WO1999056130A1

WO1999056130A1 - Method for assaying a plurality of analytes

Info

Publication number: WO1999056130A1
Application number: PCT/GB1999/001336
Authority: WO
Inventors: Ian James; David Perrett; Russell Hart; Barbara Scheuer; Nancy Schmidt
Original assignee: Queen Mary & Westfield College; Assay Designs, Inc.
Priority date: 1998-04-29
Filing date: 1999-04-29
Publication date: 1999-11-04
Also published as: AU3720899A; GB9809160D0

Abstract

A method and a device are provided for the quantitation of multiple analytes in single assay without cross-contamination of assay results. The assay can be used to determine the presence or absence of a plurality of analytes from a sample by using immunoassay, ELISA or DNA probe based assay techniques.

Description

METHOD FOR ASSAYING A PLURALITY OF ANALYTES

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.

It its simplest embodiment, 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 β-galactosidase are commonly used as labels in immunoassays due to their high catalytic activity and the variety of available substrates. For example 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

requirements, regardless of the difficulty in differentiating multiple responses. 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 ¹²⁵Iodine and ⁵⁷Cobalt 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.

Other approaches to such assays have concentrated upon the chemistry of the detection molecules, the methods of attachment of antibody or binding partner to one or more solid phases or manufacture of multianalyte devices. 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. There have also been reports of 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). Methods for site specific attachment of binding agents within a single solid phase support have been described (WO 94/27137) involving the coupling of thiol groups on the capture molecules to photo activatable affinity crosslinkers. WO 95/24649 describes techniques where different species of oligonucleotides attached at spatially separated locations on a single solid phase are used to capture antibodies with complementary oligonucleotide sequences with the binding or capture occurring 'in solution' rather 3

than on the surface of the solid phase.

The quantitation of bound analytes can be achieved either simultaneously or sequentially. Where 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. In practice due to limitations in instrumentation measurements are essentially made within distinct spectral regions. For example 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. However in many instances complex mathematical processing of data is required to discern overlapping signals. While the narrowing of 'windows' enables increased specificity of detection it can also result in a loss of energy and sensitivity. The number of possible analytes determined simultaneously using colourimetric, fluorimetric, time resolved fluorescence or radioisotopic techniques are restricted by the practicalities of differentiating responses when two or more labels are used.

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.

However 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. The 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)).

The spatial separation of different capture antibodies allows the same label to be used for quantitation of all analytes and this has led to the development of multianalyte microspot immunoassays (Ekins, R. P., & Chu, F. W., Clin. Chem., 37 (11) 1955-1967 (1991); Ekins et al Clin. Chim. Acta. 194 91-114 (1990)) . However their potential application may be limited by the need for very specific antibodies and the difficulty in optimising the assay range for all analytes because of the fixed sample volume. Cross contamination of microspots with binding reagents from neighbouring microspots is also a potential problem giving rise to false positives. Essentially the number of analytes which could be detected are balanced against he increased risk of failure of an analyte either in quality control or during assay, leading to need for reanalysis and diminishing commercial returns (Kricka, L. J., Clin. Chem. 38 (3) 327- 328 (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. Prior to the present invention, a combination of two or more detection systems including enzyme, chemiluminescent or bioluminescent detection 5

systems has not been reported for a multianalyte format. The system now described fulfils these goals and permits sequential detection of multiple bound labels.

According to a first aspect of the present invention there is provided a method for sequentially assaying for the presence of a plurality of analytes in a single sample, the method comprising the steps of:

(a) introducing to a test assay device a sample solution comprising a plurality of analytes, where a plurality of corresponding capture means are present in the test device to capture the analytes in the sample;

(b) introducing to the test assay device a plurality of corresponding luminescent detection means for individual analytes to be assayed for in the sample;

(c) washing the assay test device to remove excess detection means;

(d) analysing the test assay device to assay for a first analyte by assaying for the luminescent detection means for the first analyte;

(e) quantitating the amount of an analyte in the sample based upon detection of a luminescent label specific for an individual analyte; and

(f) repeating steps (d) and (e) for the second and subsequent analytes to be assayed for as appropriate;

wherein residual bound detection means are not washed from the test assay device between the detection and quantitation of individual analytes in the sample and in which the luminescent labels to detect the analytes are triggered independently in 6

sequence of lability and in which the signal from the previous label has been dissipated prior to analysis of the next label in sequence.

In principle, 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.

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.

Where the source is a biological source, 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.

In assays which are used to detect a plurality of analytes in a sample from 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. Where the animal is a mammalian 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.

The invention is equally applicable to methods of detection of samples from transgenic (non-human) animals which have produced to express one or more heterologous protein in a body fluid, e.g. milk, blood or in a tissue, or organ, or a cell. It should be noted that the term "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.

In assays of a sample of a bodily fluid from an animal, the plurality of analytes may be proteins, for example, immunoglobulins, in particular, immunoglobulins of the classes IgA, IgG or IgM, or subtypes thereof. Alternatively, 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), or nucleic acid (for example, DNA, RNA, cDNA, rRNA, tRNA, including antisense nucleic acid and ribozymes) or 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

immune system of the animal in whose bodily fluids the yeast, bacteria, prion or virus is present. Accordingly, 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.

Where the sample is obtained from a chemical source, 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.

Where the sample is obtained from an environmental source, 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).

Methods in accordance with the present invention are used to analyse a single sample using the detection of a luminescent label indicative of the presence of the plurality of analytes present in the sample. 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. 9

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.

Methods in accordance with the present invention utilise a 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-

0442231. US-A-5103838 or WO 95/02822. 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-,

64-, or 96-well plates , or multiples thereof, constructed from plastics materials. Such plates permit ready automation of the assay and high throughput using computer- controlled analysers. Alternatively, 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, Eppenddorf™ 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 chip" in respect of DNA probe-based assays

(Mirzabekov, A., Trends Biotechnol. 12 27-32 (1994); Marshall, A. & Hodgson, J., Nature Biotechnology 16 27-31 (1998)).

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

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. Suitably the primary capture means is an antibody, preferably a monoclonal antibody, and 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

in a sample which is based on immunocapture and competitive immunoassay, the 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. 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. Alternatively, if the assay is based on enzyme-linked immunosorbent assay (ELISA), the detection means can comprise a luminescent label which binds to the captured analyte in the test assay device. In some situations, where primary and secondary capture means are employed, 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. Where the luminescence of the detection means is ion dependent, an excess of the appropriate ion may be introduced in a suitable salt solution. For example, where the label aequorin is used, the luminescence can be triggered by the introduction of divalent metal ions, preferably Group II metal ions, e.g. calcium ions (Ca²⁺). Alternatively, where the luminescent label is an enzyme an appropriate substrate can be introduced. For example, for alkaline phosphatase (ALP) the substrates CDP-Star™, LumiPhos 530™, Lumigen APS-3, Lumigen APS-5 luminescent substrates and related formulations; for 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

(1979)). 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

invention.

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.

Where a 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.

For optimum performance the labels are therefore triggered in sequence of lability. The addition of a second or subsequent trigger for the second or subsequent label does not have to necessarily destroy the luminescence of the first label as the trigger for the first label preferably will have exhausted all the bound or available first label. 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. For example, aequorin followed by acridinium esters, where the reactions may be completed within a couple of seconds. Alternatively, the chemiluminescent label may be the first to be measured followed by the bioluminescent label. For quantitation of more than two analytes, 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

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.

Methods in accordance with the present invention therefore offer several advantages over those already known in the art. 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. Significantly, 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.

According to a second aspect of the invention there is provided 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.

According to a third aspect of the present invention, there is provided 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

phosphatase (bALP) and the 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.

According to a fourth aspect of the present invention, there is provided a method for assaying for the analytes B₁₂ 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.

It is believed that methods in accordance with the present invention can find application in the analysis of samples containing a plurality of analytes characteristic of many diseases.

According to a fifth aspect of the present invention, there is provided a kit for the multiple detection of analytes in a single sample comprising an assay test device as defined above and luminescent detection means.

Preferred aspects for the second and subsequent aspects of the invention are as for the first aspect mutatis mutandis.

The present invention will now be further described with reference to the accompanying Examples and drawings which are provided for the purposes of illustration only and are not to be construed as being limiting upon the invention. In the Examples, reference is made to a number of drawings which are as follows:

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).

FIGURE 2 shows the standard curve for pyridinoline from Example 1. The x- 16

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 17β-estradiol from Example 2. The x- axis shows the concentration of 17β-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).

Examples

General Experimental Methods and Materials: 1.1 Source of Antibodies to Analytes

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 17β Estradiol was obtained from Assay Designs Inc.

1.2 Coating of Star Tubes with Goat anti-mouse IgG

To produce tubes capable of binding mouse antibodies, Maxisorb Star tubes (Nunc) were coated with goat anti-mouse IgG (GxM) specific for the Fc region using the 17

following protocol. To avoid possible cross-contamination all procedures were carried out using disposable labware.

GxM at 10.4mg/ml (Chemicon International, Harrow, UK, Cat# API 27) was diluted to a concentration of 1 Oμg/ml in coating buffer comprising, 1 OmM phosphate, 1 OmM

NaCl pH 7 and following mixing left to stand for 5 minutes. Volumes were adjusted accordingly for the number of tubes to be coated. 250μl of antibody solution was added to each tube and allowed to stand for 15-24 hours at room temperature. The antibody solution was aspirated to waste and 300μl of blocking buffer comprising 50mM Tris, 0.15M NaCl/KCl, 5% sucrose, 1% BSA, 0.1% azide, pH 8, added immediately. Tubes were covered and allowed to stand for 4-24 hours. The blocking buffer was aspirated to waste and aspiration completed for complete removal. Tubes were dried for 18-24 hours in a drying chamber, then removed and packed in Ziploc bags with desiccant, only when the humidity in the chamber fell below 20%. Tubes were labelled according to coating and stored at 4°C until use.

1.3 Coating of Microtitre Plates with Goat anti mouse IgG

To produce 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 Oμg/ml in coating buffer comprising lOmM phosphate, lOmM NaCl, pH 7 and following mixing left to stand for 5 minutes.

Volumes were adjusted accordingly for the number of plates to be coated. 250μL of antibody solution was added to each well, plates wrapped with cling film and allowed to stand at room temperature for 15 to 24 hours. The antibody solution was aspirated to waste and plates patted on paper towelling to dry. 250μL of blocking buffer 18

comprising of 50mM Tris,0.15M NaCl/KCl, 5% Sucrose, 1% BSA. 0.1% azide, pH 8.0 was then added to each well. Plates were wrapped and allowed to stand at room temperature for 4-24 hours. The blocking buffer was aspirated to waste, and aspiration repeated for complete removal. Plates were dried by patting vigorously on paper towelling and transferred to a drying chamber. Plates were taken and packed with desiccant in Ziploc bags, only when the humidity in the chamber fell below 20%. Plates were labelled according to coating and stored at 4°C until use.

1.4 Coating of Microtitre Plates with Goat anti mouse IgG and Goat anti rabbit IgG

To produce microtitre plates capable of binding mouse and rabbit antibodies the manufacturing procedures used were as described above except that goat anti rabbit IgG (GxR) (Chemicon International) was included in the coating buffer at a final concentration of 1 Oμg/ml, in addition to GxM at 1 Oμg/ml.

1.5 Activation of Antigens with N-hydroxysuccinimide

(a) Activation of Progesterone 3-o-carboxymethyloxime (PCMO) 10.5mg of PCMO (Cat. #P3277, Sigma Chemical Co, St. Louis), 21.4mg Dicyclocarbodiimide (DCC, Cat. #D8000-2) and 12.4mg N-hydroxysuccinimide (NHS, Cat. #13097-2) (Aldrich Chemical Co) were each dissolved in separate 1ml aliquots dry dimethyl formamide dried and stored desiccated in sealed glass vials at - 20°C until use.

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.

(b) Activation of 17β Estradiol-6-carboxymethyloxime (17βE₂CMO) 19

5mg 17βE₂CMO was dissolved in 1ml dry DMF and other reagents prepared as above. The activation reaction was carried out with a 10 fold molar excess of DCC and 1.1 molar excess of NHS relative to 17βE₂CMO. The reaction was stirred overnight in a sealed vial at room temperature in a desiccator. The 17βE₂CMO-NHS ester was stored at -20°C until required.

1.6 Conjugation of Antigen-NHS Esters to Detection Systems

(a) 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 lOμl 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.

(b) Conjugation of pyridinoline to Acridinium C₂ NHS ester

Pyridinoline (Metra Biosystems Inc. USA Lot 3G112) was reacted with a 3 molar excess of acridinium C₂ NHS ester (Assay Designs Inc., USA, Cat#90600). The conjugation mixture was vortexed and incubated for 30 minutes at room temperature.

The reaction was stopped by the addition of concentrated HC1 and the resultant conjugate stored at -20°C until use. 20

(c) Conjugation of PCMO-NHS ester to Aequorin

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. A 5 molar excess of PCMO-NHS ester (synthesis described above) was conjugated to Aequorin. The conjugation mixture was stirred at room temperature for

30 minutes and the reaction terminated by the addition of lOμl of 10% lysine. The PCMO-Aequorin conjugate was isolated after passing through a G15 desalting column equilibrated and eluted with lOmM Hepes, 0.2M NaCl, 2mM EGTA pH 7. 1ml fractions were collected and an aliquot of each assayed for aequorin activity using a tube lurninometer. 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.

1.7 Conjugation of 17βE₂CMO-NHS ester to Alkaline phosphatase Calf intestinal alkaline phosphatase (AP) (Biozyme, Cat. #ALPI-12G,) at a cone. Of

14.73mg/mL in 50% Glycerol was diluted in borate buffer pH 8.5 to a cone. Of 2mg/ml. 17βE₂CMO-NHS ester (synthesis described previously) was added in an 80 fold molar excess. The conjugation mixture was stirred at room temperature for 30 minutes and the reaction terminated by the addition of lOμl of 10% lysine. The 17βE CMO AP conjugate was isolated after passing though 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. lml fractions were collected and an aliquot of each assayed for AP activity using p- nitrophenol phosphate as substrate. Fractions containing AP activity were pooled, aliquoted and stored at -20°C until use.

1.8 Additional Reagents

(a) Antibody and Conjugate Diluent Buffer 21

Both antibody and conjugate were diluted in buffer comprising 50mM Tris, 2mM EGTA, 15mM NaCl, O.lmM zinc sulphate, 4.9mM MgCl₂, 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 (2.5μl/ml) and conjugate buffer blue by the addition of blue food colouring (1.25μl ml).

(b) Washing Buffer

Wash buffer comprised of 50mM NaCl, 0.1% sodium azide, 0.05% Tween 20, lOOμM zinc sulphate, 4.9mM MgCl₂, lmM EGTA, pH 8, with HC1.

(c) Acridinium Trigger Reagents

To elicit a response from any bound acridinium label, 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.

(d) Aequorin Trigger Reagent

To elicit a response from any bound Aequorin label, 25-75μl of trigger solution comprising of 50mM Tris, lOOmM CaCl₂, 15mM sodium azide, pH 7 was added. The signal was read immediately for 2 seconds.

(e) AP Substrates

Several substrates for AP were used including p-nitrophenol phosphate APS3 and

APS5 (Lumigen Inc.), CDP-Star (Lot #3633, Tropix Inc.) and Lumiphos 530™.

(f) AP-Aequorin Reagent

To enable the combined triggering of Aequorin and activation of AP labels 25mM calcium acetate was added to CDP-Star AP substrate and mixed prior to injection. 22

1.9 Dilution of Antibodies and Conjugates

All dilutions of antibodies and conjugates were made with diluent buffer as described above.

2.0 Equipment

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.

Example 1 : Sequential Assay of Bone Turnover Markers (Bone Specific Alkaline Phosphatase fbALPl and Pyridinoline) Procedure

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. 23

Results

The 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.

Table 1: Bone Specific ALP data

Concentration Replicate (pg/ml) A | B Average Std. cv%

0 992 1014 1003 16 1.6

2 7283 6919 7101 257 3.6

80 279982 301282 290632 15061 5.2

,40 437162 439788 438475 1857 0.4

CV% = % coefficient of variation; Std. Standard deviation

Table 2a: Pyridinoline data

Replicate

A B J Average 1 Std. cv%

TC 595087 596063 595575 690 0.1

NSB 128 117 123 8 6.3

TC Total counts; NSB = Non-specific binding Table 2b: Pyridinoline data

J Cone. 1 Replicate

(pg/ml) A B Average Std. CV% %Bound

0 4448 4024 4236 300 7.1 0

0.5 3690 3690 3690 0 0.0 87.11

2 3368 3699 3534 234 6.6 83.42

10 3150 3150 3150 0 0.0 74.36

80 1188 1135 1162 37 3.2 27.42

200 582 614 598 23 3.8 14.12

1000 248 244 246 3 1.1 5.81

24

The standard curves for bone specific ALP and pyridinoline are shown in Figures 1 and 2.

Example 2: Sequential Assay of Progesterone and 17β Estradiol The assay involves the simultaneous capture of both monoclonal and polyclonal antibodies to two steroids simultaneously, progesterone and 17β-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).

Procedure

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 17β-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.

At the end of the immunological binding reaction, 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 Ca²⁺ ions and CDP-Star, a rapid ALP substrate. The aequorin signal is generated by the Ca²⁺ 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 17β- estradiol using polyclonal and monoclonal capture are shown below in Tables 3a, 3b, 4a and 4b and in Figures 3 and 4. 25

Table 3a: Progesterone data

—————— — Replicate

A B Average | Std. | cv%

TC 146871 147258 147065 j 274 0.2

NSB 554 597 576 30 5.3

Bo 15329 14603 14966 513 3.4

Blank 334 326 330 6 1.7

Results for A and B are given in terms of relative light units; Std. = Standard deviation; TC = Total Counts; NSB = Non-specific binding; Bo = Maximum signal bound.

Table 3b: Progesterone data

1 Cone. Replicate |

(pmol/ml) A 1 ^B Average Std. CV% j %Bound I

10000 1335 1314 1325 15 1.1 5.2

4000 2391 2293 2342 69 3.0 12.3

1000 6571 7405 6988 590 8.4 44.6

500 8494 9299 8897 569 6.4 57.8

125 13386 12664 13025 511 3.9 86.5

31.25 15394 14178 14786 860 5.8 98.7

CV = coefficient of variation (%)

Table 4a: Estradiol data

1 Replicate ge Std.

^A j B 1 Avera | cv%

NSB 13832 14926 14379 774 5.4

Bo 69889 71364 70627 1043 1.5

Blank 6543 7401 6972 607 8.7

26

Table 4b: Estradiol data

J Cone. 1 Replicate

I (pmolml) A B Average Std. CV% %Bound

8000 22238 20995 21617 879 4.1 12.9

2000 27919 27248 27584 474 1.7 23.5

500 36502 33289 34896 2272 6.5 36.5

125 45056 43990 44523 754 1.7 53.6

31.25 56458 57976 57217 1073 1.9 76.2

1.95 66187 66613 66400 30, 05 92.5

» I

The binding curve for progesterone is shown in Figure 3 and the binding curve for estradiol is shown in Figure 4.

Example 3: Primary Acridinium Trigger and Quenching of CDP-Star Generated

Luminescence

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. 20μl of ALP conjugate dilutions (Assay Designs Inc., USA Lot# 97S011) were incubated with lOOμl of CDP-Star substrate for 45 minutes and the resultant luminescence determined (Table 5 - replicates A and B). lOOμl of primary acridinium trigger was then injected into the wells (without) removal of the ALP luminescent product) and the luminescence again determined.

27

Table 5

ALP Generated Luminescence Average I RLU Post Primary %Loss RLU (RLU/Well) J Acridinium

A j B Trigger

223893 225994 224943.5 346 99.8

107048 110651 108849.5 349 99.7

55305 55701 55503 451 99.2

30181 29584 29882.5 378 98.7

RLU Relative Light Units

The results show that addition of the acridinium trigger solution reproducibly quenched the ALP generated luminescence (>99%) to background levels.

Other 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.

Claims

28CLAMS

1. A method for sequentially assaying for the presence of a plurality of analytes in a single sample, the method comprising the steps of:

(c) washing the assay test device to remove excess detection means;

wherein residual bound detection means are not washed from the test assay device between the detection and quantitation of individual analytes in the sample and in which the luminescent labels to detect the analytes are triggered independently in sequence of lability and in which the signal from the previous label has been dissipated prior to analysis of the next label in sequence. 29

2. A method as claimed in claim 1, in which the analyte is detected by immunoassay, ELISA, a DNA probe based assay or a DNA/protein shift assay.

3. A method as claimed in claim 1 or in claim 2, in which the sample is obtained from a biological source, a chemical source or an environmental source.

4. A method as claimed in claim 3, in which the biological source is obtained from a plant, animal, fungal, yeast, viral or bacterial species.

5. A method as claimed in claim 4 in which the sample is obtained from an animal.

6. A method as claimed in claim 5, in which the animal sample is a bodily fluid

7. A method as claimed in claim 6, in which the body fluid is blood, urine, sweat, saliva, tears, milk, semen, synovial fluid, cerebrospinal fluid, amniotic fluid, tissue exudate at site(s) of infection, or a tissue extract or hydrolysate.

8. A method as claimed in any one of claims 5 to 7, in which the animal is a mammal.

9. A method as claimed in claim 8 in which the analytes to be determined are immunoglobulins, enzymes, hormones, steroids, metabolic markers, diagnostic markers, drug or nucleic acid.

10. A method as claimed in claim 9, in which the immunoglobulin is from the immunoglobulin classes IgA, IgG or IgM, or subtypes thereof.

11. A method as claimed in any one of claims 1 to 10, in which the luminescent 30

label is a chemiluminescent label.

12. A method as claimed in any one of claims 1 to 10, in which the luminescent label is a bioluminescent label.

13. A method as claimed in any one claims 1 to 10 in which the first analyte is detected by a bioluminescent label and the second analyte is detected by a chemiluminescent label.

14. A method as claimed in any one claims 1 to 10 in which the first analyte is detected by a chemiluminescent label and the second analyte is detected by a bioluminescent label.

15. 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.

16. A device as claimed in claim 15 in which the capture means are bound to the surface of the device.

17. A kit for the multiple detection of analytes in a single sample comprising an assay test device as defined above and luminescent detection means.