GB2385123A

GB2385123A - Assay and dipstick type device for detecting target moieties in suspect samples

Info

Publication number: GB2385123A
Application number: GB0229038A
Authority: GB
Inventors: Michael Frederick Sanders
Original assignee: UK Secretary of State for Environment Food and Rural Affairs
Current assignee: UK Secretary of State for Environment Food and Rural Affairs
Priority date: 2001-12-13
Filing date: 2002-12-13
Publication date: 2003-08-13
Anticipated expiration: 2022-12-13
Also published as: GB2385123B; AU2002350951A1; GB0229038D0; WO2003050556A3; WO2003050556A2; GB0129776D0

Abstract

A method and associated device for detecting a target moiety, such as pathogenic organisms, in a suspect sample comprises: (a) immersing in a suspect sample a solid support with a detection zone thereon which contains a first specific binding partner, such as an antibody, which binds the target moiety; (b) removing the support from the sample; (c) immersing the support into solution / suspension of a labelled second binding partner which binds and is present at an amount to bind all of the first specific binding partner; (d) detecting for labelled material in the detection zone on the support; and (e) relating this result to the presence of target moiety in the sample. This last stage optionally utilising calibration curves. The method is particularly useful as a rapid test for pathogenic microorganism such as anthrax spores (<I>Bacillus anthracis</I>) which may be present at high concentrations.

Description

Assay Device and Method The present application relates to a method for

conducting an assay for a target moiety such as a microorganism, which is 5 present at relatively high concentrations in a sample, as well as to assay devices and kits for use in the method.

The detection of moieties such as proteins on a variety of solid supports is well known in the art. Many such tests are in the 10 form of "dipstick" assays which rely on lateral flow of liquid sample containing the moiety along a membrane, where they encounter labels, labelled binding partners and/or immobilized binding partners, in a sequence whereby a detectable visible signal is developed on the membrane. Such methods are 15 advantageous in that they provide rapid results, and may be used by unskilled operators in almost any location.

Such techniques are only applicable however where the moiety being detected can be mobilised relatively easily, so that it 20 can flow through pores in the membrane. Thus such methods are less useful where the moiety to be detected is large, such as microorganisms or microorganism spores. Furthermore, high levels of bacteria in a sample may also inhibit such tests, making dilutions of sample necessary before the test can be 25 operated successfully. Although generally bacteria will be present in most samples at relatively low concentrations, there are instances, in particular where plant pathogens and particularly food spoilage organisms are concerned, where they may present in very high concentrations. Examples of such 30 organisms include bacteria from the Erwinia, Pseudomonas and Xanthomonas strains.

The detection of microorganisms and the like may be carried out in homogenous assays such as those described in US Patent No. 35 5888725. A problem commonly encountered in such assays is that the concentration of the organism in a sample is generally very low. Thus it may be necessary to incubate the sample for a significant period of time prior to conducting the assay in

order to culture the organism to detectable levels. This delay may be unacceptable in many situations where public health is at risk, for example, where biological warfare or terrorism is suspected. Alternatively, amplification techniques such as those described in Murphy et al., "The use of adenylate kinase for the detection and identification of low numbers of micro-organisms". In Proceedings of the gth International Meeting on Bioluminescence 10 and Chemiluminescence, Woods Hole, USA, J. Wiley & Co., Chichester, 1997, may be employed. However such methods require a significant level of skill to operate and are generally conducted in laboratory conditions.

15 Many detection techniques rely on the detection of bacterial or viral DNA or RNA using amplification reactions such as the polymerase chain reaction (PCR). However, some organisms such as those that form spores (e. g. Bacillus anthracis), may be difficult to break open to liberate detectable cellular 20 components. In such cases, it may be necessary to grow out the spore to provide vegetative cells for PCR or other molecular biological methods, which will increase the time taken to obtain the results, potentially to an unacceptable level.

25 The rapid detection Bacillus anthracis is currently very important in the field of public safety and likely to remain so

for some considerable period of time. Bacillus anthracis has been investigated for its potential as a biological warfare agent and possibly used as such for some time. More recently it 30 has been used as a terrorist weapon and has been targeted towards the largely unprotected civilian population.

In the case of terrorist attacks, it has been presented as the dried spore of the organism, as vegetative cultures are not 35 viable for long periods of time. The spore form of presentation is able to cause the inhalation form of the disease, and once infection has occurred, medical intervention once symptoms have appeared is of little use. It has been estimated that there is

only a 6 hour window in which to administer effective treatment after the initial inhalation of spores.

To cause infection large numbers of spores are usually required 5 (104) and for this reason the organism has been presented as a powder derived from a dried spore crop. Such powders include a very high concentration of anthrax spores. However there have been cases of "hoax" threats where attempts have been made to pass off innocuous white powders as anthrax spores.

There is therefore a need to provide a reliable test method for potential hazards such as anthrax containing powders, which can be operated easily, if necessary outside a laboratory, and rapidly, preferably in less than one hour. The method must also 15 have a low level of false positive or negative results According to the present invention there is provided a method for detecting the presence of a target moiety in a sample, said method comprising: 20 a) immersing in a liquid sample suspected of containing said moiety, a detection zone on a solid support, said detection zone having a first specific binding partner, which specifically binds said target moiety, immobilized therein, said first specific binding partner being present in the detection zone an 25 amount which binds up to a predetermined concentration of said target moiety, b) removing said solid support from the sample, c) subsequently immersing the detection zone on the solid support in a solution or suspension of a labelled second 30 specific binding partner which binds said first specific binding partner, wherein the amount of said labelled second specific binding partner is at least sufficient to bind to all said first binding partner, d) detecting the presence of labelled material in the detection 35 zone on said solid support; and e) relating the result of (d) to the presence of target moiety in said sample.

The method of the invention is simple to operate and should give rapid results in situations where high concentrations of organisms such as pathogenic bacterial spores are present, such as might be found in a suspect package.

When the sample contacts the solid support, any of the target moiety present binds to the first specific binding partner in the detection zone, blocking at least some of the first specific binding partner from further binding. Where there is sufficient 10 target moiety, all of the binding sites in the detection zone on the support will be occupied in this way. The solid support with the target moiety bound thereto is then separated from the sample and subsequently immersed in a second liquid containing a labelled second specific binding partner, which binds the first 15 specific binding partner.

Where the sample contained none of the target moiety, all the binding sites on the solid support will still be available, and will therefore bind the labelled second specific binding 20 partner. As a result a detectable signal equivalent to all available binding sites being occupied by labelled material (i.e. a maximum signal), will be generated in step (d), indicating a 100% negative result.

25 However where there is a sufficient concentration of target moiety in the sample to bind to all the available first binding partner, there will be no site for the labelled second specific binding partner to occupy, and thus no signal will be generated, indicating that the sample contains a concentration of target 30 moiety at least equal to the predetermined concentration.

At intervening concentrations of target moiety in the sample, some of the first specific binding partner on the solid support will be available to bind to the second specific binding 35 partner, but in a reduced amount. The assay may therefore be operated in a semi-quantitative fashion by detecting the reduction in the signal and thus the reduction in the amount of labelled second specific binding partner which may be bound to

the solid support, as a result of exposure to the sample. This is best achieved by calibrating the assay device with known quantities of labelled second binding partner.

5 The method of the invention can be suitably applied to the detection of a moiety for which a specific binding partner may be identified. Thus it may be used to detect proteins, polypeptides or microorganisms including bacteria, bacterial spores and viruses (for which specific binding partners may be lo antibodies or binding fragments, or receptors or the like) as well as nucleic acid sequences such as DNA and RNA (where the first specific binding partner will comprise complementary nucleic acid sequences which hybridise to the target sequence).

15 However, the method is suitably applied to the detection of relatively large moieties, such as microorganisms, and in particular bacteria and bacterial spores.

For example, they may comprise anthrax spores such as those used 20 in terrorist attacks, where powders containing the spores have been delivered through the mail.

Alternatively the may comprise plant pathogens, which may be present in relatively high concentrations in infected plant 25 tissue, Such pathogens may be viruses, but in particular are bacteria such as Erwinia, Pseudomonas and Xanthomonas species.

For example, there are three major bacterial diseases of tomato bacterial speck caused by Pseudomonas syringae pv. tomato, 30 bacterial spot caused by Xanthomonas campestris pv. vesicatoria, and bacterial canker caused by Clavibacter michiganense subsp.

michiganense. Using the method of the invention, the precise cause of disease in plant tissue can be readily and rapidly determined, and these three related diseases distinguished.

Over seven Erwinia species and sub-species have been reported to occur in Hawaii alone (Raabe, et al., 1981. Checklist of Plant Diseases in Hawaii. Hawaii Institute of Agriculture and Human

Resources, College of Tropical Agriculture and Human Resources, University of Hawaii (Information Text Series 022). These are Erwinia carnegiana which causes Bacterial Spot in watermelon (Citrullus vulgaris), Erwinia carotovora which causes Bacterial 5 Soft Rot in for instance, garden beans (Phaseolus vulgaris); Erwinia carotovora subsp. atroseptica which causes Black Heart and Soft Rot in particular in potato (Solanum tuberosum); Erwinia carotovora subsp. carotovora which causes bacterial Soft rot in particular in shallot (Allium ascalonicum), onion (Allium 10 cepa), Allium sp., broccoli, cauliflower (Brassica oleracea var.

botrytis) and cabbage (Brassica oleracea var. capitata), Erwinia chrysanthemi which causes Bacterial Blight in florist's chrysanthemum (Chrysanthemum morifolium); Erwinia herbicola which causes Leafspot in syngonium (Syngonium podophyllum), Pink 15 Disease in pineapple (Ananas comosus) and Purple Stain; Erwinia Sp. which affects watermelon (Citrullus vulgaris) and also Fire blight of apple, although this may be caused by Erwinia amylovora. 20 It is also possible to use the method of the invention in the rapid detection of medically important bacteria. Examples of such bacteria include Streptococcus pneumoniae, Neisseria meningitidis, Salmonella sp, Staphylococcus aureus, Haemophilus influenzee type b(Hib) Escherichia cold (particularly those 25 strains containing the K1 polysaccharide), Listeria monocytogenes Enterococci, nonenterococcal group D streptococci, -hemolytic streptococci, and other gram-negative enteric organisms (eg, Klebsiella sp, Enterobacter sp. and Citrobacter diversus) Bacteremia and septic shock are closely related conditions.

Bacteremia denotes bacteria in the bloodstream. Septic shock is sepsis with hypoperfusion and hypotension refractory to fluid therapy. Sepsis refers to a serious infection, localized or 35 bacteremic, that is accompanied by systemic manifestations of inflammation. Sepsis due to bacteremia is often called septicemia; this often imprecisely used term is now being

discouraged. The more general term, systemic inflammatory response syndrome, recognises that several severe conditions (e.g., infections, pancreatitis, burns, trauma) can trigger an acute inflammatory reaction, the systemic manifestations of 5 which are associated with release into the bloodstream of a large number of endogenous mediators of inflammation. Diagnosis depends on isolating bacteria from blood and avoiding contamination with bacteria from the skin.

10 Sample may suitably be prepared by taking a sample of any suspect material such as powder, or contaminated plant or other organism, and suspending this in water or an aqueous liquid.

This liquid may contain suspending agents, surfactants or detergents if necessary to achieve good suspension of the target 15 moiety. In the case of a blood sample, this may be centrifuged or filtered if necessary to provide a usable serum sample.

Where pathogenic organisms such as anthrax spores or the like are employed, it would be possible to set the concentration of 20 first binding agent on the support to be such that any concentration above levels which are known to cause infection (i.e. more than 104 spores per sample) will give a lOOt positive result (zero signal). Thus the test will provide a rapid method for detecting risk to persons exposed to any suspect powder or 25 the like, and will allow antibiotic treatment to begin immediately. The first specific binding partner suitably comprises an antibody or a binding fragment thereof, which is specific for 30 the target moiety such as the microorganism. Antibodies may be monoclonal or polyclonal.

The solid support is suitably in the form of a single self-

supporting unit such as a slide or membrane, for instance, a 35 nitrocellulose membrane, which can be readily moved from one sample tube to another and if necessary also into a detector, either manually or using an automated device.

It is suitably prepared by immobilizing suitable first specific binding partners thereon using conventional methods, such as by spraying or covalent or other bonding, in a detection zone.

The amount of first specific binding partner applied in this way 5 is suitably measured to ensure that similar amounts are applied to similar batches of the assay device.

Once applied, any remaining binding sites on the support are suitably blocked by immersion in a blocking solution as is known 10 in the art. For example, unused sites on nitrocellulose membranes may be blocked with polyvinylalcohol as described in GB-A-2204398.

For rapid field use, such as in the case of suspected terrorism,

15 the device is suitably a dipstick type device comprising a nitrocellulose membrane, if necessary on a backing support to make it self-supporting, or within a protective housing or cover such as are well known in the art. The construction of the support must be such however that the detection zone containing 20 the immobilized first binding agent is immersible in the sample solution or suspension. This area should then be accessible for detecting signal, for example by being visible to the eye, or for inclusion in a detection device such as a fluorimeter or luminometer. Suitably, the support is subject to washing between steps (b) and (C) and/or (c) and (d). Step (c) of the method of the invention is suitably effected by immersing the detection zone on the solid support into a solution or suspension of the second 30 specific binding partner so as to allow any available first specific binding partner to bind to it.

The nature of the labelled second specific binding partner will vary depending upon the nature of the target moiety and the 35 first specific binding partner. Suitably, where the target moiety is a microorganism cell or spore, the labelled second specific binding partner will comprise a labelled cell or spore.

Where these comprise a pathogenic organism, the cell or spore is suitably attenuated or inactivated.

Alternatively, the labelled second specific binding partner will 5 comprise a labelled protein in particular an antigen which binds the first specific binding partner. In particular, such forms will comprise a microsphere coated with the antigen. The microsphere is conveniently a labelled microsphere of a size similar to that of the microorganism or spore which forms the 10 target moiety, so that it will bind in a similar fashion to that of the microorganism or spore and thus mimic the binding of the target moiety. A wide variety of microspheres, for example hydrophobic polymeric microspheres are available for example from Bangs Labs. These include florescently labelled 15 microspheres.

Suitably the label used in the labelled second specific binding partner is a visible label, such as a fluorescent label, or a chemiluminescent label as well as coloured particulate labels 20 such as coloured latex particles, or gold or silver particles.

However, other labels such as those which need to be "developed" may be employed. These will include enzymes used in conventional ELISA tests, such as horse-radish peroxidase and phosphatase, which form coloured soluble reaction products on 25 contact with the enzyme substrate.

In a particular embodiment, the label is a fluorescent or chemiluminescent label, which may be detected using a fluorimeter or luminometer, capable of detecting the intensity 30 of the signal generated. This will allow a more accurate assessment of the actual concentration of the target moiety in the sample to be made. Fluorescent labels are available from a variety of sources and include fluorescein, Sybrgreen, SybrGold TM, Cy5, rhodamine dyes, as well as pyridyloxazole dyes such as 35 Cascade yellow and dapoxyl dyes and dyes based on the 6,8-

difluoro-7-hydroxycoumarin fluorophore such as Marina blue and Pacific blue dyes available from Molecular Probes.

Preferably, the results obtained in step (d) of the method of the invention will be compared with results obtained using known concentrations of labelled second binding partner. Such results are suitably formulated into a calibration graph. In this way 5 any nonspecific binding of the labelled second binding partner to the support would be detected and this could act as the baseline for the results. Thus, detectable levels between the baseline and 100% saturation of the indicator would provide an indication of the quantity of target moiety present.

Thus the method is based on a dipstick type of assay, which is portable and easy to use. In particular, a substrate such as a dipstick or slide would be covered with stable antibody to for example Bacillus anthracis spores. As described above, this 15 substrate would be so engineered as to contain a fixed number of binding sites and as such only able to bind a finite number of spores. As part of the test system or 'kit' a preparation of biologically inactive Bacillus anthracis spores would be available. These inactive spores would be labelled with a 20 suitable marker (luminescent or fluorescent for example). If the substrate was exposed to these labelled spores a signal would be obtained which would be relatively constant at the 100% spore saturation level. If however the substrate were pre exposed to unlabelled Bacillus anthracis spores, as might be 25 found in a suspect sample, some or all of the binding sites would be occupied. If the substrate, with now reduced binding sites, was then exposed to the labelled spores a reduction in signal would be observed and a presumptive identification of Bacillus anthracis could be made.

Such kits provide a further aspect of the invention. Thus the invention further provides a kit for conducting a method as described above, said kit comprising a solid support having immobilized thereon in a detection zone, a predetermined amount 35 of a first specific binding partner which binds a target moiety, said detection zone being arranged such that it is immersible in a liquid sample, and an excess of a labelled second specific

binding partner which binds said first binding partner and which mimics said target moiety.

Other components used in the method such as suspending agents, 5 detergents or surfactants, as well as suitable containers for sample and/or suspension of second specific binding partner may also be included in the kit.

The method and kits of the invention can be used as a 10 preliminary assessor of risk, to allow early treatment to begin, where positive results are found. The test results obtained in this way may then be confirmed or otherwise using for example laboratory based tests, or PCR methods, which may take too long in some instances.

The invention will now be particularly described by way of example with reference to the accompanying diagrammatic drawings in which: 20 Figure 1 illustrates schematically, the assay of the invention wherein the sample contains a high concentration of analyte, and Figure 2 illustrates schematically, the assay of the invention wherein the sample contains a moderate concentration of analyte.

Example 1

An antibody able to bind a target bacterial cell or spore is applied to a substrate in the form of a slide or dipstick, able to be inserted into a measuring device. The measuring device is 30 suitably an instrument containing a UV or visible light source designed to deliver a wavelength able to excite a dye or other marker. The instrument will also contain a detector able to measure the light emitted from the excited dye.

35 The substrate is then calibrated with either inactivated bacterial spores or cells labelled with fluorescent dye or fluorescently labelled microspheres coated with the target antigen.

Substrates are prepared and calibrated in batches and will be designed to be consistent within each batch. Each substrate will be labelled to saturation point, washed and measured. This result will give the 100% saturation/detection value of the 5 signal.

Thereafter, blank antibody-labelled substrates are exposed to the target organism or spore in suspension. The substrate is washed and exposed to same, labelled substance described above 10 in relation to the calibration process.

After washing the substrate is placed in a detector and the signal at the excitation wavelength of the label or dye is measured. Any signal less that the 100% saturation signal level 15 or other predetermined level will indicate the presence of the target due to blocking of the binding sites.

An assay of this type is illustrated schematically in Figures 1 and 2. A solid substrate (1) has immobilized thereon in a 20 detection zone, a quantity of antibody (2) that is specific for a target analyte such as a bacterial sport. The substrate is calibrated as described above, so that the concentration of analyte, which will bind to it, is known.

25 The substrate (1) is then immersed in a sample (3) which includes a relatively high concentration of bacterial spores (4) in suspension in water. The substrate is maintained in the sample for a sufficient period of time to allow the spores to bind to the antibodies (2). In view of the high concentration 30 of spores (4) in this sample, all the available sites within the detection zone on the substrate (1) become blocked.

The substrate is removed from the sample 3, if necessary washed, and subsequently immersed in a solution of labelled inactivated 35 spores (6). These spores would also bind the antibodies (2), if there were any available for binding. However, in this instance, all the binding sites have been blocked.

When the substrate is removed from this solution, no signal from the label (6) is visible. It may be necessary to insert the substrate into a signal reading device such as a fluorimeter or luminometer as described above.

It may be concluded from this result therefore that the concentration of the spores in the sample (3) is above the level set by the calibration of the substrate. In particular, it may be above a level where infection results, and therefore, 10 prophylactic or therapeutic treatments may be administered to anyone who has been exposed to the contents of the sample.

In the assay illustrated in Figure 2, the sample contains less spores (4), which means that after incubation and removal of the 15 substrate from the sample, some antibody sites (2) on the substrate (1) are still available. As a result, immersion in the second solution results in the adherence of some labelled material (6) to the available sites. These will be detected in the signal reading device.

If there were no spores (4) present in the test sample, a stronger signal would be generated in the second solution.

Therefore this provides a rapid and reliable method for judging 25 exposure and risk to potentially hazardous samples.

Claims

Claims

1. A method for detecting the presence of a target moiety in a sample, said method comprising: 5 a) immersing in a liquid sample suspected of containing said moiety, a detection zone on a solid support, said detection zone having a first specific binding partner, which specifically binds said target moiety, immobilized therein, said first specific binding partner being present in the detection zone an 10 amount which binds up to a predetermined concentration of said target moiety, b) removing said solid support from the sample, c) subsequently immersing the detection zone on the solid support in a solution or suspension of a labelled second 15 specific binding partner which binds said first specific binding partner, wherein the amount of said labelled second specific binding partner is at least sufficient to bind to all said first binding partner, d) detecting the presence of labelled material in the detection 20 zone on said solid support; and e) relating the result of (d) to the presence of target moiety in said sample.

2. A method according to claim 1 wherein the amount of 25 labelled material detected in step (d) is related to the amount of target moiety in the sample.

3. A method according to claim 1 or claim 2 wherein the target moiety is a protein, a polypeptide, a microorganism or a nucleic 30 acid sequence.

4. A method according to claim 3 wherein the target moiety is a microorganism.

35 5. A method according to claim 4 wherein the microorganism is a bacteria or bacterial spore.

6. A method according to claim 5 wherein the microorganism is anthrax spores.

7. A method according to claim 4 wherein the microorganism is 5 a plant pathogen.

8. A method according to claim 5 wherein the microorganism a medically important bacteria.

10 9. A method according to claim 4 wherein the microorganism is a pathogenic organism, and the predetermined concentration of first binding agent on the support is equivalent to a concentration known to cause infection in a human or animal.

15 10. A method according to any one of the preceding claims wherein the first specific binding partner comprises an antibody or a binding fragment thereof, which is specific for the target moiety. 20 11. A method according to claim 10 wherein the first specific binding partner is a monoclonal antibody.

12. A method according to any one of the preceding claims wherein the solid support is in the form of a single self-

25 supporting unit.

13. A method according to claim 12 wherein the solid support is a slide or membrane.

30 14. A method according to claim 13 wherein the membrane is a nitrocellulose membrane.

15. A method according to claim 14 wherein binding sites on the membrane not occupied by the first specific binding partner are 35 blocked to binding.

16. A method according to any one of the preceding claims wherein the solid support is subject to washing between steps (b) and (c) and/or (c) and (d).

5 17. A method according to any one of the preceding claims wherein in step (c), the detection zone on the solid support is immersed in a solution or suspension of the second specific binding partner such as to allow any available first specific binding partner to bind to it.

18. A method according to claim 4 wherein the labelled second specific binding partner is a labelled cell or spore.

19. A method according to claim 18 wherein the labelled cell or 15 spore is attenuated or inactivated.

20. A method according to claim 4 wherein the labelled second specific binding partner is a labelled protein which binds the first specific binding partner, and which is attached to a 20 microsphere.

21. A method according to any one of the preceding claims wherein the label used in the labelled second specific binding partner is a visible label.

22. A method according to claim 21 wherein the label is a fluorescent label or a chemiluminescent label.

23. A method according to claim 22 wherein the label is 30 detected using a fluorimeter or luminometer, capable of detecting the intensity of the signal generated.

24. A method according to any one of the preceding claims wherein the results obtained in step (d) are compared with 35 results obtained using known concentrations of labelled second binding partner.

r 25. A method according to claim 24 wherein the results obtained using known concentrations of labelled second binding partner are formulated into a calibration graph.

5 26. A kit for conducting a method as claimed in any one of the preceding claims, said kit comprising (i) a solid support having immobilized thereon in a detection zone, a predetermined amount of a first specific binding partner which binds a target moiety, said detection zone being arranged such that it is immersible in 10 a liquid sample, and (ii) an excess of a labelled second specific binding partner which binds said first binding partner and which mimics said target moiety.

27. A kit according to claim 26, which further comprises a 15 suspending agent, detergent or surfactant.

28. A kit according to claim 26 wherein the target moiety is an anthrax spore.

20 29. A solid support having immobilized thereon in a detection zone, a predetermined amount of a first specific binding partner, which binds a target moiety, said detection zone being arranged such that it is immersible in a liquid sample.

25 30. A method for detecting a target moiety in a sample, substantially as hereinbefore described with reference to the Examples.