CA1183080A - Use of synthetic bifunctional ligand for the immunometric determination of the concentration ratio of two solutes - Google Patents

Use of synthetic bifunctional ligand for the immunometric determination of the concentration ratio of two solutes

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Publication number
CA1183080A
CA1183080A CA 398267 CA398267A CA1183080A CA 1183080 A CA1183080 A CA 1183080A CA 398267 CA398267 CA 398267 CA 398267 A CA398267 A CA 398267A CA 1183080 A CA1183080 A CA 1183080A
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CA
Grant status
Grant
Patent type
Prior art keywords
solute
solid phase
bound
component
antigenic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired
Application number
CA 398267
Other languages
French (fr)
Inventor
William F. Coulson
Terence S. Baker
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Celltech R&D Ltd
Original Assignee
William F. Coulson
Terence S. Baker
Celltech Limited
Celltech Therapeutics Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
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Abstract

Abstract The use of a synthetic bifunctional ligand for the immunometric determination of the concentration ratio of two solutes The specification describes an immunochemical method for detecting the fertile period of the menstrual cycle, by determining the ratio, in urine of the two hormonal metabolites oestrone-3-glucuronide (E13G) and pregane-diol-3-glucuronide (PD3G). The method depends of deter-mining how an immunocomplex dissociates in the presence of the two metabolites. The immunocomplex comprises a solid phase with antibodies to PD3G attached, and bifunctional ligand (E13G and PD3G both covalently bound to bovine serum albumin) bound to these antibodies, finally E13G antibodies are bound to the E13G moieties of the bifunctional ligand.

The immunocomplex is incubated with urine. The meta-bolites in the urine compete for the antibodies so the immunocomplex partially dissociates. The solid phase is washed and the number of exposed E13G moieties remaining depends on the ratio of E13G to PD3C in the urine. These are determined with enzyme labelled E13G antibodies.

Description

~3~8~
:L -`le llSf' of rl 'iyrl~het.ic l3~ cl,j,o~ ,l lir!arld i'or the lmm~ nlllol:rie ~oS~t!T~ i,rl~ ioll of tbo (`onccrl~;ral,:ir~rl l`~at,io o~ o 'iolllt~
__.. . . ,_ _ _ _ _ . .,._,_ __ _ _.. , ._ _, .. _ _ _ _ . _ .. __, _, __ ~rlt,~?(!-~!-r-~t-lorl The speciFicatit)ll descrihes a i-,;in~ple techn~ t, (a kit) for tletsrmin,ing reliably thrJ feItil~ per,iocl of the l~urnclr rnenstrual cycle. ~-;ut~l~ a kit: ~i],l be suit:ab:l,e for horrle use, to enable a WOlliall t,o de~errl;ine ~herl srle i5 fertile and so avoid CClnCeption by p~riod:ic abstlnarlce f'ronl L~ ,intercourse. Convsarsa:ly the ab:iJity ~,o pred,ict OVlJ-; lation will be valuable in cases ~ ere conception i5 d 3Si red.

It is proposed to llSC a techni(lue in ~Ihich thl3 ratio 15 of two steroid rnetabolitec,~ in urine, is rneasured directly; a signal being generated in direct proportion ~l to the value o-l~ tilis ratie. The t~lo metabolites are:
s 3-hydroxy-oestra-1,3,5 (10) - trielle-17-orle 3-~-D-i glucupyranosiduroni.c acid (trivial name: oestrone-3-n ~lucurorlide or E]~G) and 5~-pregnane-3~-v~,2~~diol 3~-~-D-glucupyranosidurollic acid (trivial name: pregncanediol-' 3-glucuronide or PD3G). The signal ~li].l be a colour produced by an enzyme-labelled antibody. lhe novelty ~ of the spproach lies in the use of a mixed steroid j, J g].ucurorlide-protej,n immunocomplex to measure directly a stero;d/steroid ratio. Thj,s principle can be applied to the measurelnerlt of any solute/solute ratio.
~, .
Materials Mixec,~ steroid ylucuronicle-protein complex.
The scheme is based on the preparat:ion o~ a macro-s molecule containiny both oestrone-glucurorliSda and I pregnanedio]-glucuronide cova:Lently linked to a protein, 9uch as bovil~e serum albumir, (BSA~
~,,, - ,' ~

........ . . .... .. - .- --ni de)~.
.~j Ihe metl-locl used wi.l.l be~ thr mixerJ arlllyclJ.~-ide r~ cti.on 5 ac describecllwitll Lhe clifferer-~ce tha~ two stero.i.cls glucurnnides insteLld of one wou.l.cl br.~ .involved ~ thr reaction. Tllc adval-ltage oF usl.ny a protej.rl, such as bovine serurn albumi.n is ttlat -the nulTIbl-r of rnolccll1.es o~ each ste].~old ~J:Lucuron.i.cle per molecu:l.e of 13S~ cal~l be 10 independently varied by adjuc.ti.rlg the stoich.iomrtry of reagents. It is interlcJed to synthesise a rarl~Je of mixecl stcroid glucurorlide-BS~ cornF)lexes wi-th ~aryin~
ratios of oestrone-glucuronide to pregnanediol-g:lucuro-nide. Such mixed steroi.d glucuronitle--BSA complexes will 15 be capable oF sirnultaneously binding both oestrorle-glucuronide antibodies and p:reananediol-glucuronide ant1bodies. The complex with an op-timum ratio will finally be se:Lected after tes-ting in the reaction scherne set out be10w.

Ant sera ImrnunoglobuLin enric~hed fracti.ons of both oestrone-3-gluc-uronide and pregnanediol~ gl-lcuronide antisera will be used.

2~
Oestrone g].ucuronide antiserum labelled with the enzyme "horse radish perox.idase" prrpared by 2 modification of the method using cJlutaraldehydeas thr-~ cross linl<:ing eagent2. An alternative enzyme label all<a1ine phospha-I 30 tase w:i.ll also be investigated.
t Solid Phase .
- Polystyrene test tubes (50 x 6mrn) obtained clean and sterile from Sterilin Ltd wll.l be use(i as a solid phase.
The antibody coated tubes are robust and can be stored

3~ empty, in a sealed contailler, at -~0C for many months, without .l.oss of an-tibody activ;ity. Alternatlve soli.d phase supports ~`~ill he investigated SUCil as filter paper strips, to which antibody is c~valentl.y bound~

~' ~ ]. () c t~ cl ~
The rncthotll-,roposecl :is in two pa:rts. (1.) prr?paratiorl of a sol.:icl phase :ilnlnurlocomp~.e~x (lig.l); (2) the testir-lg o~ a sample for a [-:13G~PI~(; rca~.i.o (Fi (1) Pre~ t~l_,_orl ~ r ~t~ r ~ ,t~ c~e ~ r~ o~nr~
1. Coatil-lg of tubes w.il,l-l prerlllcln~cl;.o,l-gl(lcuroll.itJe antiser lJ m .
Z. Bufier wash.
. 10 3. Incut~a~ior\ of ant,ibocly coated tubes w:itll the rnixed steroid glucurorl:ide--BSA complex (i(? E~.~G-~S~-P~G).

4. Buffer wash.

5. Incubati.on of coated -tubes with oestrone glucuro~
nide antiseIum.

6. Buffer wash.

7. Store empty, but sealed at ~20C ullt.i.l required.
. (2) Us~ of ab,ove preparatiorl to test fnr the E13G/PD3G
, `l 1. Add (d:iluted) urine to immurlocomplex coateci tubes.
. ~ 20 Il~cubat:e.
, ~ 2. Buffer wash.
3. Add excess enzyrne ].abellecl oestronr-~,-glucuronide , : anti.sera.
4. ~uffQr wash.
2~ 5, Measure arnoullL of label, by additi.on of subs~rates followed by colori.metry.
. NB. Excess.reayent i.n step 3 ~i.ll enab.l.e thls to be ~. a very ~hort step. Also ar,y non-specific interfering ,. factors from the uri.ne sample will be washed away therefore en~.yme blanks should be minirnal.
Rationale lle reaction end point ~lill be determineci by tlle amount ~ of resi.dual labelled oestrcne g].ucuronide antihody bound : . ~5 to the sn.licl phase~ and this.will be proport,ional to the number of exposed oestrone glucuron:ide residues on the soli.d phase after ineutJation wit,h dilutrld urine. This in turn is dependent on two factors. (:L) t;he number of :' ... .......... ~ .. , .. ...... . ~ .. . . . .. . . .. . . ........... . ..

~:1.3(; r~ r(Jp(!~iorll:L t;o tll~ (oll(r.~r~ itirl of oestrogen gluc~ o~ in tlle ~.lr:i.nr ~ mp.L~.~, si.ncr,, fl,e oesi:rolle (Jluc-1rollicle w:i.l.l compctj.t:i.ve:1y d:i~)J.ac(- nc~
:Lahe] 1ed OeStrOr1e (J1UCU:rC)rl:j (I(e .1ll~ j L)O(J1eS IJ~OIII t;-O j mlrlll~nocumple~x (2) the~ cJ-Irlt oF :i.mmul)oco1lll)lr?x hour~ to tile solicl ph..arle wil.l. br invc3rC;c]y propolt::iona:l t(J the collcelltral;io,- O r prcynanetii.o]. g.luc:urorl:icle in ttlo urllle sample~ as fret3 prt-3cJrl.lnecliol g:Lucurol-l:i.de l~rill colllF)r~
t.ively displace BS~ :Li.nl<ecl prc(lanecl:io1. 9I IJCllrOnj CIe t`rOm the solicd phast-~F).rfcJnarlediol g:l.ucllrorlidt3 ~-arlt.ibocl:i.ec;.

It f'olJows that, for ralTIples containi.l-g high concell'tra-.tions of oestrone glucuronide and low concentraL:ions - of pregnanedlol glucuronide (periovulatory phase) maxi--.- I5 mum binding of l~belJ.ed antibody will be ahjeved i.e.
high El3G/~D3~'. r~-tio wil:L give a high sigr7a.l.
.
l~hree other extreme conditiorIs are possible:
(l) I.-ow oestrone gluc~lronide -~ low preganediol glucu-2û roni.de (ear].y fol.licular phase);
(2) low oestrone glucuronide -~ hi.gh pregrlanediol glucu-ronlde;
(3) high oestrone glucuronide -~ high pre3narlediol glucuronide (micl luteal phase).
Under conditio1-l (l), un].abelled oestrone glucuronide antibodies will not be displaced frorn the immunocornplex3 ` no labelled oestrone glucuron.ide antibodies can bind .~ to the solid phA-~e and a low signal will result. Under ~ 30 conditions (2) and (3) the immurlocomplex will be cleaved ~ .
f'rom the solid phase Altogether, no oestrogen residues will remain on tne solid phase and no signal will be poss:ible.

The above schernt-~ h.7s A number nf advant.-lges.
(l~ Colour oevelopnle,-7t occurs ~hen the test is pos:itive.
~- (2~ Interf'er:ing fac-tnrs from ttlt3 urine sample are eli.rninated by wasl-lincJ after absorption 0l7tO solid phase.
.

~3~ 3 .. .

( 3 ) r ~l e t f ~ J f ~ l ~ r ~ ` l l t: f l ~ l 1 1 r' i. ~ l ( ` V O
n~` ~J 5~ <l~ir~ t~ lrli.~ 3 L~rl~ r~ t~
U S C! O I ~I S i lllFI 1. C' ~ C~ ? ' i t ~ .

Optirni~atioll ~uld evcllu.~'ir3rl of thc kit The kit w:ill be opt-ilnisecl :in teJIllt; of 'turlin~3' lll~
co]our sign<ll rcspnllse to corrcF,I-~nrlcl wjth l;he r~rl-Je oF oestrone-3-JJucuror~ e/pIegn.-lllccl:iol-3-g:luc(lronjclfJ
1~) ratios thaL occur ir) a normal mel-lstruc-l:L c~ycl~ le followlng var:iables wi]L be invrC;tig~teci in order to ; achieve this encl:
; (a) Variation oF the n-lrnber oF residues of oestrone-3_91ucuronide arld/or pregnanecliol-3-glucuronide per molecule n-F mi~ecl steroicl-protein cor-,p1ex.
(b) Variatjon in the dilution of pregnallediol-3-glu-curonide antiserunlused to coat the solid phaseA
(c) Variation in the characteristics of the ur!labelletl antisera, e.g. high versllslow affinity antiserurn.
(d) Variation in the ch.lracteristics of tl,e en~yme-`~ labelled antiserum.
(e) Variation in the nature of the en~ylne label and/
or sub~trates.

It is intended to examine urines from a lar~e number of menstr(lal cycles using tl)e kit. Usinr7 a simFJle colour chart, signiFicani: measuIes in colour intensity will be recorded and correlated with the fertile period.
.
Preliminary clata cn the ElPG~PD3G ratio frorrl nine normal menstrual cycles by conventional analyses have shown that the ratio increases 2 to 5 folcl from the mean follicular phase baseline value (days - 12 to - 6) to the pre-ovlllatory peak value. D:ifferences in chrorno-pho~e cnncel7trations of this rnal-lnitude are re~dily detec~
~` ted by the human eye therefore in theory the kit i.8 feasibio.

However it has been observecl that the absolute value of the ratio can differ from woman to woman. For the nine subjects examined the follicular phase baseline ratio (mean of days - 12 to - 6) ranges from 0.01 to 0.03 and the pre-ovulatory peak value (days - 2 to 0) ranges from 0.02~ to 0.156. In ~erms of a kit this indicates that an absolute colour endpoint may not be suitableg rather a relative coLour charlge would mark the end point.

It is envisaged that the kit would contain a colour chart, graduated in increa~3ing intensities of colour~
For each woman a f~llicular phase baseline of colour intensity would be recorded on the chart. As mid cycle approaches an increase in colour intensity by a certain number of graduations on the chart would be taken as a positive result.

In thisspecification the term "irreversible bond"
means a bond~ the dissociation constant of which is lower than the dissociation constant of an immunorhemical bond9 In particular the bonds betw0en the solid phase and the second antibody have a lower dissociation constant than the dissociation constants of the immunochemical bonds which bind the first and second antibodies to the ligand molecules.

References 1Samarajeewa P b Kellis. A.E. (1975) Biochem.J.151,369-376 25 2Van Weemen B.b Schuurs.A. (1974) FEBS Letters 4~15.

~.

Claims (15)

The embodiments of the invention in which an exclusive property of privilege is claimed are defined as follows:
1. A reagent system for use in determining the con-centration ratio of a first antigenic solute to a second anti-genic solute, comprising a first and a second component, the first component comprising;
a solid phase, a first antibody capable of reversibly binding to the first antigenic solute, a second antibody capable of reversibly binding to the second antigenic solute, a ligand molecule comprising one or more molecules of the first antigenic solute and one or more molecules of the second antigenic solute covalently bound to a support molecule, wherein the second antibody is irreversibly bound to the solid phase, the ligand molecule is capable of being reversibly bound to the solid phase by the reversible binding of the second antibody with one or more molecules of the second antigenic solute bound to the support molecule and the first antibody is capable of being reversibly bound to the ligand molecule by the reversible binding of the first antibody with one or more molecules of the first antigenic solute bound to the support molecule, thereby forming an immunocomplex bound to the solid phase, and the second component comprising;
a labelled antibody having substantially the same antigenic specificity as the first antibody.
2. A reagent system according to claim 1 wherein the support molecule is a protein.
3. A reagent system according to claim 2 wherein the protein is bovine serum albumin.
4. A reagent system according to claim 1, 2 or 3, wherein the first and second antibodies are immunoglobulin enriched fractions of antisera to the first and second antigens respectively.
5. A reagent system according to claim 1, 2 or 3 wherein the labelled antibody is labelled with an enzyme.
6. A reagent system according to claim 1, 2 or 3, wherein the enzyme labelled antibody is capable of producing a visible signal in conjunction with a substrate.
7. A reagent system according to claim 1, 2 or 3, wherein the labelled antibody is labelled with horseradish per-oxidase.
8. A reagent system according to claim 1, 2 or 3, wherein the first and second antigenic solutes are steroids or steroid metabolites.
9. A reagent system according to claim 1, wherein the first antigenic solute is 3-hydroxy-oestra-1,3,5(10)-triene-17-one 3-.beta.-D-glucupyranosiduronic acid (oestrone-3-glucuronide).
10. A reagent system according to claim 1, wherein the second antigenic solute is 5.beta.-pregnane-3.alpha., 20.alpha.-diol3.alpha.-.beta.-D-glucupyranosiduronic acid (pregnanediol-3-glucuronide).
11. A reagent system according to claim 9 or 10 when used in a method for the detection of the fertile period of the menstrual cycle.
12. A process for the determination of the concentra-tion ratio of a first antigenic solute to a second antigenic solute using the reagent system according to claim 1, 2 or 3 comprising the steps of:
placing a sample containing a first and second antigenic solute in contact with the first component of the reagent system, thereby causing competitive dissociation of the immunocomplex, the equilibrium of which dissociation depends upon the ratio of the concentration of the first and second solutes present in the sample, washing the first component with a buffer solu-tion, thereby removing immunocomplexes not bound to the solid phase, placing the labelled antibody in contact with the first component thereby allowing the formation of an immunocomplex with non-complexed molecules of the first antigenic solute on the ligand molecule, washing the first component with a buffer solution, thereby removing immunocomplexes not bound to the solid phase, and measuring the amount of labelled antibody associated with the solid phase.
13. A process for the determination of the concen-tration ratio of a first antigenic solute to a second anti-genic solute using the reagent system according to claim 1, 2 or 3, wherein the first and second antibodies are immuno-globulin enriched fractions of antisera to the first and second antigens respectively and comprising the steps of:
placing a sample containing a first and second anti-genic solute in contact with the first component of the reagent system, thereby causing competitive dissociation of the im-munocomplex, the equilibrium of which dissociation depends upon the ratio of the concentration of the first and second solutes present in the sample, washing the first component with a buffer solution, thereby removing immunocomplexes not bound to the solid phase, placing the labelled antibody in contact with the first com-ponent thereby allowing the formation of an immunocomplex with non-complexed molecules of the first antigenic solute on the ligand molecule, washing the first component with a buffer solution, thereby removing immunocomplexes not bound to the solid phase, and measuring the amount of labelled anti-body associated with the solid phase.
14. A process for the determination of the concen-tration ratio of a first antigenic solute to a second anti-genic solute using the reagent system according to claim 1, 2 or 3, wherein the labelled antibody is labelled with an enzyme and comprising the steps of:
placing a sample containing a first and second anti-genic solute in contact with the first component of the re-agent system, thereby causing competitive dissociation of the immunocomplex, the equilibrium of which dissociation de-pends upon the ratio of the concentration of the first and second solutes present in the sample, washing the first component with a buffer solution, thereby removing immunocomplexes not bound to the solid phase, placing the labelled antibody in contact with the first com-ponent thereby allowing the formation of an immunocomplex with non-complexed molecules of the first antigenic solute on the ligand molecule, washing the first component with a buffer solution, thereby removing immunocomplexes not bound to the solid phase, and measuring the amount of labelled anti-body associated with the solid phase.
15. A process for the determination of the concen-tration ratio of a first antigenic solute to a second anti-genic solute using the reagent system according to claim 1, 2 or 3, wherein the enzyme labelled antibody is capable of producing a visible signal in conjunction with a substrate and comprising the steps of:
placing a sample containing a first and second anti-genic solute in contact with the first component of the re-agent system, thereby causing competitive dissociation of the immunocomplex, the equilibrium of which dissociation de-pends upon the ratio of the concentration of the first and second solutes present in the sample, washing the first component with a buffer solution, thereby removing immunocomplexes not bound to the solid phase, placing the labelled antibody in contact with the first com-ponent thereby allowing the formation of an immunocomplex with non-complexed molecules of the first antigenic solute on the ligand molecule, washing the first component with a buffer solution, thereby removing immunocomplexes not bound to the solid phase, and measuring the amount of labelled anti-body associated with the solid phase.
CA 398267 1982-03-12 1982-03-12 Use of synthetic bifunctional ligand for the immunometric determination of the concentration ratio of two solutes Expired CA1183080A (en)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0225054A1 (en) * 1985-10-30 1987-06-10 Celltech Limited Binding assay device
EP0327843A1 (en) * 1988-02-08 1989-08-16 Hygeia Sciences, Inc. Positive step immunoassay
US5500350A (en) * 1985-10-30 1996-03-19 Celltech Limited Binding assay device
US6234974B1 (en) * 1992-08-21 2001-05-22 Unilever Patent Holdings B.V. Monitoring method
US6451619B1 (en) 1994-06-29 2002-09-17 Inverness Medical Switzerland Gmbh Monitoring methods and devices for use therein
US6454726B1 (en) 1992-08-21 2002-09-24 Inverness Medical Switzerland Gmbh Monitoring method
US6585663B1 (en) 1992-08-21 2003-07-01 Inverness Medical Switzerland Gmbh Advisory method
US6951631B1 (en) 1996-09-27 2005-10-04 Inverness Medical Switzerland Gmbh Test kits and devices
US7141212B2 (en) 1993-11-12 2006-11-28 Inverness Medical Switzerland Gmbh Reading devices and assay devices for use therewith

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0225054A1 (en) * 1985-10-30 1987-06-10 Celltech Limited Binding assay device
GB2191578A (en) * 1985-10-30 1987-12-16 Boots Celltech Diagnostics Binding assay device
GB2191578B (en) * 1985-10-30 1989-11-01 Boots Celltech Diagnostics Binding assay device
US5500350A (en) * 1985-10-30 1996-03-19 Celltech Limited Binding assay device
US5604110A (en) * 1985-10-30 1997-02-18 Celltech Therapeutics Ltd. Binding assay device
EP0327843A1 (en) * 1988-02-08 1989-08-16 Hygeia Sciences, Inc. Positive step immunoassay
US4952517A (en) * 1988-02-08 1990-08-28 Hygeia Sciences, Inc. Positive step immunoassay
US6234974B1 (en) * 1992-08-21 2001-05-22 Unilever Patent Holdings B.V. Monitoring method
US6585663B1 (en) 1992-08-21 2003-07-01 Inverness Medical Switzerland Gmbh Advisory method
US6454726B1 (en) 1992-08-21 2002-09-24 Inverness Medical Switzerland Gmbh Monitoring method
US7141212B2 (en) 1993-11-12 2006-11-28 Inverness Medical Switzerland Gmbh Reading devices and assay devices for use therewith
US6451619B1 (en) 1994-06-29 2002-09-17 Inverness Medical Switzerland Gmbh Monitoring methods and devices for use therein
US6951631B1 (en) 1996-09-27 2005-10-04 Inverness Medical Switzerland Gmbh Test kits and devices
US7632460B2 (en) 1996-09-27 2009-12-15 Inverness Medical Switzerland Gmbh Test kits and devices

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