The present invention relates in general to the field of diagnostics, namely to device for the detection of at least one target in a sample, preferably in urine. More precisely, the present invention relates to a high sensitive rapid immunochromatographic test device preferably providing a unique system for the detection of more than one target in a single assay at the same time. The present invention further refers to a method for the production of the test device, to the uses of the test device for the detection of more than one disease infection such as Human Immunodeficiency virus (HIV) and Tuberculosis Lipoarabinomannan (LAM) at the same time in urine, as well as to a kit comprising the test device.
In recent years the in vitro diagnostics (IVD) industry has made enormous efforts to develop immunochromatographic tests. Such tests have found applications in both clinical and non-clinical fields1. A clinical utility of this test format has been shown for more than 150 different analytes, and many of them are target now of commercially available diagnostic products3. The wide range of applications for such devices has been reviewed1, 2.
Rapid immunochromatographic test devices, e.g. in the form of a test strip, are made up of a number of components (
Rapid immunochromatographic test devices for diagnostic purposes are easy to operate and thus do not only contribute to the comfort of professional users, e.g. medical stuff, but also allow the operation by non-professionals users, e.g. most patients.
Using rapid immunochromatographic tests, a physician can instantly not only examine a sample by himself, but also diagnose a patient on the basis of the obtained results. The main disadvantage of the one rapid immunochromatographic test is that it only detects one parameter at a time. Thus, many different rapid immunochromatographic test devices are needed to check the suspected parameters, resulting in the requirement of enormous sample volume obtained from a patient as well as cumbersome test procedures.
In addition, the use of urine samples may be attractive due to the ease of sample collection, possible cost savings, better safety (against needle-stick injuries) and higher compliance rates. Assays for this type of specimens might be a useful alternative when it is for instance difficult or impossible to perform HIV-tests in blood samples. Further, it might be possible that blood cannot be drawn for religious reasons or that difficulties may be experienced in collecting blood samples. Therefore, urinary samples are the more safety type of specimens in most of infection cases.
Thus, there is a need to develop a multi-parameter rapid immunochromatographic detection test device suitable to detect more than two types of antigens and/or antibodies in a single assay at the same time. Further there is a need in the prior art to develop a multi-parameter rapid immunochromatographic detection test device sensitive enough to detect more than two types of antigens and/or antibodies in a sample such as urine.
Thus, it is an object of the present invention to provide a rapid immunochromatographic detection test overcoming the drawbacks of the prior art, being preferably suitable for ultra sensitive detection of at least one antibody and at least one antigen, preferably in urine at the same time.
In one embodiment the present invention concerns a rapid immunochromatographic test device for the detection of at least one target in a sample, comprising
In another embodiment the present invention relates to a rapid immunochromatographic test device for the detection of at least one target in a sample, comprising
In a further embodiment the present invention concerns a method for the production of a device according to the present invention comprising the steps of
In another embodiment the present invention relates to the use of a device according to the present invention for the detection of at least two diseases in at least one sample.
In a further embodiment the present invention refers to a kit for detection of a disease comprising the device according to the present invention and a manual
Before the present invention is described in more detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step.
Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
As outlined above there is a need in the prior art to provide a new test device suitable for the detection of at least one antibody and one, preferably in urine at the same time.
In a first aspect the present invention provides a rapid immunochromatographic test device for the detection of at least one target in a sample, comprising
The first and second colloidal (gold I and II) will capture the antigen/antibody in the sample and form the complexes “target-gold conjugate I” and “target-gold conjugate II”. Preferably the targets in the sample, preferably urine, are an antigen and/or antibody.
In one preferred embodiment of the device according to the present invention the first antibody of gold I will capture the target antigen from a first site (let us consider this site as site A), the first antigen of gold II will be captured by the target antibody, the third antibody of gold III will capture the target antigen from a second site (let us consider this site as site B), and the second antigen of gold IV will be captured by the same target antibody that capture the first antigen as mentioned above.
In another preferred embodiment of the device according to the present invention each gold conjugate comprises between 1 and 6 different oligonucleotides. Preferably each gold conjugate comprises between 2 and 4 different oligonucleotides. These oligonucleotides usually have a length of about 15 to 25 nucleotides, preferably of about 20 nucleotides. Further these oligonucleotides have an amino group at the 5′ terminus, preferably conjugated with bovine serum albumin. The bonds formed between the gold and oligonucleotides are the same bonds as those usually formed between gold and antibodies/antigens, and which are known to a person skilled in the art. Preferably such bonds are driven by hydrophobic, hydrophilic and dative binding forces.
In one preferred embodiment of the device according to the present invention the first gold conjugate releasing pad contains the gold conjugates I and II. Gold conjugate I is specific for an antigen, preferably the antigen is a target in a sample. Therefore gold I is conjugated with “clone one”. Please note, that the numbering of clones are only for explanation and to recognize that always two different clones of monoclonal antibodies were used. Preferably, the two monoclonal antibodies capture the target antigen from two different sites, so they were called as a pair of monoclonal antibodies. In addition, “clone one” is specific for one site (A) of the antigen. At the same time gold 1 is conjugated with some oligonucleotides (O-1). Gold conjugate II is specific for an antibody, preferably the antibody is a target in a sample, and is conjugated with an antigen or a specific antibody for the antigen-specific antibody and is at the same time conjugated with some oligonucleotides (O-2) which are different from the oligonucleotides (O-1).
In a further preferred embodiment the second conjugate releasing pad contains the two gold conjugates III and IV. Gold conjugate III is specific for an antigen, preferably the antigen is a target in a sample, and therefore is conjugated with “clone two”, whereas “clone two” is specific for the second site (B) of the antigen. At the same time gold III is conjugated with the complementary oligonucleotides (O-3) of that in the related conjugate I in the first conjugate pad. Gold conjugate IV is specific for an antibody, preferably the antibody is a target in a sample, and conjugated with the antigen or specific antibody for the antigen-specific antibody and at the same time conjugated with the complementary oligonucleotides (O-3) of that in the related conjugate II in the first conjugate pad (
In one preferred embodiment of the device according to the present invention the rapid immunochromatographic test device for the detection of at least one target in a sample, comprises
In one preferred embodiment of the device according to the present invention the first antibody 205 of gold I will capture the target antigen from a first site 205′, the a first antigen 206 of gold II will be captured by the target antibody 206′, the third antibody 208 of gold III will capture the target antigen from a second site 208′, and the second antigen 207 of gold IV will be captured by the same target antibody 207′ that capture the first antigen as mentioned above, (
In a second aspect the present invention provides a rapid immunochromatographic test device for the detection of at least one target in a sample, comprising
Preferably, the antibodies or antigens (A-1 to A-4) differs from said first antibody or antigen. The first antibody or antigen captures the target from the sample, whereas the other antibodies (A-1 to A-4) only functions to amplify the signal by recognizing the specifically-related antibodies or antigens (A-1 to A-4). In addition, the antibodies or antigens (A-1 and A-2) conjugated with gold conjugates I and II are specifically-related to the antibodies or antigens (A-3 and A-4) of the gold conjugates III and IV, respectively.
In one preferred embodiment of the device according to the present invention the device is suitable to detect the targets in urine.
In one embodiment of the device according to the present invention the device comprises a test strip comprising
In a preferred embodiment of the device according to the present invention one capture test zone suitable to detect an antigen in a sample comprises a third antibody specific for one site of the antigen, and the other capture test zone suitable to detect an antibody in a sample comprises a second antigen or a fourth antibody.
In another preferred embodiment the antibodies or antigens within the test zones are immobilized.
The complexes “target-gold conjugate I” and “target-gold conjugate II will be captured by the specific antibodies or antigens within the test zones and therefore be kept within these test zones to form the sandwich detection (
In one embodiment of the device according to the present invention the membrane is attached by means of an adhesive to a supporting backing. Preferably an acrylic pressure sensitive adhesive as known in the art is used.
In another embodiment of the device according to the present invention the first and second gold conjugate pad are laminated between the sample pad and the membrane, wherein the two gold conjugates are separated by a divider.
In a preferred embodiment of the device according to the present invention the first 103.1 and second gold conjugate pad 103.2 are laminated between the sample pad 102 and the membrane 104, wherein the two gold conjugates are separated by a divider 110 (
In another embodiment, the device according to the present invention the first gold conjugate pad is attached between the sample pad and the membrane while the second gold conjugate pad is within the upper part of the plastic housing to be released after sample application onto the nitrocellulose membrane directly.
In one preferred embodiment of the device according to the present invention the supporting backing is a plastic backing.
In another preferred embodiment of the device according to the present invention the membrane is nitrocellulose membrane.
In one embodiment of the device according to the present invention the first or second antibody is selected from the group comprising mouse anti-HIV p24, mouse anti-HBsAg, anti-hlgG, anti-Lipoarabinomannan, anti-H. Pylori antigen, anti-Leishmania antigen, anti-Pneumonia antigen, anti-Malaria antigen, anti-Chlamydia antigen, anti-Toxoplasma antigen, anti-Schistosoma antigen, HIV 1 antibody, and HIV 2 antibody.
In a preferred embodiment of the device according to the present the first or second antibody is a monoclonal or polyclonal antibody, preferably a monoclonal antibody.
In another embodiment of the device according to the present invention the first antigen is selected from the group comprising conjugate of HIV antigen, conjugate of hepatitis C antigen, HIV 1 antigen, HIV 2 antigen, Lipoarabinomannan, H. Pylori antigen, Toxoplasma antigen.
In a further embodiment of the device according to the present invention the control zone comprises a non-specific capturing antibody and/or a non-specific antibody capturing protein.
In one preferred embodiment of the device according to the present invention the non-specific antibody is selected from the group consisting of anti-mouse IgG, anti-rabbit IgG, anti-goat IgG, anti-donkey IgG, Anti-sheep IgG, anti-HIV p24, anti-Lipoarabinomannan, anti-H. Pylori antigen, anti-Leishmania antigen, anti-Pneumonia antigen, anti-Malaria antigen, anti-Chlamydia antigen, anti-Toxoplasma antigen, anti-Schistosoma antigen, HIV 1 antibody, and HIV 2 antibody.
In another preferred embodiment of the device according to the present invention the non-specific capturing protein is either Protein A or Protein G.
In a preferred embodiment of the device according to the present invention the device comprises a housing comprising at least one test strip according to the present invention.
In another preferred embodiment of the device according to the present invention the housing comprises two, three, four, five, six, seven, eight, nine, or ten test strips. Preferably the housing comprises two, three, four, or five test strips, more preferably the housing comprises two or three test strips.
In one preferred embodiment of the device according to the present invention each test strip comprises at least four antibodies or antigens, wherein two of these antibodies or antigens are immobilized onto the membrane and at the same time are conjugated with the two colloidal gold conjugates and wherein the other two antibodies or antigens are conjugated with the other two gold conjugates.
In another aspect the present invention concerns a method for the production of a device according to the present invention, comprising the steps of
In a preferred embodiment of the method according to the present invention the method further comprises the steps of
wherein the solutions are added before step h) according to the method of the present invention.
In another preferred embodiment of the method according to the present invention the method further comprises the steps of
In another aspect the present invention relates to the use of a device according to the present invention for the detection of at least one disease in at least one sample.
In one preferred embodiment of the use of the device according to the present invention preferably two diseases are detected at the same time in the same sample.
In one preferred embodiment of the use according to the present invention the antibody in one sample (e.g. specimen) and the antigen in another sample (e.g. specimen) is detected. For example, in the case two test according to the present invention are used, Lipoarabinomannan-antigen can be detected in urine, while anti-lipoarabinomannan is detected in serum (
In another preferred embodiment of the use according to the present invention the antibody and antigen are detected in the same sample (specimen). For example, HIV antibodies and the HIV p24 antigen are detected in the same serum sample (specimen) using a device of two different strips (
In another embodiment of the use of the device according to the present invention the sample is obtained from a human.
In one preferred embodiment of the use of the device according to the present invention the sample is selected from the group comprising of whole blood, serum, plasma, saliva, and urine. Preferably the sample is urine.
In another preferred embodiment of the use of the device according to the present invention the disease detected in said sample is selected from the group consisting of HIV, Hepatitis A, Hepatitis B, Hepatitis C, H. Pylori, Leishmania, Schistosomiasis, Malaria, Pneumonia, Toxoplasmosis, Tubercolosis and Chlamydial infection.
In a further aspect the present invention refers to a kit for detection of a disease comprising the device according to the present invention and a manual.
In one preferred embodiment of the kit according to the present invention the kit further comprises an assay buffer. The assay buffer can be any buffer known in the art suitable for the use of whole blood samples. Preferably in the case of whole blood samples Tris buffer is used, more preferably 0.1M Tris buffer having a pH of 7.5 and comprising a preservative. Any preservative known by a person skilled in the art can be used, preferably sodium azide and even more preferably 0.01M sodium azide is used.
The following example illustrate the present invention without, however, limiting the same thereto.
a: shows top and side views of a typical rapid-flow immunochromatographic test device known in the art in the form of a test strip 101 comprising a sample pad 102, a conjugate pad 103, a membrane 104, an absorbent pad 105, an adhesive 106, a supporting backing 107, a test zone 108, and a control zone 109.
b: shows top and side views of a preferred embodiment of a rapid-flow immunochromatographic test device according to the present invention in the form of a test strip 101 comprising a sample pad 102, a first conjugate pad 103.1, a second conjugate pad 103.2, a membrane 104, an absorbent pad 105, an adhesive 106, a supporting backing 107, two test zones 108a and 108b, a control zone 109, and the conjugates divider 110.
a: shows the schematically view of a preferred embodiment of the colloidal gold conjugates I to IV according to the present invention, wherein gold I is conjugated with an specific antibody 205 suitable to detect the target from one side (A) and two different oligonucleotides 209 and 210, gold II is conjugated with a specific antigen 206 and two different oligonucleotides 211 and 212, gold III is conjugated with another specific antibody 208 suitable to detect the antigen from the other side (B) and two different oligonucleotides 209′ and 210′, and gold IV is conjugated with another antigen 207 and two different oligonucleotides. The oligonucleotides of gold I and II are complementary to the oligonucleotides of gold III and IV, respectively.
b: shows the schematically view of the sample 213 that contains the two targets; two distinguish sites of the antibody 206′ and 207′ and that of the antigen's 205′ and 208′.
a: shows an internal view of a preferred embodiment of a test device according to the present invention suitable to detect two different targets within the same sample (specimen) from the same person (patient). In this preferred embodiment the device comprises two test strips 101.a and 101.b, wherein both test strips share the same sample pad 102, the same humidity indicator 110 and the same absorbent pad 105. Further each test strip 101.a and 101.b comprises its own gold conjugate 103.a and 103.b, sample line 108.a and 103.b and control line 109.a and 109.b.
b: shows an internal view of a preferred embodiment of a test device according to the present invention suitable to detect two different targets within two different samples (specimens) from the same person (patient). In this preferred embodiment the device comprises two test strips 101.a and 101.b, wherein each of these two strips comprises its own sample pad 102.a and 102.b, gold conjugate 103.a and 103.b, sample line 108.a and 103.b, control line 109.a and 109.b, humidity indicator 110.a and 110.b, and absorbent pad 105.a and 105.b.
a: shows an external view of a preferred embodiment of a test device 501 according to the present invention suitable to detect two targets within the same sample (specimen) from the same person (patient). In this preferred embodiment the device 501 comprises a sample application window 502, two test result windows 503.a and 503.b, two control result windows 504.a and 504.b, humidity indication window 505 and a patient ID area 506.
b: shows an external view of a preferred embodiment of a test device 501 according to the present invention suitable to detect two targets within two different samples (specimens) from the same person (patient). In this preferred embodiment the device 501 comprises two sample application windows 502.a and 502.b, two test result windows 503.a and 503.b, two control result windows 504.a and 504.b, two humidity indication windows 505.a and 505.b and a patient ID area 506.
5 mg of bovine serum albumin (BSA) was linked to each oligonucleotide and another 5 mg to the complementary oligonucleotide. Every oligonucleotide had a length of about 20 nucleotides having an amino group at the 5′ terminus. The procedure was performed according to the method described by Duncan et al. 198320 comprising the following steps:
The oligonucleotide and complementary oligonucleotide linked BSA prepared as described in Example 1 are further processed according to a procedure comprising the following steps:
(* In case of antibodies/antigens and their specific antigens/antibodies there is no need for these steps of bovine serum albumin or any other protein labelling. ** Other proteins or peptides could be used other than bovine serum albumin).
Additionally, print first sample line (e.g. anti-HIV p24, 2nd clone), second sample line (e.g. HIV p160) and control line (e.g. mixture of goat anti-p160 and goat anti-mouse IgG) onto nitrocellulose membrane, then heat dry at temperature around 50° C.
Finally, laminate cards according to the following procedure:
A. In case of Conjugate Releasing Site Laminated Within the Upper Side of the Device Plastic Housing
Lamination of cards using the first gold conjugate. Laminate card components onto the backing material with the sequence:
B. In Case of Conjugate Releasing Site Laminated Onto the Test Strip Itself Separated From the First Conjugate By a Divider
Laminate card components onto the backing material with the sequence (see
C. Alternatively
Lamination of the second gold conjugate could be applied within the plastic housing itself to ensure that the two conjugates will not propagate before release from the releasing pad and so stick within the releasing pad.
The 1st gold conjugate pad contains:
The 2nd gold conjugate pad contains:
The 1st sample line 108a (tuberculosis specific detection line) comprises anti-LAM clone 2 immobilized onto the nitrocellulose membrane. The second sample line 108b (HIV specific detection line) comprises of recombinant envelop protein gp160 immobilized onto the nitrocellulose membrane. The control line 109 is anti-mouse IgG. Sample line 108a and control line 109 turn into purple color in case of LAM availability in the sample, sample line 108b and control line 109 turn into purple color in case of HIV gp160 antibody availability in the sample, and only the control line 109 turns into purple color in case of HIV antibody and LAM antigen free sample.
There aren't any commercially available rapid tests for urinary HIV/Tuberculosis detection in urine.
The 1st gold conjugate pad contains:
The 2nd gold conjugate pad contains:
The 1st sample line 108a (tuberculosis specific detection line) comprises of anti-LAM clone 2 immobilized onto the nitrocellulose membrane. The second sample line 108b (HCV specific detection line) comprises of recombinant HCV antigen immobilized onto the nitrocellulose membrane. The control line 109 is anti-mouse IgG. Sample line 108a and control line 109 turn into purple color in case of LAM availability in the sample, sample line 108b and control line 109 turn into purple color in case of HCV antibody availability in the sample, and only the control line 109 turns into purple color in case of HCV antibody and LAM antigen free sample.
There aren't any commercially available rapid tests for urinary HCV/Tuberculosis detection in urine.
The 1st gold conjugate pad contains:
The 2nd gold conjugate pad contains:
The 1st sample line 108a (tuberculosis specific detection line) comprises of anti-LAM clone 2 immobilized onto the nitrocellulose membrane. The second sample line 108b (H. Pylori specific detection line) comprises of recombinant H. Pylori antigen immobilized onto the nitrocellulose membrane. The control line 109 is anti-mouse IgG. Sample line 108a and control line 109 turn into purple color in case of LAM availability in the sample, sample line 108b and control line 109 turn into purple color in case of H. Pylori antibody availability in the sample, and only the control line 109 turns into purple color in case of H. Pylori antibody and LAM antigen free sample.
There aren't any commercially available rapid tests for urinary H. Pylori/Tuberculosis detection in urine.
The features disclosed in the foregoing description, in the claims and/or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in divers forms thereof.
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Number | Date | Country | Kind |
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06025522.1 | Dec 2006 | EP | regional |
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/EP07/10630 | 12/6/2007 | WO | 00 | 10/29/2009 |