~ to NAD and lactate. The change in concentration of NADH can be monitored by absorbence at 3~0nm.

In the assay system with amplificatioD ( that is the assay incorporating the secondary system) the preceeding sequence takes place but in addition the secondary,system generates ATP. In this amplified system, in addition,to the PEP, ADP, NADH, PK(II) and LDH present in the non-amplified system, the following are also present: F6P, PFK
and PK(I). In the presence of ATP (generated by primary cycle) the P~K converts the ~6P into FDP. The FDP activates the PK(I ) which then is available to convert the PEP
and ADP to ATP and pyruvate. The ATP thus produced feeds back withmthe secondary system and the pyruvate is converted into lactate by LDH in the presence of NADH. This end .,reaction is monitored at 3~0nm as iD the unamplified assay, To indicate the usefulness of this amplification method the PK(II) was initially assayed by the direct reaction as ~ollows: The assay mix was as shown in Table 6 (left column) with a buffer consis*i~g of 1~ dimethyl-glutarate pH 6.8 ~nM MgCl2. All assays we~e carried out at 30C. The PK(II~,was obtained from Calbiochem and, as a.
25 result of trials, a standard amount chosen which gave the ' .-,". . ,' ' ' '' .
. - , . . . . .
- .
.
, . . :
'' ' ~ ' I' ~ l~Vl~ I
-~3- !

activity shown in the 'rable. This amount was used for a~l following experiments. PK(I) was as in previous experiments.

The assay system including the positive feed-back amplifier was composed as shown on the right hand column of Table 6. The activity attribuatable to the addition of the same amount of PK(II) as before and a~ter allowing the feedback system to get underway for seven minutes, is shown. The activity a*tribuable to PK(II) is raised eleven times over the direct method.
Table 6 _ _ Direct Feed-back assay assay . __.___ -ADP 0.4mM O.~mM
PEP l.OmM l.OmM
NADH O.lmM O.lmM
LDH 1.1 U 1.1 U
PK(II) standard standard F6P 2.Om~
P~K 1.0 U
PK~I) 10 ~1 Buffer To l.Oml To l.Oml Rate of Change __ _ _ Absorbence (OD 0.005 0.055 Units at 3~0nm per 10 minutes~
. ~ .

Using the direct method, the standard ~ount of .
.

- . . . - - . , ' ~

' .
'' 1 7 ~

-6~-PK (II) could just about be detected whereas it was quite r.eadily detectable using the feed-back amplifier.

In any situation where the PK(II) is used as a label in analogous manner to those used in the Examples hereinbefore the increased overall activity of the secondary system attributable to PK~ would clearly be of benefit in the detection of material labelled by the enzyme. Thus for example, on labelling antibody agaiDst human serum albumen with PKII), a complex is available for the detection of human serum albumen as a result of the activity of the primary enzyme tri~gering a secondary sequence which is itself capable of generating modulator for example as described iD Example 2.

Exampl_ 4 Detection of Anti-rubella antibody.

A Rubelisa 96 well plate is taken and labelled ' ' - . ,. : ' ~ ' ;

, 0~7~

as necessary. ~he wells are washed b~ filling each with ~S-Tween~fr~m a ~ash bottle and then air bubbles are removed ~rom the wells by moving the plate gently back and forth~ ~he P~S-~reen is shaken from the wells into a disposal receptacle containing a solution o~ 5% household bleach and each well refilled with P~S-~ween a~d the air bubbles removed again. ~he plate i~ then left for three minutes and then emptied again aq above. The whole process is repeated two more~times - the final time ensuring completeemptying by tapping the upside-down plate on a clean paper towel. Reconstituted serum diluent (250 ul~
is then added to each well. ~he patients' individual sera are shaken to homogeneity and then 5 ul of each serum is added to one well coDtaining rubella antigen and also to one wel~ containing control antigen. Serum is withdrawn and expelled three times in the ~ell to help mix it. It i~ of utmost importance to use different pipe~tes for -different sera. ~hree control sera (negative, low positiYe and high positive) are included with each test that i~
carried out. ~hree pairs of wells must therefDre be used for the~e controlsO If le~s than three patients' sera are to be tested at any one time it is advised that these can be positioned immediately following the test sera on the plateO However9 ~f the plate is used to its maximum capacity of 45 separate test sera then it is advised that the control samples are distributed on the plate as: high positi~e - A1 & Bt, low positive - C11 & D11; negative -.
~ r~3D.e J~f~
- - - . .. . . .
.. . ' . , . ' . . . .
.

. . .

~17()1 ~

G12 ~ H12 (5 ul of each), If fewer than 45 patient~l sera are tested it is advisable to place them with the high positive at the beginning, the lo~ positive in the middle and the negative at the end of the batch of sera tested.
Af-ter all of the sera are added the plate is placed on a Micromixer for a few minutes. ~he plate is placed in a plastic bag with a wet cotton o:r paper ple~get, the bag sealed to allow a humid atmosphere to develop and kept at roorn temperature (20-25~ f`or two hours. The plastic 1, bag is then opened and the liquids are shaken from their welIs into the disposal receptacie. The wells are then washed again as described above (with PBS-Tween, four times). Then 250 ul of 50-fold diluted conjugated anti-serum supplied with the kit is added to each well. The plate is incubated as above in the humid bag for a further 2 hour~ at room temperature, after ~hich the liquid is shaken from the wells into the disposal receptacle and the plate washed again four times as above. The wells are now ready to be assayed for their alkaline phosphatase content~ ~o each well is added 210 ul 0.14M ethanolamine-HCl buffer at pH 9.3 and containing 5mM MgC12. The following are added to each well: 10 ul 10mM thiazolyl blue tMTT?7 10 U1 of 40mM phenazine ethosulphate (PES), 5 ul of alcohol dehydrogenase supplied by the Sigma Chemical Compan~r ~td e~pecially free of NAD ~cataloque number A3263) and 5 ul o~ 1M ethanol. NADP is then added to each well.

.. . .
~ " . ' ' . ' ' . ' . ' . .

-; .

`` 11~0:17~
.-67-~his iB done b~ adding 10 ul of a 10mM solution of IIADP
to each well. Depending on the number of samples being tested and the accuracy required this may be achieved either by adding the solution to each successive well quiclcly and then mixing on a Mioromixer or (with many samples3 by adding the solution at fixed intervals (such as a second to minutes) from one well to the next. ~he reactions are then monitored by obserYing the colour change of the solutions from pale yellow towards black visually - taking account of any differences in time of addition of the NAD~ solutions to the various wells.

~ he solution in the well initial~y coated with rubel~a antigen and to which high positive control serum was added will change colour at a much faster rate than the solution in a rubelIa antigen well to which serum containing significantly less anti-rubella antibody activity was added, and also much faster than thé well containi~g control antigen to which the high positive control æerum was added. ~ .

If required, the reaction bringing ab~ut the colour change ~an be stoppe~,after a suitable period (such as minutes to hours - depending on the æensitivity required) for example 5 mi~utes, by for example changing ' - - . ' .
' " ' ' ' .

7 0 .1 7 the pH of the solutionO To achieve this the contents of a well can be removed to a vessel, such as a ~,Oml colour-imeter cuvette, containing 0.75ml of 2M citrate buf~er at pH 4.8~ the solutions mixed and the degree of previous reaction documented by measuring the degree of colour change from pale yellow to black agai.~st a standard chart or by use of a colourimeter set to 570nm. ~his alternative has the advantage that the degree of reaction does not have to be documented at the very time it is occurring.

In anot~eralternative, the wells may be assayed for B their alkali~e phosphatase content by allowing them to first act on a standard NArP solution which is then trans-fered to another vessel for the colour generating reaction I to take placeO In this alternative~ the NAD concentration is not therefore continuously increasing whilst the colour reaction is taking place and so quantification is some what easier~ For this, 250 ul of 0014M ethanolamine buffer at pH 9.3 and containing 5mM MgC12 and 0.4mM NA~P
is added to each well to be assayed.. ~he plate is incubated ~0 at room temperature ~or a time such as minutes to hours (depending on the sensiti~it~ required) for example 5 minutes.
At the end of this period the coDtents o~ each well are added to suitable vessels containi~g 10 ul o~ 10mM M~, - 10 ul of 40mM PES, 5 ul of alcohol dehydrogenase~(A ~ 63), 5 ul o~ 1M ethanol in 0~14M ethanolamine buffer at pH 9.~
sufficient to ~k the flnal ~cl~e 1.0 I,~and t}e c ~ tents .

, miY~ed. Again the colour change of the solutio~s from pale yellow to black is monitsred either vlsually or with a colourimeter set to 570nm. The results of these alternative approaches will be found to be in accordance with that stated above i,e~ wells initially containing rubella antig~n which recei~e a serum high in anti-rubella antibody, by the methvcl of the test, will give rise to a faster development of black colour than wells which do.not meet these criteria.

-.
- . ,

Claims (35)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method for determining a ligand or receptor, which comprises carrying out an assay for the ligand or receptor, the assay requiring a labelled component, wherein the labelled component is a conjugate between (i) a ligand or a receptor and (ii) a primary enzyme that is itself capable of producing or removing a modulator for a secondary system or that is the first enzyme in an enzyme system that is capable of producing or removing a modulator for a secondary system, and determining that portion of labelled component to be determined by allowing the primary enzyme and any other enzymes in the enzyme system, to produce or remove the modulator for the secondary system, allowing the secondary system to function in the presence or absence as appropriate of the modulator, and determining a product of the secondary system.

2. A method as claimed in claim 1 which comprises carrying out an assay for the ligand or receptor, the assay requiring a conjugate between the ligand or receptor and a primary enzyme that it is itself capable of producing a modulator for a secondary system and determining that portion of labelled component to be determined by allowing the primary enzyme to function so that said modulator is produced, whereby the secondary system is caused to function, and determining a product of the secondary system.

3. A method as claimed in claim 2 which is adapted for the determination of an antigen.

4. A method as claimed in claim 2 which is adapted for the determination of an antibody.

5. A method as claimed in claim 2 wherein the modulator is a co-factor for a secondary system that is capable of generating co-factor.

6. A method as claimed in claim 5 wherein the secondary system comprises a cycle capable of generating the co-factor.

7. A method as claimed in claim 5 wherein either:
(a) the primary system comprises phosphofructokinase and the secondary system comprises pyruvate kinase type 1.
(b) the primary system comprises non-regulated pyruvate kinase and phosphofructokinase and the secondary system comprises pyruvate kinase type I.

8. A method as claimed in claim 2 wherein the modulator is generated in (a) a secondary system comprising one enzyme catalyzed reaction and one non-enzyme catalyzed chemical reaction (b) a secondary system that does not comprise any enzyme catalyzed reactions or (c) a secondary system comprising two enzyme catalyzed reactions.

9. A method as claimed in claim 8 wherein a reaction involves nicotinamide adenine dinucleotide (NAD) reduced NAD (NADH) interconversion or nicotinamide adenine dinucleotide phosphate (NADP) and reduced NADP (NADPH) interconversion.

10. A method as claimed in claim 2 wherein the secondary system has all components except the modulator present in optimal amounts before the modulator is allowed to contact the secondary system.

11. A method as claimed in claim 8 wherein a reaction involves NAD/NADH interconversions for which NAD is the modulator.

12. A method as claimed in claim 11 wherein the NAD is produced from NADP by the action of a conjugate between a phosphase and a ligand or receptor.

13. A method as claimed in claim 12 wherein the conjugate is one between alkaline phospatase or an acid phosphatase and an antibody.

14. A method as claimed in claim 1 wherein the modulator is a co-factor for a secondary system that is capable of generating the co-factor.

15. A method as claimed in claim 14 wherein the secondary system comprises a cycle capable of generating the co-factor.

16. A method as claimed in claim 15 wherein the cycle comprises the interconversion of nicotinamide adenine dinucleotide (NAD) and reduced nicotinamide adenine dinucleotide (NADH).

17. A method as claimed in claim 16 wherein the modulator is NAD.

18. A method as claimed in claim 17 wherein the modulator is obtained by dephosphorylation by the primary system.

19. A method for determining a ligand and or receptor which method comprises carrying out an assay for the ligand or receptor, the assay employing a labelled component wherein the labelled component is a conjugate between a ligand or receptor and a phosphatase capable of producing NAD which is a modulator for a secondary system which interconverts NAD and NADH and determining that portion of labelled component to be determined by allowing the conjugate between the ligand or receptor and phosphatase to produce NAD, allowing the secondary system to function in the presence of NAD, and determining a product of the secondary system.

20. A method as claimed in claim 19 wherein the NAD is produced from NADP.

21. A method as claimed in claim 19 wherein the interconversion of NAD and NADH is accompanied by the oxidation of an oxidizable reagent and by the reduction of a reducible reagent at least one of which oxidation and reduction is accomplished enzymatically.

22. A method as claimed in claim 19 wherein the interconversion of NAD and NADH is accompanied by the oxidation of an oxidizable reagent and by the reduction Or a reducible reagent both of which oxidation and reduction is accomplished enzymatically.

23. A method as claimed in claims 21 or 22 wherein the reducible reagent is a tetrazolium salt which is reduced and produces a colour change.

24. A method as claimed in claim 21 wherein the enzyme causing oxidation of the oxidizable reagent is a dehydrogenase.

25. A method as claimed in claim 24 wherein the dehydrogenase is an alcohol dehydrogenase.

26. A method as claimed in claim 19 wherein the conjugate is between a phosphatase and an antibody.

27. A method as claimed in claim 19 wherein the conjugate is between a phosphatase and an antigen.

28. A method as claimed in claim 26 or 27 wherein the phosphatase is an alkaline phosphatase.

29. A method as claimed in claim 26 or 27 wherein the phosphatase is all acid phosphatase.

30. A method as claimed in claim 1 wherein the enzyme is a phosphatase and the phosphatase linked to a ligand or receptor is used to produce NAD which starts a NAD/NADH cycle a product of which is determinable.

31. A method as claimed in claim 30 wherein the NAD is produced from NADP.

32. A method as claimed in claim 30 or 31 in which the cycle is accompanied by the reduction of a tetrazolium compound which produces a detectable change.

33. A method as claimed in claim 2 wherein the modulator is a substrate for a secondary system that is capable of generating the substrate.

34. A method as claimed in claim 33 wherein the secondary system comprises a cycle capable of generating the substrate.

35. A method as claimed in claim 34 wherein the cycle involves phosphorylation and dephosphorylation.