NZ268623A - Device for the detection of a analyte present in a body fluid - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number £68623 New Zealand No. 268623 International No.

TO BE ENTERED AFTER ACCEPTANCE AND PUBLICATION Priority dates: 14.07.1993; Complete Specification Filed: 14.07.1994 Classification:^) G01N33/543.96 Publication date: 24 October 1997 Journal No.: 1421 NEWZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of Invention.

Test device NO DRAWINGS Name, address and nationality of applicant(s) as in international application form: CORTECS LIMITED, The Old Blue School, Lower Square, Isleworth, Middlesex TW7 6RL, England New Zealand No. International No. 268623 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of Invention: Test device Name, address and nationality of applicant(s) as in international application form: CORTECS LTD, of The Old Blue School, Lower Square, Isleworth, Middlesex TW7 6RL, England WO 95/02822 PCT/GB94/01522 268623 TEST DEVICE The present invention relates to a device for detecting the presence of analyte in a sample of a body fluid, in 5 particular a sample other than a serum sample. Such analytes are typically immunoglobulin molecules but may also be other proteins.

Immunoglobulin molecules, or fragments thereof, are known 10 to occur in such non-serum body fluids as saliva and urine. In particular, the immunoglobulin molecules of classes IgA, IgG and IgM predominate in saliva. Saliva and urine are secreted body fluids and collection of samples is easily and simply carried out.

In the field of medical diagnostics, there are many tests and devices known for detecting analytes in samples of a particular body fluid. These tests include biochemical assays of the amount and/or presence of particular 20 proteins or immunoglobulins; so called 'sandwich assays' for immunoglobulins and the use of electrophoresis to identify individual classes of immunoglobulins.

Devices used to detect analytes such as immunoglobulins 25 in saliva include various probes consisting of a stick with an absorbent or adsorbent pad of material at one end. Such probes are disclosed in EP-A-0418739, EP-A-0442231 and US-A-5103838. The probes of the prior art additionally include detection systems capable of 3 0 identifying antibodies in saliva.

Detection systems used in the past on diagnostic probes of this type have employeu enzyme-linked assays or particle-agglutination assays to produce visibly WO 95/02822 PCT/GB94/01522 detectable results.

However, diagnostic test devices have suffered from an important: disadvantage. All immunodiagnostic tests 5 relying on antibody-antigen binding are subject to the occurrence of false negative results in any one particular sample. In the classic enzyme-linked immunosorbent assay, an antigen which will specifically bind with the anf.ibody of interest is bound to a solid 10 base. A sample cor'-.aining the antibody of interest is added and forms a specific complex with the bound antigen. Unbound antibody is then, washed off and a labelled antibody is added to detect the complexes formed on the surface of the test device. This assay, however, 15 is only reliable when quite large amounts of analyte are present in the sample. Samples of urine and saliva which a clinician might wish to assay generally contain levels of antibody which may be 2 or 3 orders of magnitude lower than the levels of antibody found in serum or plasma 20 samples. Antibody concentration in urine and saliva samples is influenced by several factors, and shows natural variability. The antibody concentration may be lowered when urinary output is increased through ingestion of large volumes of fluid prior to sampling, or 2 5 when saliva production has been artificially increased through stimulation by citric, or other acids.

This variability can give rise to false-negative results, where the concentration of antibody in the sample is 3 0 insufficient to give a positive reaction in an enzyme- linked assay or similar test procedure. False negative results can result in the incorrect diagnosis of a medical condition by a clinician. Where the test is for the presence of an antibody to a certain pathogenic 2686 bacteria or virus or for antibodies associated with autoimmune diseases or allergic conditions, a false-negative result due to the use of an inadequate sample can have devastating consequences for the patient and cause the subject unnecessary distress. This is especially so in the case of Human Immunodeficiency Virus (HIV) infection. Recently, it has been reported that the United States Food and Drug Administration (FDA) has decided that all test devices in the immunodiagnostic field must indicate the viability of the sample.

The present invention seeks to solve this problem by providing a device with an inbuilt means for detecting false negative results arising from use of an invalid sample.

In a first aspect of the present invention, there is provided a device for the detection of an analyte present in a body fluid of an animal species, the device comprising a substrate and on the substrate in discrete locations: (a) a mobile labelled first specific binding molecule which binds to any characteristic molecule of the species being tested to form a binary complex; wherein the characteristic molecule is a molecule which is typically found in the type of sample being tested, which will be present in detectable concentrations if the sample is viable and which is chosen such that the analyte forms a portion of or is a subset of the characteristic molecule; (b) a means for detecting the analyte; and Pr. (c) an immobilised second specific binding molecule which binds to the binary complex.

The advantage of this device is contained in the final step of binding the characteristic molecule to a second 4 SEP 1997 4 immobilised specific binding molecule. This step ensures that a sufficient quantity of characteristic molecule is present to give meaningful results and therefore that particular test can be readily verified. The overall 5 concentration of characteristic molecule in a test sample is easily quantitated and if the level is sufficiently high then the risk of a false negative result is significantly reduced.

GB-A-2204398 relates to an assay device which also contains a mobile labelled first specific binding molecule, a means for detecting analyte and an immobilised second specific binding molecule. However, it differs significantly from the device of the present 15 invention because the first specific binding molecule is specific for the analyte rather than for a characteristic molecule of the species being tested as is the case with the present invention. This means that using the device of GB-A-2204398 it is not possible to determine whether 20 the sample is viable and, thus, to eliminate false negatives.

In this specification, the term 'binary complex' is used to define the complex formed by the specific binding 25 molecule bound to a particle on the substrate and a characteristic molecule which may include analyte molecules. The analyte may be an antibody.

The term 'mobile', when used in connection with the first 3 0 specific binding molecule, indicates that the first specific binding molecule is not immobilised on the substrate.

The term 'characteristic molecule' refers to a molecule which is typically found in the type of sample to be tested and which will therefore be present in detectable concentration if the sample is viable. For example, in saliva samples, it is usual to find detectable quantities 5 of IgG, and IgG would therefore be a suitable characteristic molecule for saliva samples. The first specific binding molecule would, in that case, bind specifically to all antibodies of the IgG isotype.

The device of the present invention is suitable for the detection of an analyte in any body fluid but it is especially useful for overcoming the problems of detecting analytes in saliva and urine samples where the concentrations of analytes tend to vary. It is also 15 useful for other body fluids such as blood.

The analyte to be tested by the device of the present invention may be an antibody to a bacterial, viral, fungal or protozoal pathogen for example Helicobacter 20 pylori, the Human Immunodeficiency viruses (HIV 1 and 2) , a Salmonella spp., a Streptococcus species, polio virus, hepatitis A, hepatitis B surface antigen or hepatitis B core antigen, Candida spp., Aspergillus spp., Entamoeba spp. or Giardia spp. The analyte may also be an antibody 25 to a tissue or species-specific antibody associated with autoimmune diseases, or allergic conditions.

Alternatively, the analyte may be an antigen, for example a pathogen protein or a hormone, blood group 3 0 characterising protein or any other protein or glycoprotein which may require detection. Nucleic acids can also be detected using the device of the present invention. 6 The analyte should also form a portion of the characteristic molecule, or be a subset of characteristic molecules, which bind to the first specific binding molecule to form binary complex.

Thus, when the analyte is an antibody of a particular isotype, for example IgG, IgM, IgE or IgA, the characteristic molecule will be any antibody of that isotype and the first specific binding molecule will be 10 selected so that it binds to all antibodies of that isotype. Similarly, if the analyte is a glycoprotein, the first specific binding molecule may be a lectin which binds to all glycoproteins present in the sample and labels them and if the analyte is a nucleic acid, a 15 ' suitable first specific binding molecule would be a labelled low specificity nucleic acid probe.

Because the analyte is a subset of the characteristic molecule, a portion of the labelled binary complex will 20 contain the analyte and this is a particularly advantageous feature of the invention as it eliminates the need for more than one detectable label.

In a preferred embodiment the device provided by this 25 invention is designed for use in diagnosing medical conditions suffered by humans.

As mentioned above, the first specific binding molecule will often be an antibody which will preferably be of the 3 0 IgG cr IgM isotype. Other isotypes of antibody may be similarly so used if required. The first specific binding molecule may be of polyclonal or monoclonal origin, and may be either an intact molecule or a specific binding-site containing fragment derived by 7 enzymatic or chemical cleavage, or obtained through the application of recombinant DNA technology or peptide synthesis.

The first specific binding molecule may be labelled with any convenient label. The label may, in particular, allow the specific binding molecule to be visualised, or otherwise detected, when immobilised. Particles constitute preferred labels for this purpose. Particles 10 coupled to the first specific binding molecule on the test device may be of any known type, for example, latex, colloidal metal, liposomes or polystyrene. Chromophores, fluorophores, and/or other labels may alternatively or additionally be used.

The means for detecting the analyte on the test device may suitably comprise one or more discrete areas comprising an immobilised binding molecule specific for the analyte. Because the analyte forms a part of or is 20 a subset of the characteristic binding molecule, it will already be bound to the detectably labelled first specific binding molecule and will therefore be detectable once it has been immobilised in the detection zone.

It is particularly useful to provide several analyte detection areas on the device since this allows quantitation of the analyte. Thus, if the analyte is present only in small amounts, it will interact with the 3 0 immobilised binding molecule in the first detection area, but there will be no surplus analyte which can pass to the further detection areas. If more analyte is present, some surplus may pass to the next detection area and so on so that the amount of analyte present will be 8 indicated by the number of detection areas in which it is detected.

Alternatively, two or more immobilised binding molecules 5 specific for different analytes may be deposited on discrete areas of the test device, permitting the simultaneous detection of the presence of two or more different analytes in the same sample.

When the analyte is one of the antibodies discussed above, the immobilised binding molecule in the detection area will generally be an immobilised pathogen protein, carbohydrate or other fragment, or a human tissue-derived protein, carbohydrate or other fragment, or a non-human 15 species-derived protein, carbohydrate or other fragment.

The immobilised binding molecule may also be an anti-idiotypic antibody.

When the analyte is a protein, the immobilised binding 2 0 molecule may be a monoclonal or polyclonal antibody which binds specifically to the protein or some other binding molecule specific for the particular protien to be detected. For nucleic acids, a suitable immobilised binding molecule would be a high specificity nucleic acid 25 probe.

Binary complexes which contain a characteristic molecule other than the analyte will not be immobilised or otherwise removed at location (b) , where the analyte is 3 0 detected and will pass to location (c), which functions as a verification station. A second specific binding molecule immobilises the complexes, which are then detectable by means of the label. The second specific binding molecule may specifically bind any part of the 9 complex. It is preferred for the second specific binding molecule to bind the characteristic molecule; so, for example, if the characteristic molecule is a human antibody isotype, the second specific binding molecule may again be anti-human antibody such as anti-human IgG. Other suitable binding molecules are as discussed above in relation to the first specific binding molecule and will again be chosen depending on the analyte and the characteristic molecule.

The device may further comprise a sample receiving portion of adsorbent material continuous with or separate from the substrate. This flexibility of construction permits maximum utility of operation depending on how the 15 sample is to be applied.

The substrate material of the test device may be composed of paper or membrane. The substrate material may also optionally be a carbohydrate and may preferably be a 20 nitrocellulose material.

It is preferred that the device be constructed so that body fluid (or components of it) can flow to locations (a) , (b) or (c) successively, for example by capillary 25 action. Other means of delivering body fluid to the locations are within the scope of the invention.

In a preferred aspect of the invention, as mentioned above, sample is wicked along the membrane or pa.per 3 0 substrate by capillary action. No additional solvent is required although particular sample diluents may increase the speed at which a result is obtained, or enhance the signal obtained with a positive test result.

WO 95/02822 PCT/GB94/01522 In a second aspect of the present invention there is provided a method for the detection of one or more analytes present in the body fluid of an animal, the method comprising: (i) adsorbing a sample onto an adsorbent substrate; (ii) bringing the sample into contact with a labelled specific binding molecule such that one or more characteristic molecules of the tested species present in the sample bind to the specific binding molecule to form a binary complex (wherein characteristic molecule is as defined in claim 1); • (iii) bringing the binary complex comprising the analyte in contact with an immobilised analyte-specific binding molecule such that the binary complex comprising analyte binds to the analyte-specific binding molecule to give a detectable reaction product; and bringing the binary complex not comprising the analyte into contact with an immobilised specific binding molecule which reacts with the binary complex to form a visibly detectable product.

The detectable products in steps (iii) and (iv) are preferably visibly detectable, as they are when the label 30 referred to in step (ii) is a particle.

Preferred features of the second aspect of the invention are as for the first aspect, mutatis mutandis. (iv) N.Z. PATENT OFFICE - 4 SEP 1997 11 The invention will now be further described by way of example with reference to the following drawing in which: FIGURE 1 shows a perspective view of a device in 5 accordance with the invention.

Referring now to Figure 1, a sampling device 10 comprises a substrate 12 formed from an adsorbent material, typically nitrocellulose paper, and, attached to the 10 substrate, a sample receiving portion 14. On the substrate is positioned a first area 16 in which is contained an anti-human IgG antibody coupled to a particle. The particles may be of any known type, for example latex, colloidal metal, liposomes or polystyrene. 15 Sample detection areas 18, 20 contain antibodies specific for the analyte immobilised on the nitrocellulose substrate 12. A particularly preferred analyte which this device may be used to detect is antibody to H. pylori and, therefore, in that case, the detection areas 20 18, 20 would contain an immobilised antigen specific for H. pylori antibody.

The final area on the device is the verification area 22 which contains immobilised anti-human IgG. This may be 25 the same as the anti-human IgG which is bound to the particles but this is not necessarily so.

In use, a sample is placed on the sample receiving portion 14 and, because of the adsorbent nature of the 3 0 substrate 12, the sample wicks along the substrate until it reaches the first area 16. At this point, any human IgG present in the sample binds to the anti-IgG coupled to the particles to form a binary complex. The binary complex continues to wick along the substrate 12 until it

Claims (20)

WO 95/02822 PCT/GB94/01522 12 encounters the first sample detection area 18. In this area, binary complex containing analyte binds to the immobilised antibody to form a discrete visible clustering on the sample detection area 18. Unbound 5 binary complex continues to pass along the substrate until it reaches the second sample detection area 20 where any remaining analyte-containing binary complex will be immobilised to form a second discrete visible clustering. Finally, remaining binary complex passes 10 along the substrate until it reaches the verification area 22, at which point it will bind to the anti-IgG in this area and will form a further discrete visible clustering. This visible signal in the verification area is an indication that the initial sample contained a 15 sufficient amount of human IgG for the sample to be viable and any positive result to be relied upon. Clearly, therefore, the device of the present invention is simple to use and provides a quick and easy method of 20 verifying that the sample is a viable one. 268623 13 CLAIMS

1. A device for the detection of an analyte present in a body fluid of an animal species, the device comprising a substrate and on the substrate in discrete locations: (a) a mobile labelled first specific binding molecule which binds to any characteristic molecule of the species being tested to form a binary complex; 10 wherein the characteristic molecule is a molecule which is typically found in the type of sample being tested, which will be present in detectable concentrations if the sample is viable and which is chosen such that the analyte forms a portion of or 15 is a subset of the characteristic molecule; (b) a means for detecting the analyte; and (c) an immobilised second specific binding molecule 20 which binds to the binary complex.

2. A device as claimed in claim 1, wherein the body fluid is saliva, urine or blood. 25

3. A device as claimed in claim 1 or claim 2, wherein the analyte is an antibody to a bacterial, viral, fungal or protozoal pathogen, an antibody to a tissue or species-specific antibody associated with autoimmune diseases, or allergic conditions, an antigen, or a 30 nucleic acid. 35

4. A device as claimed in claim 3, wherein the N.Z. RUENT OFFICE - 4 SEP 1997 268623 14 pathogen is Helicobacter pylori, the Human Immunodeficiency viruses (HIV 1 and 2) , a Salmonella spp., a Streptococcus species, polio virus, hepatitis A, hepatitis B surface antigen or hepatitis B core antigen, 5 Candida spp., Aspergillus spp., Entamoeba spp. or Giardia spp,

5. A device as claimed in claim 3, wherein the antigen is a pathogen protein or a hormone, blood group 10 characterising protein or any other protein or glycoprotein which may require detection.

6. A device as claimed in any one of claims 1 to 5, wherein the characteristic molecule is an antibody, a 15 protein or a nucleic acid.

7. A device a& claimed in claim 6, wherein the antibody is of the IgG, IgM, IgE or IgA isotype.

8. A device as claimed in any one of the preceding 20 claims wherein the first and/or the second specific binding molecule is an antibody, a lectin or a nucleic acid probe.

9. A device as claimed in any one of the preceding 25 claims wherein the species is human.

10. A device as claimed in any one of the preceding claims, wherein the first specific binding molecule is labelled with a particulate label. 30

11. A device as claimed in claim 10, wherein the particulate label is latex, colloidal metal, liposomes or polystyrene particles. 35 12. A device as claimed in any one of the preceding claims, wherein

N.Z. PAT ENT OFFICE - 4 SEP 1997 . ■ -,/<•-n 26862 15 the means for detecting an analyte comprises one or more discrete areas comprising an immobilised binding molecule specific for the analyte. 5

13. A device as claimed in claim 12, wherein the immobilised binding molecule in the detection area is an immobilised pathogen protein, carbohydrate or other fragment, or a human tissue-derived protein, carbohydrate or other fragment, or a non-human species-10 derived protein, carbohydrate or other fragment, a monoclonal or polyclonal antibody or a high specificity nucleic acid probe.

14. A device as claimed in any one of the preceding claims further comprising a sample receiving portion of is adsorbent material continuous with or separate from the substrate.

15. A device as claimed in any one of the preceding claims wherein the substrate is of paper or membrane 20 material.

16. A device as claimed in claim 15, wherein the paper or membrane substrate is a carbohydrate material.

17. A device as claimed in claim 15, wherein the 25 substrate is nitrocellulose.

18. A method for the detection of one or more analytes present in the body fluid of an animal, the method comprising: 30 (i) adsorbing a sample onto an adsorbent substrate; (ii) bringing the sample into contact with a 35 labelled specific binding molecule such that r_... ——j i i

- 'I': L "lb '37 208623 16 one or more characteristic molecules of the tested species present in the sample bind to the specific binding molecule to form a binary complex (wherein characteristic 5 molecule is as defined in claim 1); (iii) bringing the binary complex comprising the analyte in contact with an immobilised analyte-specific binding molecule such that 10 the binary complex comprising analyte binds to the analyte-specific binding molecuje to give a detectable reaction product; and (iv) bringing the binary complex not comprising 15 the analyte into contact with an immobilised specific binding molecule which reacts with the binary complex r.o form a visibly detectable product. 20 19. A method as claimed in claim 18, wherein in step (iv) the immobilised specific binding molecule reacts with the characteristic molecule of the binary complex (wherein characteristic molecule is as defined in claim 1) .

20. A device as defined in claim 1 for the detection of an analyte present in a body fluid of an animal species substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawing. 21, A method as defined in claim 18 for the detection of one or more analytes present in the body fluid of an animal substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawing. by the authorised agents i ' ;rrT .. - — 1/-» rr :f'-i I L'rnu^ - A SEP 1997 AJ ft ENO OF CLAIMS