WO2000043785A1 - Ameliorations apportees a des dosages par deplacement - Google Patents

Ameliorations apportees a des dosages par deplacement Download PDF

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Publication number
WO2000043785A1
WO2000043785A1 PCT/EP1999/010456 EP9910456W WO0043785A1 WO 2000043785 A1 WO2000043785 A1 WO 2000043785A1 EP 9910456 W EP9910456 W EP 9910456W WO 0043785 A1 WO0043785 A1 WO 0043785A1
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WO
WIPO (PCT)
Prior art keywords
analyte
binding partner
solid support
interest
binding
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PCT/EP1999/010456
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English (en)
Inventor
Steven Howell
Gerhard Nebe Von Caron
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Unilever Plc
Unilever Nv
Hindustan Lever Limited
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Application filed by Unilever Plc, Unilever Nv, Hindustan Lever Limited filed Critical Unilever Plc
Priority to AU21036/00A priority Critical patent/AU2103600A/en
Publication of WO2000043785A1 publication Critical patent/WO2000043785A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]

Definitions

  • This invention relates to a method of performing an assay and to apparatus for performing the assay method of the invention.
  • assays which make use of the specific binding properties of certain molecules to detect the presence of an analyte of interest in a sample.
  • assays involve the specific binding between immunoglobulins (such as antibodies or functional binding fragments thereof) and haptens or antigens to which the immunoglobulins bind.
  • immunoglobulins such as antibodies or functional binding fragments thereof
  • haptens or antigens to which the immunoglobulins bind.
  • assays include enzyme-linked immunosorbent assays (ELISAs) and radio-immunoassay (RIA).
  • binding partner in order to detect binding between the analyte of interest and a binding partner having specific binding affinity therefor, it is usual for the binding partner to be labelled.
  • Known labels include enzymes, radio- labels, fluorescent or chemiluminescent labels, electroactive labels (such as redox labels) and coloured particles (e.g. latex beads).
  • a refinement of assays of the general nature outlined above relates to "displacement" assays.
  • the presence of an analyte of interest in a sample typically causes the displacement either of a labelled binding partner or a labelled ligand from a pre-existing binding partner/ ligand complex.
  • the amount of displaced labelled substance will be proportional to the concentration of the analyte of interest in the sample.
  • EP 0,324,540 discloses assays designed to measure the amount of free ligand (rather than complexed ligand, which complexed ligand is typically protein-bound) in biological samples such as plasma or serum.
  • the assay method requires the use of a
  • signal reagent which is labelled monoclonal antibody.
  • the monoclonal binds to free ligand, which is in competition with a ligand analogue (which analogue does not bind to the natural ligand complexing proteins present in the sample).
  • the analogue is immobilised (e.g. on particles or beads).
  • the analogue is selected to have a lower affinity than the ligand for the anti-ligand monoclonal antibody.
  • the assay thus works on the principle of immuno-competition, the presence of free ligand in the sample serving to decrease the amount of labelled antibody which becomes associated with the ligand analogue.
  • WO 91 /05262 discloses a device and method for detecting the presence of molecular analytes in a fluid (especially e.g. steroids, and other low molecular weight analytes).
  • aqueous biological samples are drawn along a test strip by capillary action.
  • a first binding means which is an anti-analyte antibody.
  • the labelled analyte e.g. analyte /enzyme conjugate
  • the present invention relates to an improvement of displacement type assays.
  • the invention provides a method and assay device for performing displacement assays having increased sensitivity (i.e. the ability to detect lower concentrations of analyte of interest and/ or to detect the same concentration of analyte within a shorter time period) compared to prior art methods and devices.
  • the invention provides a method of detecting the presence of an analyte of interest in a sample, the method comprising the steps of:
  • the method is such that the presence of one molecule of the analyte of interest can cause the specific displacement from the first support of a plurality of molecules of binding partner.
  • a large amount of binding partner can, in effect, be transferred from the first support to the second support, such that there is a great amplification of the "signal" provided by a single analyte molecule, such that the present invention confers greatly improved sensitivity compared with prior art assays.
  • steps (b) and (c) will therefore preferably be effected a plurality of times during performance of the invention.
  • the invention provides an assay device for detecting the presence of an analyte of interest in a sample, the device comprising: a first solid support having reversibly immobilised thereon a binding partner having specific binding activity for the analyte of interest, the binding partner being displaced from the first solid support in the presence of the analyte of interest and forming a binding partner/analyte complex; a second solid support bearing a capture moiety, the capture moiety having a specific binding affinity for the binding partner which is greater than that of the analyte of interest for the binding partner, such that the binding partner may be captured by the capture moiety and the analyte of interest displaced from the binding partner/analyte complex; means for conducting the binding partner/analyte complex from the first solid support to the second solid support; and means for conducting the displaced analyte from the second solid support to the first solid support.
  • the method and device of the invention may be used qualitatively or quantitatively.
  • the sample will generally be in fluid form, conveniently (but not necessarily) as a liquid (e.g. aqueous solution, or comprise a sample of body fluid, such as blood, serum, plasma, urine, saliva, sweat, semen or tears) .
  • a liquid e.g. aqueous solution, or comprise a sample of body fluid, such as blood, serum, plasma, urine, saliva, sweat, semen or tears
  • the analyte of interest may be any molecule, such as a drug, hormone, protein, carbohydrate, nucleic acid (DNA, RNA or chimera), enzyme, antibody, antigen and the like.
  • the analyte may be of a macromolecular or particulate nature, such as bacteria or virus particles, or other micro-organism (fungus, yeast, chlamydia, fungal or bacterial spore, etc), or allergen (e.g. house dust mite faeces).
  • bacteria or virus particles or other micro-organism (fungus, yeast, chlamydia, fungal or bacterial spore, etc), or allergen (e.g. house dust mite faeces).
  • analytes which may be present in the human body or body fluids, such as hormones, especially sex and/ or fertility hormones and analogues thereof, such as estrone-3-glucuronide (E3G), pregnanediol-3-glucuronide (P3G), human chorionic gonadotrophin (hCG), luteinising hormone (LH), and follicle stimulating hormone (FSH).
  • hormones especially sex and/ or fertility hormones and analogues thereof
  • E3G estrone-3-glucuronide
  • P3G pregnanediol-3-glucuronide
  • hCG human chorionic gonadotrophin
  • LH luteinising hormone
  • FSH follicle stimulating hormone
  • antigens that may be present on solid surfaces that may, or may not, be taken up into solution (e.g. biofilms in plant and machinery, or residues left on surfaces - e.g. after cleaning, especially in the food processing industry).
  • the reversibly immobilised binding partner and the analyte are conveniently members of a specific binding pair.
  • specific binding pairs e.g. DNA and DNA-binding proteins; complementary strands of nucleic acids; ligands and their receptors; antigens and antibodies thereto.
  • the binding partner is a protein, preferably an immunoglobulin (e.g. antibody) or a functional binding fragment or variant thereof, which term relates to, inter alia, Fv, scFv, Fab, Fab 2 HCV, bispecific antibody, and chimeric molecules comprising one or more of the aforementioned binding fragments, and the like.
  • the binding partner may be reversibly immobilised to the first solid support in any one of a number of ways, which will be apparent to the person skilled in the art (e.g. as described in "Protein Immobilisation” 1991, [ed. R. F. Taylor] Marcel Dehler, Inc. New York).
  • the binding partner may conveniently be removed from the solid support by application of particular chemicals (e.g. solutions, such as 50mM glycine, buffered to a very low pH [pH2] or 50mM diethylamide buffered to a very high pH [pH 12] and the like) but, under conditions in which the assay is performed (such as those generally found in biological systems (e.g.
  • the binding partner is reversibly immobilised on the first solid support is of lower affinity than that of the binding partner for the analyte of interest. It will be apparent to those skilled in the art that this condition can readily be met by judicious selection of an appropriate binding partner molecule and/ or selection of the manner in which the binding partner is reversibly immobilised to the first solid support. Conveniently this is achieved by immobilising on the first support (e.g. via covalent interactions) an analogue of the analyte of interest.
  • the binding partner is then allowed to bind (comparatively loosely) to the analogue of the analyte (e.g. via non-covalent interactions), so as to immobilise reversibly the binding partner to the first solid support.
  • the binding affinity between the binding partner and the capture moiety is greater than that between the binding partner and the analyte of interest. Again, judicious selection of the capture moiety and binding partner, and/ or optimisation of the assay conditions, will readily allow this condition to be met.
  • Binding of the binding partner to the capture moiety preferably excludes the possibility of simultaneous binding of the binding partner to the analyte. Conveniently this is achieved by requiring the capture moiety to bind to the same binding site on the binding partner (but with greater affinity) as that occupied by the analyte or to an overlapping site. Alternatively, binding of the binding partner to the capture moiety may induce a conformational change in the binding partner, which conformational change alters or abolishes the binding site occupied by the analyte.
  • the capture moiety is an analogue of the analyte of interest.
  • the capture moiety is advantageously immobilised on the second solid support, typically by means of covalent interactions and/or by adsorption. Again, methods of achieving this are well known to those skilled in the art. Accordingly, in preferred embodiments, the invention will involve the use of two analogues of the analyte of interest: a "low affinity analogue” immobilised on the first solid support and a "high affinity analogue” (the capture moiety) borne on the second solid support.
  • the affinity of the binding partner for the analyte of interest is at least about 5, more desirably at least about 10, and no more than about 100, more desirably no more than about 20 times greater than the affinity of the binding partner for the "low affinity analogue" on the first solid support.
  • the affinity of the binding partner for the "high affinity analogue" i.e. the capture moiety
  • the affinity of the binding partner for the "high affinity analogue” is at least about 5, more desirably at least about 10, and no more than about 100, more desirably no more than about 20 times greater than the affinity of the binding partner for the analyte of interest.
  • Typical useful affinity values for the various interactions are 10 " ⁇ M (low affinity analogue /binding partner), 10 " ⁇ M (binding partner/analyte) and 10" ⁇ M (binding partner/ high affinity analogue) respectively.
  • the binding partner may be reversibly immobilised on the first solid support by interaction (with relatively low affinity) with a mimotope, the mimotope itself immobilised (by conventional means) on the first solid support.
  • the capture moiety at the second solid support may conveniently take the form of a mimotope (although one having a relatively high affinity interaction with the binding partner).
  • Mimotopes are molecules (generally peptides) which "mimic" the structure of epitopes recognised by antibodies. A useful discussion of mimotopes, their synthesis, and their uses, is provided by Cortese et al, (1995 Current Opinion in Biotechnology 6, 73-80).
  • the first and second solid supports may be substantially similar or identical in nature, or may be different.
  • Solid supports which may be of use in the invention are generally well known and include, for example synthetic plastics materials, microtitre assay plates, latex beads, filters comprising cellulose or synthetic polymeric materials, glass or plastics slides, dipsticks, capillary fill devices and the like.
  • the method and device of the invention are such that the "capture" step may be performed using generic components.
  • the method and device of the invention can be used to test for the presence and/ or concentration of any analyte of interest.
  • this may be achieved by use of a chimeric binding partner, in which one portion is retained (regardless of the identity of the analyte of interest) which interacts with the generic capture moiety.
  • a generic, modular arrangement facilitates production of the assay device and reduces cost.
  • the binding partner may comprise a detectable label (e.g. enzyme, radioactive label, fluorescent or chemiluminescent label, or electroactive (redox) label or a particulate, coloured or uncoloured, e.g. latex bead or gold sol).
  • a detectable label e.g. enzyme, radioactive label, fluorescent or chemiluminescent label, or electroactive (redox) label or a particulate, coloured or uncoloured, e.g. latex bead or gold sol.
  • presence or absence of the binding partner at the first and/ or second support can be detected indirectly - if the binding partner is unlabelled it can compete with labelled binding partner added at the end of the assay, the presence of analyte /interest being indicated in such an embodiment by reduction in binding of labelled binding partner to the second support, or by an increase of labelled binding partner binding to the first support.
  • the first and/ or second support may comprise part of a mass-dependent biosensor (e.g. acoustic wave or evanescent wave type sensors, or surface plasmon resonance ["SPR"] detectors, all of which are known to those skilled in the art - see, for example, EP 0,341,927; EP 0,416,730; EP 0,453,224; and Jonsson et al, 1991 Bio/Techniques II, 620- 627).
  • a mass-dependent biosensor e.g. acoustic wave or evanescent wave type sensors, or surface plasmon resonance ["SPR"] detectors, all of which are known to those skilled in the art - see, for example, EP 0,341,927; EP 0,416,730; EP 0,453,224; and Jonsson et al, 1991 Bio/Techniques II, 620- 627).
  • the apparatus of the invention may take any one of numerous forms, depending on the precise nature of the assay being performed (e.g. the identity of the analyte of interest, the binding partner and capture moiety; the type of solid support; whether the binding partner is labelled; etc).
  • the first and second surfaces may be physically very close to each other (of the order of a millimetre or so apart), possibly inter-digitated, during performance of at least some steps of the assay.
  • the apparatus may comprise a capillary-fill test device in which a liquid sample may be drawn into the device by capillary action along a suitably-proportioned capillary inlet. Capillary-fill devices which may be adapted for use in the present invention are disclosed, for example, in US Patent 5, 141 ,868.
  • the first solid support comprises a synthetic plastics peg which is proportioned as to fit within a well of a microtitre plate, there being only a small separation between the peg and the sides of the well.
  • the second support is formed by the microtitre plate, the well being coated with the capture moiety.
  • a microtitre plate may constitute the first support and a peg provides the second support.
  • an array of a plurality of pegs may be used, the spacing of the pegs being such that the array can be inserted into a corresponding plurality of wells in the microtitre plate. The presence of the analyte of interest in a liquid sample in the well causes displacement of binding partner from the first support.
  • the displaced binding partner may then diffuse across the small gap between the pegs and the side of the well, so as to be captured by the capture moiety.
  • analyte displaced from the binding partner/analyte complex can readily diffuse across the narrow gap, so as to displace a further binding partner molecule.
  • the pegs are removed from the wells.
  • the wells and/ or pegs are then typically washed with appropriate washing buffer (e.g. PBS) and the presence of binding partner detected.
  • the binding partner may conveniently be labelled (e.g. with an enzyme label), and detection can thus be accomplished very simply (e.g. by means of an ELISA-type protocol).
  • the physical separation between the first and second supports is too great for diffusion to provide a reasonably practicable means for conducting the binding partner from the first to the second support and/ or the analyte from the second support to the first support.
  • fluid especially, liquid
  • the direction of fluid movement may be bi-directional (e.g.
  • fluid movement within the fluid communication means being from the first support to the second support for a period of time, then with flow in the opposite direction for a time, with as many changes of direction as deemed appropriate). More preferably, the fluid movement within the fluid communication means is uni-directional.
  • the fluid communication means forms a loop, in one part of which displaced binding partner is transported from the first support to the second support, and in the other part of which displaced analyte is "recycled" by transport from the second support to the first support.
  • the first and /or second support forms part of a mass-dependent biosensor (e.g. SPR or evanescent wave-type biosensor).
  • Fluid movement within the fluid communication means is typically assisted by means of one or more pumps of a conventional nature (e.g. peristaltic pump or the like) .
  • the fluid communication means may also comprise one or more valves and one or more inlet ports for intake of sample, reagents, buffers etc., and one or more outlet ports for removal of fluids.
  • the apparatus of the invention may also comprise computerised control means and/ or computerised data processing means.
  • FIGS 1 and 4 are schematic illustrations of two embodiments of performing the method of the invention.
  • Figures 2 and 3 are sensorgrams (arbitrary Response Units against time) of results obtained using apparatus in accordance with the invention.
  • Figure 5 is a bar chart showing results obtained in an Assay performed in accordance with the method of the invention.
  • Example 1 Detecting Estrone 3-Sulphate (E3S) using a mass-dependent biosensor
  • E3S Estrone 3-Sulphate
  • FIG. 1 Two surfaces are prepared as follows.
  • a first support (2) has attached to its surface a low affinity analogue (4) of the analyte (6).
  • a binding partner (8) (in this case a monoclonal antibody raised against the analyte) is then reversibly immobilised on the first surface (2) by interaction with the low affinity analogue (4).
  • a second support (10) has attached to its surface a high affinity analogue (12) of the analyte which acts as a capture moiety.
  • the assay is performed by passing sample containing the analyte (6) across the first support (2), where it displaces the binding partner (8), as it has a higher affinity for it compared with the low affinity analogue (4).
  • the binding partner/analyte complex (14) so formed is then contacted with the second surface (10) where a second displacement event occurs, as the high affinity analogue (12) has a higher affinity for the binding partner (8) than does the analyte (10).
  • binding partner molecules are transferred from the surface of the first support (2) to the surface of the second support (10) where they are detected; free, unbound analyte molecules (6) are regenerated.
  • the solution is then re-contacted with the first support (2) to displace further binding partner molecules. In this way a single analyte molecule (6) can cause the displacement of a plurality of binding partner molecules (8) from the first support (2) .
  • estradiol 3-glucuronide and estrone 3-glucuronide Coupling estradiol 3-glucuronide and estrone 3-glucuronide to a biosensor CM5 chip
  • CM5 biosensor chip (Biacore AB) was docked in the Bialite biosensor (an SPR-type device) fitted with an upgrade kit (Biacore AB) and the flow rate of hepes buffered saline (HBS, Biacore AB) was set to 10 ⁇ l/min.
  • the sensor chip surface was then activated by two sequential injections (40 ⁇ l and 20 ⁇ l) of a 1 , ethyl-3-[dimethylaminopropyl] carbodiimide (EDC) N- hydroxysuccinimide (NHS) activating solution made up as described by the manufacturer (Biacore AB).
  • a solution of ethylenediamine (20 % v/v) was injected (60 ⁇ l) across flow cells 1 and 2.
  • the flow path was then changed to flow across flow cell 2 and estrone 3-glucuronide (E3G, preincubated in EDC/NHS activating solution for 7 min) was injected (60 ⁇ l).
  • the flow path was again changed to flow across flow cell 1 and estradiol 3-glucuronide (ED3G, preincubated in EDC/NHS activating solution for 7 min) was injected (60 ⁇ l).
  • the monoclonal antibody (MAb) used as a binding partner in the following experiment (MAb 4101) was prepared according to procedures known in the prior art. A method of generating monoclonal antibodies is described by Gani et al (1994 J. Steroid Biochem. Molec. Biol. 48, 277-282) and this method can be adapted to produce a relevant antibody. A suitable monoclonal antibody can be selected on the basisof its relative affinity for the analyte and analyte analogues.
  • BiacoreTM 2000 biosensor Biacore AB, Sweden
  • Biacore AB Biacore AB
  • a panel of closely related analogues e.g. steroid analogues commercially available from Sigma Chemical Co.
  • a commercially available anti-estrone glucuronide monoclonal antibody from Wallaceville Animal Research Centre, New Zealand
  • MAb 4101 used was raised against E3G but showed cross reactivity to a number of E3G analogues.
  • the sensor chip prepared as described above was docked in the Bialite biosensor fitted with an upgrade kit and the flow rate of HBS was set to 10 ⁇ l/min.
  • the flow path was set to cross flow cell 1 and MAb 4101 was injected across the sensor surface (40 ⁇ l of a 10 ⁇ g/ml solution).
  • the Bialite biosensor was primed in 1.5 ml HBS in a closed system.
  • the closed system comprised the waste outlet pipe being fed back into the buffer reservoir used to supply the syringe pump for the instrument. This ensured that after liquid had passed through the biosensor it was fed back into the filling reservoir of the syringe pump, thus, when the syringe was next filled the same solution passed through the instrument once more (i.e.
  • Displacement of the MAb 4101 by E3S can be observed from flow cell 1 and displaced MAb 4101 (occurring as a MAb4101-E3S complex) can be seen to undergo a second displacement such that the MAb 4101 is displaced from E3S and binds to E3G at flow cell 2.
  • the MAb 4101 accumulates at surface 2 where it is detected.
  • the initial displacement of the MAb 4101 from flow cell 1 (ED3G) is dependent on the presence of E3S. This can be seen in figure 3 as, when the method was repeated but no E3S was introduced into the system, MAb 4101 was substantially retained at the surface of flow cell 1.
  • this example shows how E3S (analyte) can be detected using two displacement events occurring in close proximity to each other.
  • the example is illustrated schematically in Figure 4.
  • the principle is essentially similar to that illustrated in Figure 1 , and corresponding components are denoted by use of the same reference numerals.
  • Two surfaces are prepared as follows.
  • a first support (2) has attached to its surface a low affinity analogue (4) of the analyte (6) .
  • a binding partner (8) (in this case a monoclonal antibody raised against the analyte) is then reversibly immobilised on the first support (2).
  • a second support (10) has attached to its surface a high affinity analogue (12) of the analyte (6).
  • the high affinity analogue (12) acts as a capture moiety.
  • the two supports (2) and (10) are set up in such a way that there is a small gap between them.
  • the assay is performed by placing a sample containing the analyte (6), (E3S) in between the two supports.
  • analyte (6) displaces the binding partner (8) as it has a higher affinity for it compared with the low affinity analogue (4).
  • the binding partner/analyte complex (14) so formed then diffuses across to the second support (10) where a second displacement event occurs as the high affinity analogue (12) has a higher affinity for the binding partner (8) than the analyte (6).
  • the analyte molecule (6) is regenerated in a free unbound state and can diffuse back to the first support (2) where it can once again displace a binding partner molecule (8).
  • binding partner molecules (8) are transferred from the first to the second support, where they can be detected by conventional means.
  • the second support is a nylon peg, which fits in the wells of a microtitre plate (the first support) .
  • EDC l-Ethyl-3-(3-dimethylaminopropyl) carboiimide
  • EDC l-Ethyl-3-(3-dimethylaminopropyl) carboiimide
  • ED3G 10 mg/ml also in dimethylformamide
  • NHS N-hydroxysuccinimide
  • the NHS/EDC/ED3G mixture was slowly added dropwise to an ovalbumin solution at 4 °C with constant stirring and then incubated overnight under the same conditions.
  • the conjugate was desalted into phosphate buffered saline (PBS) using a PD 10 column.
  • An E3G-ovalbumin conjugate was prepared using the same protocol except that E3G (10 mg/ml in dimethylformamide) was used in place of ED3G.
  • Approximately 200 nylon coated cavity pegs were mixed with 200 ml of PBS containing 0.01 % sodium azide (PBSA) for 30 min. The pegs were drained and 200 ml glutaraldehyde (2 % (v/v)) was added and mixed for 60 min. The pegs were then drained and 200 ml dH 2 O was added and the pegs were mixed for 5 min. This dH 2 O washing step was repeated twice more and then the pegs were washed three times in PBSA. The pegs were drained and 200 ml E3G-ovalbumin at 20 ⁇ g/ml in PBSA was added and the pegs mixed.
  • PBSA sodium azide
  • the monoclonal antibody used (MAb) in the following experiment (MAb 4101) was prepared according to procedures known in the prior art.
  • the MAb 4101 was raised to E3G but showed cross reactivity to a number of E3G analogues.
  • the order of cross reactivity was such that MAb 4101 bound to E3G > E3S > ED3G.
  • PBSA containing 0.1 % Tween 20 was added to the wells (200 ⁇ l per well) of a microtitre plate (Greiner HB) and incubated for 1 hour at room temperature.
  • the solution in the wells was replaced with 200 ⁇ l MAb 4101 (1 :500 in PBSTA) and E3G-ovalbumin pegs were then inserted into the wells and incubated for 1 hour at room temperature.
  • Pegs were then washed in PBSTA and inserted into wells, pretreated with PBSTA, containing 200 ⁇ l anti-mouse IgG-alkaline phosphatase conjugate (1 : 1000 in PBSTA). Following a 1 hour incubation at room temperature the pegs were washed in PBSTA and inserted into wells containing 200 ⁇ l Sigma 104 phosphatase substrate solution. The absorbance of the substrate solution in the wells was measured at 405 nm after 1 hour at room temperature. It was found that the MAb 4101 bound satisfactorily to the E3G-ovalbumin coated pegs.
  • ED3G-ovalbumin conjugate 200 ⁇ l was incubated in the wells of a microtitre plate (Greiner HB) for 2 h at room temperature. The wells were then washed with PBSTA and the MAb 4101 (100 ⁇ l of a 1 : 1000 dilution in PBSTA) was incubated in the ED3G-ovalbumin adsorbed wells for 16 h at 4 °C. The wells of the plate were then washed with PBSTA. Detection of estrone 3-sulphate (E3S) by double displacement of a monoclonal antibody
  • E3S (100 ⁇ l of 1 mg/ml in PBSTA) was incubated in ED3G-ovalbumin/MAb 4101 loaded wells each containing an E3G-ovalbumin peg for 2 hours at room temperature. The pegs were removed from the wells and both the wells and the pegs were washed with PBSTA. A solution (100 ⁇ l) of rabbit anti-mouse IgG-alkaline phosphatase conjugate (1 : 1000 in PBSTA) was incubated in the wells for 1 hour at room temperature.
  • the pegs were placed in wells of a separate microtitre plate (which had been blocked by pretreatment for 1 hour with 200 ⁇ l PBSTA) containing 100 ⁇ l of rabbit anti-mouse IgG-alkaline phosphatase conjugate (1 : 1000 in PBSTA). After a 1 hour incubation at room temperature the rabbit anti-mouse IgG- alkaline phosphatase conjugate solution was removed from the original wells and the pegs and original wells were washed with PBSTA. 100 ⁇ l Sigma 104 phosphatase substrate solution was then added to the original wells and to the wells of a new microtitre plate into which the pegs were placed. The absorbance at 405 nm was determined after incubating the wells for 4 hours at room temperature. In this way the MAb 4101 could be located, either still bound to the original well or displaced at the E3G- ovalbumin peg.
  • Figure 5 is a bar chart, showing % of the MAb 4101 bound in the presence (left hand side) or absence (right hand side) of E3S substrate.
  • the MAb 4101 still bound to the wells of the original microtitre plate (i.e. undisplaced from the first solid support) is indicated by the solid bars.
  • the MAb 4101 bound to the pegs i.e. displaced from the first support and captured on the second support is denoted by the partially shaded bars.

Abstract

Cette invention se rapporte à un procédé et à un dispositif de dosage permettant de réaliser des dosages par déplacement avec une sensibilité accrue. Ce dispositif de dosage comprend en particulier un premier support solide sur lequel est immobilisé de façon réversible un partenaire de liaison possédant une activité de liaison spécifique pour un analyte d'intérêt, ce partenaire de liaison pouvant être déplacé depuis le premier support solide en présence de l'analyte d'intérêt et formant un complexe partenaire de liaison/analyte; un second support solide portant une fraction de capture, laquelle possède une affinité de liaison spécifique pour le partenaire de liaison qui est supérieure à celle de l'analyte d'intérêt pour le partenaire de liaison, de telle sorte que le partenaire de liaison puisse être capturé par la fraction de capture et l'analyte d'intérêt déplacé depuis le complexe partenaire de liaison/analyte; un organe permettant de guider le complexe partenaire de liaison/analyte depuis le premier support solide jusqu'au second support solide; et un organe permettant de guider l'analyte ainsi déplacé depuis le second support solide jusqu'au premier support solide.
PCT/EP1999/010456 1999-01-25 1999-12-23 Ameliorations apportees a des dosages par deplacement WO2000043785A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU21036/00A AU2103600A (en) 1999-01-25 1999-12-23 Improvements in or relating to displacement assays

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP99300518 1999-01-25
EP99300518.0 1999-01-25

Publications (1)

Publication Number Publication Date
WO2000043785A1 true WO2000043785A1 (fr) 2000-07-27

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020078999A1 (fr) * 2018-10-15 2020-04-23 F. Hoffmann-La Roche Ag Dosage immunologique permettant de mesurer le peptide c

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4746631A (en) * 1985-05-09 1988-05-24 Ultra Diagnostics Corporation Immunoassay method, device, and test kit
US4803170A (en) * 1985-05-09 1989-02-07 Ultra Diagnostics Corporation Competitive immunoassay method, device and test kit
WO1992016841A1 (fr) * 1991-03-12 1992-10-01 E.I. Du Pont De Nemours And Company Procede de dosage par liaison specifique au moyen d'un ligand detachable
WO1997044664A1 (fr) * 1996-05-23 1997-11-27 Unilever Plc Ameliorations relatives aux tests d'immunodetection specifiques

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4746631A (en) * 1985-05-09 1988-05-24 Ultra Diagnostics Corporation Immunoassay method, device, and test kit
US4803170A (en) * 1985-05-09 1989-02-07 Ultra Diagnostics Corporation Competitive immunoassay method, device and test kit
WO1992016841A1 (fr) * 1991-03-12 1992-10-01 E.I. Du Pont De Nemours And Company Procede de dosage par liaison specifique au moyen d'un ligand detachable
WO1997044664A1 (fr) * 1996-05-23 1997-11-27 Unilever Plc Ameliorations relatives aux tests d'immunodetection specifiques

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020078999A1 (fr) * 2018-10-15 2020-04-23 F. Hoffmann-La Roche Ag Dosage immunologique permettant de mesurer le peptide c

Also Published As

Publication number Publication date
AU2103600A (en) 2000-08-07

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