WO1999056130A1 - Procede de dosage de plusieurs analytes - Google Patents

Procede de dosage de plusieurs analytes Download PDF

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Publication number
WO1999056130A1
WO1999056130A1 PCT/GB1999/001336 GB9901336W WO9956130A1 WO 1999056130 A1 WO1999056130 A1 WO 1999056130A1 GB 9901336 W GB9901336 W GB 9901336W WO 9956130 A1 WO9956130 A1 WO 9956130A1
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WIPO (PCT)
Prior art keywords
analytes
sample
assay
label
analyte
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PCT/GB1999/001336
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English (en)
Inventor
Ian James
David Perrett
Russell Hart
Barbara Scheuer
Nancy Schmidt
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Queen Mary & Westfield College
Assay Designs, Inc.
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Application filed by Queen Mary & Westfield College, Assay Designs, Inc. filed Critical Queen Mary & Westfield College
Priority to AU37208/99A priority Critical patent/AU3720899A/en
Publication of WO1999056130A1 publication Critical patent/WO1999056130A1/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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

  • the present invention relates to the use of simultaneous assay techniques that enable the measurement of multiple analytes in a single sample.
  • the concept of multianalyte determination in a single sample is well documented in the scientific and patent literature.
  • the potential benefits include the reduction in analytical error, the use of less sample, increased precision particularly where the data from several analytes are combined and obvious speed and cost savings.
  • the detection of multiple analytes can occur either simultaneously or in a sequential manner.
  • the ratio of absorbances obtained at 262 and 280nm, commonly used to determine RNA / DNA concentration could be considered to represent a multianalyte assay format.
  • the evolution of multianalyte assays involving an initial immunological isolation of analytes onto one or more solid phase has been dependent on characterisation of classes of molecules with high specific activity for use as labels to tag analytes or binding agents and the development of sensitive and selective instrumentation.
  • Enzymes such as horseradish peroxidase, alkaline phosphatase and ⁇ -galactosidase are commonly used as labels in immunoassays due to their high catalytic activity and the variety of available substrates.
  • WO 89/06802 describes a dual enzyme assay and other scientific publications have described the use of such systems with chromogenic (Blake et al Clin. Chem. 28 1469-1473 (1982)) and fluorogenic substrates (Dean et al Clin. Chem. 29 1051-1056 (1983)) .
  • the further application of multiple enzyme systems appears to be limited by the compromising assay conditions necessarily employed, due to differing pH optimums and other specific enzyme 2
  • Dual and triple analyte immunoassays using enzyme labelled analytes and binding reagents have been developed by Biosource International. These assays involve a common immuno-isolation procedure, but the enzyme conjugates are added sequentially, only after the measurement and removal of the previous label by poisoning of the enzyme and removal of the chromophore.
  • US- A-3952091 describes an assay for simultaneous multiple radioimmunoassay using Iodine as a label for quantitation and this concept was developed further as described in US-A-4146602 for the simultaneous radioimmunoassay of folate and vitamin B 12 where 125 Iodine and 57 Cobalt were used as tracers.
  • Other examples of assays using radiolabelled analytes or binding agents are described in US-A-4332784 and also in US-A-4504587 which relates to hybrid detection systems involving radioisotopes with fluorescence.
  • WO 95/17672 and US-A- 4598044 relate to methods to enhance the stability and efficiency of chemiluminescent labels with the intention of their use in immunoassay.
  • multianalyte immunocapture procedure which involve the capture of different analytes using specific binding agents each localised to specific areas of the solid phase support (WO 94/25855; WO 89/01157) or using a mixture of solid phases or microspheres coated with specific binding agents (US-A-5028545; WO 94/21823).
  • the quantitation of bound analytes can be achieved either simultaneously or sequentially.
  • the detection systems reporting each analyte generate signals simultaneously, either by the addition of a chemical triggering agent, as a result of an incident energy source or radioisotope decay
  • the different signals produced can be processed using gating techniques, i.e. the simultaneous measurement of energy at different wavelengths in the electromagnetic spectrum.
  • gating techniques i.e. the simultaneous measurement of energy at different wavelengths in the electromagnetic spectrum.
  • radioisotopes can be differentiated by the energy of emitted particles
  • colourimetric, fluorescent and luminescent labels can be differentiated by the spectral characteristics and wavelengths of maximum absorption or emission.
  • complex mathematical processing of data is required to discern overlapping signals.
  • Molecules with inherent chemiluminescent or bioluminescent properties offer the potential for high sensitivity detection and an all or nothing response. As light is generated from the addition of a chemical or biochemical trigger the background noise is essentially instrument related. Triggering of certain luminescent tags results in rapid and transient emission of energy while others used in enzyme reactions result in prolonged emission.
  • chemiluminescent detection systems are commonly used in single analyte immunoassays. Their use in multianalyte systems has focused on the chemical modification of existing labels, for example, WO 9421823 describes the synthesis of a series of chemiluminescent compounds based on the derivatisation of an acridinium 4
  • nucleus each having different spectral characteristics, differing in wavelength of maximal emission by 60-80nm.
  • the selective derivatisation of analytes or binding agents with these compounds enables the simultaneous assay or two or more analytes captured onto different paramagnetic particles.
  • concentration of bound analytes is related to the energy emitted at different wavelengths specific to the chemiluminescent derivatives used.
  • EP-A-0478626 describes the esterification of acridinium compounds to produce labels with fast and slow light emission. Detection relies on the resolution of emitted light relative to time. Time resolved fluorescence techniques can be used for quantitation of multiple analytes.
  • the lanthanide chelates (europium-samarium, europium-terbium) are ideally suited, having narrow fluorescence emission bands and lack overlap between emission spectra (Kakabakos et al Clin. Chem. 38 (3) 338-342 (1992)).
  • a key prerequisite for the sequential detection of multiple bound labels is that the signal from the previous label be dissipated prior to triggering and analysis of the next label in sequence.
  • a combination of two or more detection systems including enzyme, chemiluminescent or bioluminescent detection 5
  • a method for sequentially assaying for the presence of a plurality of analytes in a single sample comprising the steps of:
  • a method according to the present invention is applicable to a wide variety of assay types, e.g. immunoassays, ELISA, DNA probe based assays, protein/DNA shift assays, enzyme activity based and related assays such as those for the measurement of glucose or adenosine triphosphate.
  • assay types e.g. immunoassays, ELISA, DNA probe based assays, protein/DNA shift assays, enzyme activity based and related assays such as those for the measurement of glucose or adenosine triphosphate.
  • Methods in accordance with this aspect of the present invention can be used to sequentially detect the presence of a plurality of analytes from any sample to be analysed.
  • the method can be used to detect two or more analytes, three or more analytes or four or more analytes.
  • the source of the sample may be biological, chemical or environmental.
  • the sample may be obtained from a plant, animal, fungal, yeast, viral or bacterial species.
  • the analytes to be assayed for may be proteins (including peptides, oligopeptides or polypeptides), carbohydrates, glycoproteins, drugs and their metabolites or other molecules of interest.
  • the method may comprise the detection of a plurality of analytes which are antigens with respect to the immune system of an animal, or the detection of corresponding plurality of antibodies in an animal.
  • the sample may be of a body fluid or of a tissue sample which has been prepared to be suitable in a method according to the present invention.
  • the sample of a body fluid may be blood, urine, sweat, saliva, tears, milk, semen, synovial fluid, cerebrospinal fluid, amniotic fluid, tissue exudate at site(s) of infection, tissue extract or hydrolysate or other bodily secretion or fluid.
  • the tissue extract or hydrolysate may be from any tissue, organ or cellular source. Sources of blood may comprise whole or fractionated blood, suitably serum or plasma.
  • the animal may of any species, preferably a 7
  • mammalian species including marsupial species.
  • the animal may be a human or other primate species.
  • Other species of interest include commercially important mammalian species such as ungulate species, for example, sheep, cattle, goats, pigs, camels, water buffalo, alpaca or llama.
  • Methods in accordance with the present invention are also applicable to samples from horses, cats, dogs, rodents, e.g. rats, mice and guinea pigs, or rabbits.
  • transgenic in relation to animals, should not be taken to be limited to referring to animals containing in their germ line one or more genes from another species, although many transgenic animals will contain such a gene or genes. Rather, the term refers more broadly to any animal whose germ line has been the subject of technical intervention by recombinant DNA technology. So, for example, an animal in whose germ line an endogenous gene has been deleted, duplicated, activated or modified is a transgenic animal for the purposes of this invention as much as an animal to whose germ line an exogenous DNA sequence has been added.
  • the plurality of analytes may be proteins, for example, immunoglobulins, in particular, immunoglobulins of the classes IgA, IgG or IgM, or subtypes thereof.
  • the analytes may comprise soluble or non-soluble enzymes, or hormones or other molecules of interest, e.g.
  • steroid hormones for example, progesterone, oestrogen, estradiol, testosterone
  • metabolic markers for example cholesterol, carnitine
  • diagnostic markers for example pyridinoline, homocysteine
  • nucleic acid for example, DNA, RNA, cDNA, rRNA, tRNA, including antisense nucleic acid and ribozymes
  • vitamins for example vitamins A, B complex, C, D, E, K.
  • Other analytes can include, but are not limited to, molecules derived from an agent such as a yeast, bacteria, prion or a virus. In some circumstances, these molecules can function as antigens with respect to the 8
  • the corresponding antibodies produced by the animals immune system can also be assayed for using a method of the present invention.
  • Antibodies may also be associated with an allergic disease or an autoimmune disease in the animal.
  • the sample may be obtained from the chemical preparation or product to assay for the presence of particular analytes of interest, especially contaminants and/or impurities.
  • Chemical sources include, but are not limited to, toxic substances and pollutants, or pharmaceutical compounds or analogues. The production of molecules by biofermentation of yeasts or through cell culture may also be regarded as chemical sources for the purposes of the present invention. Also included in chemical sources of samples are medicines; cosmetics; diagnostic assay standards; research materials and/or chemicals.
  • the sample may be obtained from, for example, an industrial or domestic water supply (mains or source), industrial or domestic sewage, food and/or beverage products; farm produce (animal, plant or products prepared therefrom).
  • an industrial or domestic water supply mains or source
  • industrial or domestic sewage, food and/or beverage products farm produce (animal, plant or products prepared therefrom).
  • Luminescence is a physical property of a substance which is the emission of light under the influence of a physical agent.
  • Chemiluminescence is the emission of light by chemical reaction without appreciable temperature increase.
  • Bioluminescence is the phosphorescence of living vegetable, microbial or animal organisms. In the present invention this term is used to refer to luminescent labels derived from a biological source.
  • the luminescent label can be chemiluminescent label or a bioluminescent label.
  • Chemiluminescent labels include, but are not limited to, luminol, acridinium esters or sulphonamides, or other suitable compounds which can be triggered to luminesce or enzyme/substrate producers of chemiluminescence based on enzymes such as horseradish peroxidase (HRP), alkaline phosphatase (AP) or urease.
  • Bioluminescent labels include, but are not limited to, aequorin or luciferases such as from the firefly
  • Renilla reniformis and related species that utilise ATP Renilla reniformis and related species that utilise ATP.
  • test assay device in which the assay is carried out.
  • the test assay device may be any suitable construction, for example probe devices or "dip-stick" devices as described in EP-A-0418739, EP-A-
  • Such probe devices comprise a stick with an absorbent or adsorbent pad of material at one end. Samples are placed on the pad for detection in situ. The so-called “dip-stick” devices also detect an analyte in situ but are dipped into a liquid sample directly rather than sample being applied to the device.
  • Other suitable test assay devices include multi-well plates, e.g. 4-, 8-, 16-, 32-,
  • the method of the present invention is also suitable for use in test assay devices consisting of a single sample well, e.g. a test-tube, EppenddorfTM tube or capillary tube, or on a sold support surface, such as a membrane, which can be suitably constructed from any inert material, e.g. nitrocellulose, cellulose, polyethylene, polypropylene, polyvinylchloride or silicon.
  • test assay devices consisting of a single sample well, e.g. a test-tube, EppenddorfTM tube or capillary tube, or on a sold support surface, such as a membrane, which can be suitably constructed from any inert material, e.g. nitrocellulose, cellulose, polyethylene, polypropylene, polyvinylchloride or silicon.
  • Such solid supports could therefore be used as equivalents of multi-welled devices and could be used to accommodate one or more assays sites, i.e. the device could act as a "chip", for example a "DNA
  • Step (a) of the method defined in the first aspect of the invention involves introducing to a test assay device a sample comprising a plurality of analytes, where a plurality of 10
  • the corresponding capture means are present in the test device to capture the analytes in the sample.
  • the corresponding capture means may be bound to the surface of the test assay device or may be bound to a particle or a bead present in the test assay device, which may or may not already be present in solution.
  • the capture means may therefore be added to the device prior to introduction of the sample.
  • the particle or bead may be an agarose, plastics, glass, silicon or other inert substance and may also be magnetic. Where the capture means are bound to the surface of the test device or to a particle or to a bead, the binding may be by any generally suitable chemical means.
  • the capture means present in the test assay device are correspondent to the analytes to be determined, i.e. the capture means can specifically capture the analytes for which they are specific.
  • the capture means may be antibodies, polyclonal or monoclonal antibodies, i.e. immunoglobulin molecules, or fragments or variants thereof. Fragments of immunoglobulins include, but are not limited to, Fab, F(ab') 2 , V H , V L fragments and variants can include chimaeric immunoglobulins or fragments where the molecule is derived from more than one animal species, e.g. mouse-human, rabbit- goat etc.
  • the capture means may also include other specific binding molecules such as biological receptor molecules, or fragments or variants thereof, or streptavidin, avidin or variants thereof. Other binding molecules may be prepared using class or molecule specific imprinting techniques or molecular design programmes.
  • the capture means may also comprise a binary capture process which involves the use of a primary capture means in solution to capture the analyte and where the complex of analyte and primary capture means is then captured by a secondary capture means which is bound to the surface of the test assay device.
  • the primary capture means is an antibody, preferably a monoclonal antibody
  • the secondary capture means is an antibody specific for the primary antibody.
  • the capture means may also comprise a nucleic acid sequence or oligonucleotide for specific hybridisation to the analyte. For example, in an assay to determine the presence of a plurality of analytes 11
  • test assay device could be coated with goat anti-mouse antibodies to capture murine monoclonal antibodies complexed to the particular analyte whose presence is being assayed for in the sample.
  • goat anti-mouse antibodies to capture murine monoclonal antibodies complexed to the particular analyte whose presence is being assayed for in the sample.
  • Other variations on this scheme can be carried out depending upon the analyte to be assayed.
  • Step (b) of the method defined in the first aspect of the invention involves introducing to the test assay device a plurality of corresponding luminescent detection means for individual analytes to be assayed for in the sample. Steps (a) and (b) can, of course, be carried out together if so desired.
  • the luminescent detection means may comprise a luminescent label as previously described.
  • the detection means can suitably comprise a known amount of a labelled analyte so as to permit the analysis of the presence of the unlabelled captured analyte in the sample to be assayed by competitive immunoassay.
  • the detection means can comprise a luminescent label which binds to the captured analyte in the test assay device.
  • the detection means may form a complex with the captured analyte.
  • the detection means may also be selected to detect the presence of a captured analyte complex, for example where the analyte is captured by a secondary capture means which is in turn captured by a primary capture means, it may be the secondary capture means which is detected to quantitate the analyte in a stoichiometric manner.
  • Step (c) of the method defined in the first aspect of the invention involves washing the assay test device to remove excess detection means.
  • the washing step may comprise washing the device with any suitable solvent or solution to remove excess detection means. Washing can be carried out with water or more preferably with a buffered solution of salts to increase the solubility of the excess detection means. Step (c) may also be repeated if necessary to achieve the removal of excess detection means. 12
  • Step (d) of the method defined in the first aspect of the invention involves the analysis of the test assay device to determine the presence or the absence of a first analyte by assaying for the luminescent detection means for the first analyte.
  • the step of analysing may be carried out by the introduction of a trigger or a substrate to activate the luminescent detection means.
  • an excess of the appropriate ion may be introduced in a suitable salt solution.
  • the luminescence can be triggered by the introduction of divalent metal ions, preferably Group II metal ions, e.g. calcium ions (Ca 2+ ).
  • the luminescent label is an enzyme an appropriate substrate can be introduced.
  • the substrates CDP-StarTM, LumiPhos 530TM, Lumigen APS-3, Lumigen APS-5 luminescent substrates and related formulations;
  • horseradish peroxidase substrates such as hydrazides e.g. luminol, with activators such as p-iodophenol and related compounds can be used.
  • Step (e) of the method defined in the first aspect of the invention involves the quantitation of the presence or absence of an analyte in the sample based upon detection of a luminescent label specific for an individual analyte.
  • the quantitation of the amount of analyte present in a sample or the determination of the absence of an analyte from a sample can be achieved using any suitable means for the quantitation of the particular label used, i.e. for the detection of a coloured light emission a spectrophotometer can be used (Wampler, J. E., Measurements and Physical Characteristics of Luminescence in "Bioluminescence in Action", ed. P. J. Herring, Academic Press New York (1978); Whitehead et al Clinical Chemistry 25 1531-1546
  • Examples of suitable machines include Wallac Victor and Wallac LB96V plate readers and Lumitube and Berthold Clinilumat Tube Luminometer counters.
  • Steps (d) and (e) can be repeated for the second and subsequent analytes to be assayed for as appropriate as defined in step (f) of a method of this aspect of the present 13
  • the signal from the previous label can be dissipated prior to analysis of the next label in sequence by means of allowing the luminescence to decrease by substrate exhaustion, by quenching the luminescence by the introduction of specific quenching agents or by poisoning the enzyme responsible for the luminescence by the introduction of a metabolic poison.
  • bioluminescent label is used as the first label
  • its triggering must not effect the bound second or subsequent label, i.e. the labels used are preferably compatible.
  • a preferred method in accordance with the present invention is therefore to provide a first bioluminescent label triggered by an ion to detect the first analyte and a second chemiluminescent label triggered chemically to detect the second analyte.
  • a bioluminescent label triggered by an ion to detect the first analyte
  • a second chemiluminescent label triggered chemically to detect the second analyte.
  • the chemiluminescent label may be the first to be measured followed by the bioluminescent label.
  • the ordering of labels may be appropriately modified to achieve the object of the present invention.
  • the skilled person will also be able to select labels to match their specific activity to the expected concentration of analyte, i.e. a high specific activity label with an analyte at an expected low concentration.
  • the quenching of enzyme generated luminescence with acridinium triggers may be dependent upon the substrate used and can be selected for appropriately.
  • the susceptibility of individual enzyme species to poisoning may require specific ordering of the triggering, i.e. that the luminescent labels to detect the analytes are triggered independently in sequence of lability. For example, HRP 14
  • ALP generated luminescence should be measured prior to ALP generated luminescence where the ALP substrate contains azide. Simultaneous addition of triggers and substrates may also be carried out in methods in accordance with the present invention, e.g. Ca and ALP substrate may be added simultaneously where the enzyme substrate CDP-Star has sufficient lag-time to allow for differentiation of the primary bioluminescence.
  • the method permits the ability to immuno-isolate a single or several different species of antibody; the ability to bind low and high molecular weight analytes; the ability to undertake quantitation using hybrid detection systems including enzyme/substrate, bioluminescent and chemiluminescent detection; the ability to quench luminescence from previous detection systems in order to allow sequential detection without the need to remove excess reagent between determinations.
  • a method of the present invention has no effect on assay performance compared to single analyte determination. Full automation of the assay methods is also permitted by the utilisation of available assay technology.
  • a multianalyte assay test device comprising a plurality of specific capture means present in the device which capture corresponding individual analytes in a sample containing a plurality of analytes, wherein the analytes are detected sequentially by luminescent detection means for individual analytes to be assayed for in the sample and wherein residual detection means are not washed from the test assay device between the detection and quantitation of individual analytes in the sample and the signal from the previous label has been dissipated prior to analysis of the next label in sequence.
  • a method for assaying for bone turnover markers characteristic of the disease osteoporosis in a patient sample in which the analytes are pyridinoline and bone specific alkaline 15
  • bALP phosphatase
  • assay method is as described for the first aspect of the present invention.
  • Other suitable analytes characteristic of bone turnover may also be selected for assay in accordance with this aspect of the invention.
  • a method for assaying for the analytes B 12 and folate in a patient sample which are characteristic of the disease anaemia and the assay method is as described for the first aspect of the present invention.
  • kits for the multiple detection of analytes in a single sample comprising an assay test device as defined above and luminescent detection means.
  • FIGURE 1 shows the standard curve for bone specific ALP from Example 1.
  • the x-axis shows the concentration of bone specific ALP in terms of units/litre of bALP and the y-axis shows Relative Light Units (RLU) (xlOOO).
  • RLU Relative Light Units
  • FIGURE 2 shows the standard curve for pyridinoline from Example 1.
  • axis shows the concentration of pyridinoline in pmol ml and the y-axis shows %B/Bo.
  • the abbreviation %B/Bo refers to %bound/maximum signal bound (in a competitive assay the maximum signal corresponds to the zero antigen calibrator).
  • FIGURE 3 shows the standard curve for progesterone from Example 2.
  • the x- axis shows the concentration of progesterone in pg/ml and the y-axis shows %B/Bo.
  • the abbreviation %B/Bo refers to %bound/maximum signal bound (in a competitive assay the maximum signal corresponds to the zero antigen calibrator).
  • FIGURE 4 shows the standard curve for 17 ⁇ -estradiol from Example 2.
  • the x- axis shows the concentration of 17 ⁇ -estradiol in pg/ml and the y-axis shows %B/Bo.
  • the abbreviation %B/Bo refers to %bound/maximum signal bound (in a competitive assay the maximum signal corresponds to the zero antigen calibrator).
  • the monoclonal antibodies to pyridinoline, bone alkaline phosphatase and progesterone were obtained from Metra Biosystems Inc. (Paulo Alto. CA.), the NIH (Washington, USA) and Assay Designs Inc., MI. USA) respectively.
  • the rabbit polyclonal antibody to 17 ⁇ Estradiol was obtained from Assay Designs Inc.
  • GxM at 10.4mg/ml was diluted to a concentration of 1 O ⁇ g/ml in coating buffer comprising, 1 OmM phosphate, 1 OmM
  • microtitre plates capable of binding mouse antibodies white 96 (12x8) microtitre plates (Dynex) were coated with Goat anti mouse IgG specific for the Fc region using the following protocol. To avoid possible cross contamination all procedures were carried out using disposable labware.
  • Goat anti mouse IgG (GxM) at 10.4mg/ml (Chemicon International, Harrow UK, Cat. # API 27) was diluted to a concentration of 1 O ⁇ g/ml in coating buffer comprising lOmM phosphate, lOmM NaCl, pH 7 and following mixing left to stand for 5 minutes.
  • GxR goat anti rabbit IgG
  • PCMO Progesterone 3-o-carboxymethyloxime
  • the activation reaction was carried out with a 2 fold molar excess of DCC and 1.1 molar excess of NHS relative to PCMO.
  • the reaction mixture was stirred overnight in a sealed vial at room temperature in a desiccator.
  • the PCMO-NHS ester was stored at -20°C until required.
  • PCMO-NHS ester Conjugation of PCMO-NHS ester to Acridinium C2 ester and BSA Carrier protein PCMO-NHS ester (synthesis described above) was reacted with a 40 molar excess of bovumar BSA (Intergen, Cat. #3210) dissolved in 50mM sodium terra borate, pH 8.5 (Pierce. Cat. #28384), and a 5 molar excess of acridinium C2 NHS ester (Assay Designs Inc., USA. Cat. #90600). The conjugation mixture was stirred at room temperature for 30 minutes and the reaction terminated by the addition of lO ⁇ l of 10% lysine (Sigma Chemical Co.).
  • the PCMO-BS A- Acridinium conjugate was isolated after passing through a G15 desalting column equilibrated and eluted with 50mM TBS buffer (Sigma Chemical Co. Cat. #T6789) containing 50mM NaCl, 150mM KCl, 1% (w/v) BSA and 0.1% (w/v) sodium azide. 1ml fractions were collected and an aliquot of each assayed for acridinium activity using a tube lurninometer. Fractions containing activity were pooled and aliquoted and stored at -20°C until use.
  • 50mM TBS buffer Sigma Chemical Co. Cat. #T6789
  • Lyophilised Aequorin (Sigma, St. Louis, USA) was resuspended in activation buffer comprising of lOmM Hepes, 0.2M NaCl, 2mM EDTA pH 8, to a final concentration of l.Omg/ml.
  • activation buffer comprising of lOmM Hepes, 0.2M NaCl, 2mM EDTA pH 8, to a final concentration of l.Omg/ml.
  • a 5 molar excess of PCMO-NHS ester (synthesis described above) was conjugated to Aequorin.
  • the conjugation mixture was stirred at room temperature for
  • Fractions containing aequorin activity were pooled, and diluted in storage buffer comprising of lOmM Tris, 5mM EGTA, lOmg/ml sucrose, 40mg/ml BSA pH 8, aliquoted and stored at -20°C until use.
  • Both antibody and conjugate were diluted in buffer comprising 50mM Tris, 2mM EGTA, 15mM NaCl, O.lmM zinc sulphate, 4.9mM MgCl 2 , 0.1 (w/v) sodium azide, 0.1% BSA, 0.005% SDS, pH8 with HCL.
  • Antibody buffer was coloured yellow by the addition of yellow food dye (2.5 ⁇ l/ml) and conjugate buffer blue by the addition of blue food colouring (1.25 ⁇ l ml).
  • Wash buffer comprised of 50mM NaCl, 0.1% sodium azide, 0.05% Tween 20, lOO ⁇ M zinc sulphate, 4.9mM MgCl 2 , lmM EGTA, pH 8, with HC1.
  • priming solution 1 comprising of lmM hydrogen peroxide in 0.1M nitric acid was added followed by an equal volume of trigger solution 2 comprising of 0.25% CTAC in 0.15M NaOH, sequentially and the signal read immediately for 2 seconds.
  • trigger solution comprising of 50mM Tris, lOOmM CaCl 2 , 15mM sodium azide, pH 7 was added. The signal was read immediately for 2 seconds.
  • APS5 Liigen Inc.
  • CDP-Star Lit #3633, Tropix Inc.
  • Lumiphos 530TM Lumiphos
  • Microtitre plate based luminescent measurements were performed using Wallac Victor and Wallac LB96V plate readers and tubes using Lumitube and Berthold Clinilumat Tube Lurninometer counters.
  • the assay involves the simultaneous capture of monoclonal antibodies to bALP and pyridinoline using a solid phase coated with goat anti-mouse antibody.
  • the assay principles are immunocaprure and competitive immunoassay.
  • Acridinium ester labelled-pyridinoline and two monoclonal antibodies specific for bALP and pyridinoline and different concentration standards are incubated for 3 hours at room temperature in goat anti-mouse coated tubes. After washing the bound bALP is determined by addition of Lumiphos 530, a chemiluminescent ALP substrate, and the signal read after 45 minutes for 2 seconds. This detection sequence determines the bALP specific signal.
  • Tubes are again washed and the bound acridinium labelled- pyridinoline determined by addition of trigger solutions.
  • the signal is read immediately for 2 seconds.
  • the acridinium ester detection sequence allows the concentration of bound pyridinoline to be determined.
  • the assay illustrates the co- isolation of two monoclonal antibodies and quantitation of two bound analytes on a single solid phase support following the sequential addition of substrates. Total emitted light is used for quantitation of individual analytes.
  • results of the sequential assay of bone turnover markers (bone specific Alkaline Phosphatase [bALP] and pyridinoline are shown below in Tables 1, 2a and 2b and in Figures 1 and 2. Results for A and B are given in terms of relative light units.
  • CV% % coefficient of variation
  • Example 2 Sequential Assay of Progesterone and 17 ⁇ Estradiol
  • the assay involves the simultaneous capture of both monoclonal and polyclonal antibodies to two steroids simultaneously, progesterone and 17 ⁇ -estradiol, using a solid phase coated with goat anti-mouse and goat anti-rabbit antibodies.
  • the assay uses two competitive immunoassays utilising chemiluminescent detection (CLIA).
  • Aequorin labelled progesterone and ALP labelled estradiol were incubated for 2 hours at room temperature with a monoclonal antibody to progesterone and a rabbit polyclonal antibody to 17 ⁇ -estradiol, along with different concentration standards or samples of each steroid. This incubation was carried out in microtitre plates coated with two secondary capture antibodies, i.e. goat anti-mouse and goat anti-rabbit IgG.
  • the plate is washed to remove excess unbound reagents and the aequorin is triggered by the addition of calcium ions and the ALP assay started by the co-addition of a substrate containing Ca 2+ ions and CDP-Star, a rapid ALP substrate.
  • the aequorin signal is generated by the Ca 2+ ions in the substrate and read immediately for 2 seconds. Without any washing, the CDP-Star signal is generated on the solid phase and is read in the order of addition from 5 to 15 minutes later.
  • the assay illustrates the co-isolation of two species of antibodies and quantitation of two bound analytes on a single solid phase support following a single addition of substrate. Total emitted light is used for quantitation of both analytes. There is no loss of assay sensitivity.
  • the results of the sequential assay of progesterone and 17 ⁇ - estradiol using polyclonal and monoclonal capture are shown below in Tables 3a, 3b, 4a and 4b and in Figures 3 and 4. 25
  • the binding curve for progesterone is shown in Figure 3 and the binding curve for estradiol is shown in Figure 4.
  • This example shows the quenching of the chemiluminescent signals generated by enzyme generated luminescence by the addition of reagents such as the triggers for Acridinium esters. This illustrates the ability of the primary trigger used to convert
  • Acridinium to the salt form needed for full acridinium light emission to quench certain types of ALP generated chemiluminescence.
  • 20 ⁇ l of ALP conjugate dilutions (Assay Designs Inc., USA Lot# 97S011) were incubated with lOO ⁇ l of CDP-Star substrate for 45 minutes and the resultant luminescence determined (Table 5 - replicates A and B).
  • lOO ⁇ l of primary acridinium trigger was then injected into the wells (without) removal of the ALP luminescent product) and the luminescence again determined.
  • substrate additions could be envisaged, e.g. sodium azide which could be used to terminate and quench horseradish peroxidase generated luminescence which would enable subsequent triggering of additional labels without the need for removal of a previous substrate or trigger.

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Abstract

L'invention concerne un procédé et un dispositif permettant de quantifier plusieurs analytes dans un seul dosage sans contamination croisée des résultats des dosages. Ce dosage permet de déterminer la présence ou l'absence de plusieurs analytes d'un échantillon, à l'aide de techniques de dosages immunologiques, et de dosages à base de sondes d'ADN ou d'essais immuno-enzymatiques ELISA.
PCT/GB1999/001336 1998-04-29 1999-04-29 Procede de dosage de plusieurs analytes WO1999056130A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU37208/99A AU3720899A (en) 1998-04-29 1999-04-29 Method for assaying a plurality of analytes

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GBGB9809160.6A GB9809160D0 (en) 1998-04-29 1998-04-29 Assay
GB9809160.6 1998-04-29

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WO1999056130A1 true WO1999056130A1 (fr) 1999-11-04

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012159264A1 (fr) * 2011-05-24 2012-11-29 Dai Lijun Réactif bioanalytique utilisé en phase hétérogène et procédé d'utilisation de ce réactif
CN106053443A (zh) * 2016-07-05 2016-10-26 深圳市亚辉龙生物科技股份有限公司 吖啶标记结合物及其制备方法、化学发光试剂盒
CN106124777A (zh) * 2016-07-05 2016-11-16 深圳市亚辉龙生物科技股份有限公司 吖啶标记结合物及其制备方法、化学发光试剂盒
CN106146672A (zh) * 2016-07-05 2016-11-23 深圳市亚辉龙生物科技股份有限公司 吖啶标记结合物及其制备方法、化学发光免疫检测试剂盒
CN111665353A (zh) * 2020-03-12 2020-09-15 杭州布封科技有限公司 一种紧凑型免疫分析装置及分析方法
CN117986328A (zh) * 2024-04-03 2024-05-07 江西省转化医学研究院 一种环肽发光耦合分子及其制备方法和应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2233450A (en) * 1989-06-24 1991-01-09 Univ Wales Medicine Detecting or quantifying multiple analytes
WO1992012255A1 (fr) * 1990-12-28 1992-07-23 Abbott Laboratories Determination simultanee d'analytes multiples a l'aide d'une technique de chimioluminescence heterogene a resolution temporelle
EP0522677A1 (fr) * 1991-07-10 1993-01-13 TDK Corporation Procédé pour le mesurage de la concentration d'immunoréactant au moyen luminescence électrochimique
EP0623821A2 (fr) * 1993-05-06 1994-11-09 Ciba Corning Diagnostics Corp. Essais utilisant des conjugués luminescents mixtes

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2233450A (en) * 1989-06-24 1991-01-09 Univ Wales Medicine Detecting or quantifying multiple analytes
WO1992012255A1 (fr) * 1990-12-28 1992-07-23 Abbott Laboratories Determination simultanee d'analytes multiples a l'aide d'une technique de chimioluminescence heterogene a resolution temporelle
EP0522677A1 (fr) * 1991-07-10 1993-01-13 TDK Corporation Procédé pour le mesurage de la concentration d'immunoréactant au moyen luminescence électrochimique
EP0623821A2 (fr) * 1993-05-06 1994-11-09 Ciba Corning Diagnostics Corp. Essais utilisant des conjugués luminescents mixtes

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012159264A1 (fr) * 2011-05-24 2012-11-29 Dai Lijun Réactif bioanalytique utilisé en phase hétérogène et procédé d'utilisation de ce réactif
CN106053443A (zh) * 2016-07-05 2016-10-26 深圳市亚辉龙生物科技股份有限公司 吖啶标记结合物及其制备方法、化学发光试剂盒
CN106124777A (zh) * 2016-07-05 2016-11-16 深圳市亚辉龙生物科技股份有限公司 吖啶标记结合物及其制备方法、化学发光试剂盒
CN106146672A (zh) * 2016-07-05 2016-11-23 深圳市亚辉龙生物科技股份有限公司 吖啶标记结合物及其制备方法、化学发光免疫检测试剂盒
CN106146672B (zh) * 2016-07-05 2019-11-29 深圳市亚辉龙生物科技股份有限公司 吖啶标记结合物及其制备方法、化学发光免疫检测试剂盒
CN111665353A (zh) * 2020-03-12 2020-09-15 杭州布封科技有限公司 一种紧凑型免疫分析装置及分析方法
CN117986328A (zh) * 2024-04-03 2024-05-07 江西省转化医学研究院 一种环肽发光耦合分子及其制备方法和应用

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GB9809160D0 (en) 1998-07-01

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