GB2414298A

GB2414298A - Apparatus for detecting presence of multiple antibodies.

Info

Publication number: GB2414298A
Application number: GB0423387A
Authority: GB
Inventors: Michael Strachan Walker
Original assignee: Individual
Current assignee: Individual
Priority date: 2004-05-18
Filing date: 2004-10-21
Publication date: 2005-11-23
Anticipated expiration: 2024-10-21
Also published as: GB0423387D0; GB2414298B

Abstract

The invention relates to an apparatus for use in simultaneously testing a body fluid sample for the presence of multiple antibodies, the apparatus comprising a fluid container having an inner surface with a plurality of distinct antigen-bearing locations, each of which carries an immobilised antigen and wherein the inner surface and antigen-bearing locations are arranged such that a body fluid sample placed in the fluid container contacts each of the antigen-bearing locations substantially simultaneously.

Description

24 1 4298 M&C Folio: GBP290281 Diagnostic Method and Apparatus

FIELD OF THE INVENTION

The present invention relates to a diagnostic apparatus and method for the simultaneous detection of the presence of multiple specific antibodies in a sample In particular embodiments, the invention relates to an apparatus and method for detecting antibodies to particular foodstuffs in a urine sample from a patient; this embodiment is particularly of use in assessment of food allergies and intolerances.

BACKGROUND TO THE INVENTION

There is an increasing incidence of allergic reactions to food and other comnonly-encountered environmental stimuli. Once the antigens responsible for a particular allergic reaction are known, it is possible for a sufferer to reduce or minimise their exposure to such antigens; for example, in the case of nut allergy by avoiding eating peanuts or other relevant foodstuffs containing nut-derived materials.

In order to identify particular antigens to which a subject is allergic or intolerant, it is known to assay a sample from the patient for the presence of antibodies to that antigen. Typically, the test is conducted with a blood sample from the patient, which is separated to yield plasma, diluted in an appropriate buffer to a desired concentration, and added to an immobilized food antigen within a reaction well. When an antibody to the antigen is present in the plasma, it binds to the antigen and is itself imn1obilised.

Unbound antigen is then washed away, and a labelled anti-human IgG antibody added to tl1e well; this then binds to any human IgG bound to the antigen. Detection of the label serves to confirm the presence of the appropriate antibody in the sample.

Typically a peroxidase labelled anti-IgG may be used, which may be detected by addition of hydrogen peroxide and tetramethylbenzidine as substrates for the peroxidase. Where peroxidase is present, the solution will change colour. This is a typical ELISA (enzyme-linked immunosorbent assay).

Presence of antibodies to a particular food antigen nosy be taken as indicating that the patient is allergic or intolerant to that antigen.

Conventional tests such as these suffer from a number of disadvantages. Firstly, the need to obtain a blood sample to perfonn the test can discourage individuals from taking the test, and may reduce patient compliance. In addition, the need to take blood samples, and to separate the plasma will prevent the test being suitable for non-clinical (for example, domestic) use.

Further, prior art tests typically test against a single type of antigen in each test.

This means that, if the potential allergen is unknown, many separate tests may be needed in order to determine the identity of the allergens. This is particularly problematic with food intolerance, rather than allergy, since the onset of food intolerance may be somewhat slower than a true allergy, thereby obscuring the connection between consumption of a particular foodstuffand the onset of intolerance symptoms.

Certain prior aft tests may be usable to detect multiple antibodies. For example, WO03/046563AI (Yorktest Laboratories Ltd) describes an assay based on the innobi]isation of a mixture of three allergens (wheat, milk, and egg white) on a test sticlc. A blood sample is adsorbed into the test stick and travels along the stick by capillary action, passing first through a mixture of labelled allergens, which bind to antibodies in the blood and are carried along with the flow, and then to the immobilized mixture of the three allergens. Tle antibodies will bind to the immobilized allergens, causing the labelled allergens also to be immobilised whereupon the label can be detected. However, this test relies on the use of a blood sample, and cannot detect which of the three allergens is responsible for a positive result. Further, the sample is drawn into the test stick and must pass through a number of defined zones on the stick before a result is obtained. It may be difficult for users to determine what point along the stick the sample has reached, or whether the sample has entered the stick at all.

US2002/0045195AI (Hubscher et al) describes an alternative assay which makes use of a lateral flow test strip to detect anti-IgG or anti-IgE antibodies to several potential allergens in a single sample of serum. Again, the serum sample must be drawn along the test strip to pass through each allergen sequentially, n1aking it difficult for a user to detennine wheeler the test leas worked or at what stage along the test strip the serum sample is. t

Other tests are known which make use of an immunodot strip which may carry more than one antigen. The strip must be immersed in a vessel containing a serum sample, and the user must assess whether each of the antigens produces a reaction. The immunodot strip is produced by spotting an antigen onto a nitrocellulose strip and allowing the antigen to dry.

It is among the objects of embodiments of the present invention to provide an alternative assay method or device for detection of antibodies in fluid samples. It is further among objects of certain embodiments of the invention to provide an assay method or device which are suitable for domestic, rather than clinical use.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention, there is provided an apparatus for use in testing a body fluid sample for the presence of antibodies, the apparatus comprising a fluid container having an inner surface with a plurality of distinct antigen-bearing locations each of which carries an immobilized antigen the inner surface and antigenbearing locations being arranged such that a body fluid sample placed in the fluid container contacts each of the antigen bearing locations substantially simultaneously.

The present invention allows a body fluid sample to be tested against a plurality of antigens simultaneously, thereby permitting a number of different tests to be carried out rapidly on the same sample. By 'simultaneously', it is meant that a single sample- loading action will be sufficient to allow the sample to contact the antigens. Although prior art testing in 96-well plate formats may allow multiple tests to be carried out on the same apparatus, the prior art tests require that each weld be separately loaded with a relatively small volume of sample. The present invention makes use of the surprising realization that a single sample of relatively large volume may be contacted with multiple antigens simultaneously without interfering with the assay. Clearly there may be a slight difference in time of contact of a sample with antigens as the sample is loaded and allowed to flow over the inner surface of the container; this is intended to be encompassed by the terns 'simultaneously'. The simultaneous nature of the sample / antigen contact also allows for the results of each assay to be obtained substantially simultaneously, unlike prior art devices which rely on wicking of the sample along an absorbent material. With the present invention, if the user can read the result of one assay, they will know that all results have been obtained.

The use of a large sample volume means that the apparatus is particularly suited to domestic testing, since the user does not need to dispense small volumes into multiple wells. Further, the provision of a number of distinct antigen-bearing locations allows different antigens to be detected separately, unlike the prior art tests with a number of combined antigens.

Each of the anligen-bearing locations may carry a single type of immobilized antigen, or each location may carry a defined mixture of antigens. A combination of these may also be used, such that each of a subset of the antigen-bearing locations carries a single type of antigen, while others of the locations carry a mixture of antigens.

Preferably the firmer surface of the container is substantially planar. This makes it easier to arrange for the sample to contact the antigenbearing locations simultaneously.

The locations may be defined by marks or formations on the surface; for example, the surface may be formed with a plurality of raised rings, squares, triangles or other marks or shapes thereon, each of which marks an antigen-bearing location; this allows a user to more easily identify tile location of each of the antigens, but does not significantly affect the flow of a fluid sample over the surface. Alternatively, the rings or marks may be depressions in the surface such as shallow dimples.

Preferably the container is in the form of a tray having an edge. The inner surface of the container may be defined by a lower surface of the tray, with the edge serving to retain a sample within the container.

Preferably the inner surface of the container is substantially impermeable to fluid. This ensures that the sample contacts the antigens in the liquid phase. Unlike prior art tests in which the sample is absorbed into a permeable material which carries the antigens, the relatively rapid movement of a fluid further assists the simultaneous contact of the sample with the antigens. The impermeable nature of the surface also serves to reduce capillary movement of pigment or other compounds indicative of test results (for example, from an ELISA) within the surface; this is particularly useful in view of the plurality of antigen-bearing locations on the surface.

Each of the antigen-bearing locations may be marked by indicia allowing identification of the location. For example, each location may be numbered or lettered.

Preferably the antigen-bearing locations are arranged in an array format; conveniently 48 locations are provided, in a 6 x 8 array. Of course, other arrangements may be used.

Preferably a plurality of different antigens are provided. Conveniently each antigen-bearing location carries a different antigen, although in certain embodiments of the invention duplicates may be provided. One or more of the antigens may be a control antigen; that is, one which will give a positive or negative result from the test to be used, lo ensure that the test is working correctly. The controls will of course depend on the test to be used, the body fluid used, and the antibodies to be detected.

Preferably the antigens are food antigens.

According to a further aspect of the invention, there is provided a method of testing a body fluid sample for the presence of antibodies, the method comprising the steps of: placing a body fluid sample in a fluid container having an inner surface with a plurality of distinct antigen- bearing locations each of which carries an immobilized antigen, such that the sample contacts each of the antigen-bearing locations substantially simultaneously; allowing antibodies in the sample to bind to corresponding antigens on the surface; removing unbound antibodies and the sample; and detecting the presence of bound antibodies on the surface.

The body fluid sample may be blood, serum, sputum, urine, etc. although preferably urine is used. This has the advantage that it is non-invasive to obtain, can be obtained in relatively large quantities from a subject, and requires no preparation or processing before it can be used. These properties make its use particularly advantageous for domestic testing. The present inventor has also surprisingly identified that urine contains sufficient antibodies to allow its use in testing for food intolerance or allergy. Preferably the body fluid sample is undiluted, and may be otlervise untreated, before being placed in the container. Indeed, in certain embodiments of the invention, the sample may be placed directly in the container as it is obtained from tile subject; this may be desirable in domestic testing, where users may consider it unpleasant or unhygienic to use a separate sample obtaining / transfer step.

Preferably the sample contacts the antigens at the interface between the sample and the surface; that is, the sample is not absorbed into the surface. Conveniently this is achieved by use of an impermeable surface.

Preferably each of the antigen-bearing locations carries a different antigen.

Preferably the antigens are food antigens. At least one of the locations may bear a control antigen. Preferably me antigen bearing locations are arranged in an array.

The step of removing unbound antigens and the sample may comprise washing the surface will, a wash solution. The wash solution may comprise a surfactant, for

example, Triton X.

The step of detecting bound antibodies may comprise either direct detection (where the antibodies are detected directly with a labelled detector) or indirect detection (where the antibodies are first exposed to a further antibody which is itself then detected). Preferably indirect detection is used, as this allows the signal to be amplified and thereby improves sensitivity of detection.

Detection may be achieved using labelled anti-human antibodies; conveniently anti-IgG or anti-IgE antibodies. The label may be any convenient label, for example, peroxidase. Unbound excess anti-human antibodies may then be washed away, followed by addition of a suitable substrate for the peroxidase; for example, hydrogen peroxide and tetramethylbenzidine. A suitable substrate solution may include 0.05% tetramethylbenzidhle and 0.02% hydrogen peroxide. Other detection methods and materials will be apparent to those skilled in the art.

The method may further comprise the step of comparing the fluid container after detection with a chart indicating the identity of the antigens on the surface. This allows the user to identify which antibodies have been detected in the sample.

According to a further aspect of the present invention, there is provided a kit for use in testing a body fluid sample for the presence of antibodies, the kit comprising a fluid container having an inner surface with a plurality of distinct antigen- bearing locations each of which carries an immobilized antigen, the inner surface and antigen-bearing locations being arranged such that a body fluid sample placed in the fluid container contacts each of the antigen- bearing locations substantially simultaneously; a wash solution; a detector solution; and a chromogen solution.

The kit may further comprise instructions for use. The kit may also comprise a chart indicating the identity of the antigens at each antigenbearing location.

Tle wash solution may comprise Triton hi) X. The detector solution may comprise labelled anti-human antibodies; preferably peroxidase-labelled anti-IgG or IgE antibodies.

The chromogen solution may comprise tetramethylbenzidine and hydrogen peroxide.

Conveniently the various solutions are differently-coloured; this allows easy identification by the user, and where instructions are present, they may refer to the solutions by colour.

The kit may further comprise an activator solution, for preparing the container for use. The activator solution may comprise buffered protein.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other aspects of the present invention will now be described with reference to the accompanying drawings, in which: Figure 1 shows a perspective view of an apparatus for use in testing a body fluid sample for the presence of antibodies in accordance with an embodiment of the present invention; Figure 2 shows a sectional view of the apparatus of Figure 1; Figure 3 shows an alternative apparatus; and Figure 4 shows the apparatus of Figure 2 with antigens in the antigen-bearing locations.

DETAILED DESCRIPTION OF THE INVENTION

Referring first of all to Figures 1 and 2, these show perspective and side sectional views of an apparatus for use in testing a body fluid sample, preferably urine, for the presence of antibodies to specie c antigens, preferably food antigens.

The apparatus comprises a fluid container 10 having a lower surface 12 and a number of walls 14. On tle lower surface 12 are a number of antigenbearing locations 16, each of whicl, is defined by a raised ring 18 on tle surface 12. The apparatus is formed of a moulded plastics material, Bluish is in- tperneable to fluid.

Act alternative embodiment is allows in Figure 3, where it can be seen that, in place of the raised rings 18, a number of shallow depressions 18A are present. The antige,,-beanug locations are defined by these depressions. It will be noted that neither the raised rings nor tle shallow depressions are sufficient to impede the substantially tree flow of a sample over the surface of the container.

As Carl be see', in Figure 4, the antigen-beanng locations in use contain inmobilised antigen 20. The presence of the raised rings 18 allows accurate localization of antigens during the,'anufacturi'g process, arid allows the user to clearly see the location and extent of each antigen.

In a preferred embodiment, eacl, antigen-beanug location carries a different antigen. Each antigen-beari'g location may be marked with a number or other indicia off the surface, and this may be compared Title a chart to identify which antigen is in wlicl location. A sample chart, for a 48-antigen apparatus, is given below.

1 2 -- 3 -- 4 - 1 5 6 -- Oats Cow's milk White fish Carrot Grapefruit Garlic _. - ._ _ 7 8 9 10 11 12 Wheat Soya Shell fish Orange/Lemon Melon Ginger _. . _._. .. _ 13 14 15 16 17 18 Rice Freshwater fish Kidney Bean Broccoli Pineapple Mint _.. . _ _ _ 19 20 21 22 23 24 Corn Tuna Cabbage Leek Strawberry Olive _. ... _ _ _ _ 26 27 28 29 30 Durum Beef Peas Bell peppers Nut mix Coffee wheat 31 32 33 _ 34 -- 35 36 Gluten Chicken Potato Cucumber Asparagus Tea l 37 3-8 -- 39 40 _ 41 - 42 Egg white Pork Tomato Apple Chilli Yeast 43 44-- 45 - 46 -- 47 - 48 Egg yolk Lamb Aubergine Kiwi fruit Negative Positive I Control Control To immobilise the antigens onto the surface, typically a liquid carrier containing the antigen is located on the surface at the antigen-bearing locations. The liquid is then dried on the surface, thereby immobilizing the antigen. Following drying, the immobilized antigens may be protected with a coating layer.

To perform the test, the apparatus is used as follows. Firstly, a body fluid sample is obtained and placed h1 the container; tile design of the container is such that the sample contacts each of the antigens substantially simultaneously. The container is then left for 20 minutes at room temperature, to allow antibodies in the sample to bind their corresponding antigens.

The sample is then removed from the container, for example by tipping the contents of the container into a toilet, and the container then washed three times with a wash solution including Triton tli) X. A detector solution is then added to the container; again, the design of the container is such that the detector solution can contact each of the antigen-bearing locations substantially simultaneously. The detector solution includes peroxidase labe]led anti-IgG molecules. The container is then incubated for 10 minutes at room temperature, followed by four further washes with the wash solution.

Finally, a chromogen solution containing 0.05% tetrametlylbenzidine and 0.02% hydrogen peroxide is added to the container and incubated for 1 minute at room temperature. Where antibody has bound to the antigen, the chromogen will cause a blue colour to develop. The user may then compare these with the chart to identify which antibodies are present in their sample. Control antigens will allow confirmation that the test has been performed correctly.

In variations to the test described herein, different detection mechanisms may be used, as may different labelled antibodies. For example, an immunogold-avidin conjugate may be used to detect biotin labelled antibodies. Alternatively, direct detection of bound antibodies may be performed. Other suitable labels and detection steps will be known to those of skill in the art.

It is apparent that the present invention provides a simple and robust test whicl1 may be used in a domestic, rather than clinical setting. Tile use of urine as a sample promotes greater patient compliance, and removes the need to obtain blood samples or to process the samples before testing. Further, the arrangement of the container allows a i\ single relatively large volume sample to be tested against many antigens simultaneously, thereby simplifying the testing and again making it particularly suitable for domestic use.

Claims

CLAIMS: 1. Apparatus for use in simultaneously testing a body fluid sample

for the presence of multiple antibodies, the apparatus comprising a fluid container having an inner surface with a plurality of distinct antigenbearing locations each of which carries an imnobilised antigen the inner surface and antigen-bearing locations being arranged such that a body fluid sample placed in the fluid container contacts each of the antigenbearing locations substantially simultaneously.
2. The apparatus of claim 1, wherein the inner surface of the container is substantially planar.
3. The apparatus of claim I or 2, wherein the antigen-bearing locations are def ned by marks or formations on the surface.
4. The apparatus of any preceding claim, wherein the container is in the form of a tray having an edge.
5. The apparatus of claim 4, wherein the inner surface of the container is defined by a lower surface of the tray, with the edge serving to retain a sample within the container.
6. The apparatus of any preceding claim, wherein the inner surface of the container is substantially impermeable to fluid.
7. The apparatus of any preceding claim, wherein each of the antigenbearing locations is marked by indicia allowing identification of the location.
8. The apparatus of any preceding claim, wherein a plurality of different antigens are provided.
9. The apparatus of claim 8, wherein each antigen-bearing location carries a different antigen.
10. The apparatus of claim 8 or 9, wherein one or more of tle antigens is a control antigen.
11. Tile apparatus of any preceding claim, wherein the antigens are food antigens.
12. A reflood of testing a body fluid sample for the presence of antibodies, the method comprising the steps of: placing a body fluid sample in a fluid container having an inner surface with a plurality of distinct antigen-bearing locations each of which carries an immobilized antigen, such that tle sample contacts each of the antigen-bearing locations substantially simultaneously; allowing antibodies in the sample to bind to corresponding antigens on tle surface; removing unbound antibodies and the sample; and detecting the presence of bound antibodies on the surface.
13. The method of claim 12, wherein the body fluid sample is urine.
14. The method of claim 12 or 13, wherein the sample contacts the antigens at the interface between the sample and the surface.
15. The method of any of claims 12 to 14, wherein the step of detecting bound antibodies comprises indirect detection.
16. The method of any of claims 12 to 15 further comprising the step of COnlparing the fluid container after detection with a chart indicating the identity of the antigens on the surface.
17. A kit for use in testing a body fluid sample for the presence of antibodies, the kit comprising a fluid container having an inner surface with a plurality of distinct antigen bearing locations each of which carries an immobilized antigen, the inner surface and antigen bearing locations being arranged such that a body fluid sa nple placed in the fluid container contacts each of the antigen bearing locations substantially simultaneously; a wash solution; a detector solution; and a chromogen solution.
18. The kit of claim 17, further comprising instructions for use.
19. The kit of claim 17 or 18, further comprising a chart indicating the identity of the antigens at each antigen-bearing location.
20. The kit of claims 17, 18, or 19, wherein the various solutions are differently- coloured.
21. Apparatus, method, or kit substantially as bereinbefore described with reference to the Figures.