Patent Application
20100291542
The present invention relates to a new method for rapid immunochromatographic detection. More precisely, the present invention relates to a method for rapid immunochromatographic detection of a target in a sample, wherein the target is an antibody and/or an antigen, using double sandwich immunoassay detection for sensitivity enhancement by signal amplification. The present invention further refers to rapid immunochromatographic detection device, to uses of the method for detecting diseases or specific conditions, and to a method for the manufacture of the device as well as to a kit which comprises the 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 fields (1). 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 products (3). The wide range of applications for such devices has been reviewed (1, 2).
Rapid immunochromatographic test devices, e.g. in the form of a test strip, are made up of a number of components, see
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.
However, despite the wide use of rapid immunochromatographic test devices, their suitability is still limited with regard to certain applications. Urine, for example, contains very low levels of IgG, frequently around 1 mg/l. Therefore, the detection of antibodies, e.g. directed to HIV or HCV, require very sensitive techniques. To date, the tests for antibodies in urine samples are based on ELISA and Western blot techniques, which are labour-intensive, time-consuming and need to be carried out by qualified persons. Efforts are being made to develop simple and/or rapid tests for the detection of antibody to HIV in urine specimens (4).
Oral fluid specimens consist often of saliva, which predominantly contains IgA class antibody, and oral mucosal transudates, which mostly contain IgG, and therefore also have much lower levels of IgG than serum. The levels of IgG normally found in oral fluid specimens (approximately 15 mg/l) are, however, higher than in urine specimens and innovative simple and rapid technology that has been shown to be effective for whole blood, serum and plasma, e.g. lateral flow through a chromatographic membrane, has been developed for use with these specimens (4).
Human chorionic gonadotropin (hCG) is a glycopeptide hormone produced by the placenta during pregnancy. The appearance and rapid increase in the concentration of hCG in the subject's urine makes it a good marker for confirming pregnancy. The concentration of hCG in urine increases steadily to a circulation peak of as much as 50,000 mIU/ml between the eighth and eleventh weeks.
Urine hCG levels during pregnancy are estimated to be:
1. 10-30 mIU/ml 7-10 days post conception.
2. 37,000-50,000 mIU/ml 18-11 weeks after last menstrual period.
3. <5 mIU/ml for healthy men or non-pregnant women.
In the prior art the hCG test is a chromatographic immunoassay which uses specific antibodies to selectively identify hCG in urine with a high degree of sensitivity. Elevated levels of hCG as low as 20 mIU/ml can be detected within 3 minutes.
There are several tests used to detect the presence of hepatitis B antibodies. There are also several tests that detect the presence of viral antigens. The hepatitis B surface antibody (anti-HBs) is the most common test. Its presence indicates previous exposure to HBV, but the virus is no longer present and the person cannot pass on the virus to others. The antibody also protects the body from future HBV infection. In addition to exposure to HBV, the antibodies can also be acquired from successful vaccination. This test is done to determine the need for vaccination (if anti-HBs is absent), or following the completion of vaccination against the disease, or following an active infection.
Hepatitis B surface antigen (HBsAg) is a protein antigen produced by HBV. This antigen is the earliest indicator of acute hepatitis B and frequently identifies infected people before symptoms appear. HBsAg disappears from the blood during the recovery period. In some people (particularly those infected as children or those with a weak immune system, such as those with AIDS), chronic infection with HBV may occur and HBsAg remains positive. To test for HIV is an essential component in the diagnosis and treatment of persons infected with the virus, in screening of blood for transfusion, in surveillance and in HIV/AIDS related research. Thus accurate and cost-effective testing is of great importance in combating the spread of HIV. It is imperative that tests for the diagnosis of HIV infection be as accurate as possible, given the serious ethical, legal and social issues that accompany HIV infection.
The number of people living with HIV has now risen to reach its highest level ever: close to 40 million people are living with the virus and close to 5 million people were newly infected with HIV in 2004 alone. Worldwide, the AIDS epidemic killed over 3 million people last year alone (Source: UNAIDS). Furthermore, only one in five people needing HIV prevention worldwide have access to basic prevention services and only one in ten people living with HIV has been tested for the virus.
The HI virus is most easily transmitted to others during the initial period of acute HIV infection, when the viral load (quantity of HIV RNA in the blood) is especially high and when people are not aware of being contaminated by the virus. Most HIV infections are transmitted at this stage, called primary infection. Earlier detection using ultra sensitive tests avoids missing primary infections, enabling immediate precautionary measures to be taken to help prevent the risk of HIV transmission to a non-infected partner, to an unborn child, or through blood donations or direct blood contact. Earlier detection of HIV infection also ensures the implementation of early antiretroviral therapy (ART) to slow down the progression of HIV infection, thereby improving patient care and quality of life.
The diagnosis of HIV infection is usually made on the basis of the detection of HIV antibodies and/or antigen. The diagnosis of an HIV infection can be made indirectly, i.e. through the demonstration of virus-specific antibodies. Besides such indirect diagnosis based on detection of antibodies, a direct diagnosis of HIV infection is also possible: either through the demonstration of infectious virus (using cell culture), viral antigens (p24 antigen ELISA) or viral nucleic acid (i.e. viral genome); the latter is also termed nucleic acid testing (NAT).
One important problem of HIV antibody testing is the so-called “diagnostic window”. This is the time period that elapses between the time of acquisition of HIV infection until detectable levels of antibodies are present. The switch from antibody-negative to antibody-positive is called “seroconversion”.
The most widely used screening tests are ELISAs as they are the most appropriate for screening large numbers of specimens on a daily basis, e.g. blood donations. The earliest assays used purified HIV lysates (1st generation assays). Improved assays based on recombinant proteins and/or synthetic peptides, which also enabled the production of combined HIV-1/HIV-2 assays, became rapidly available (2nd generation assays). The so-called 3rd generation or antigen-sandwich assays, which use labeled antigens as conjugate, are more sensitive and have reduced the diagnostic window period considerably (5, 6).
It is an object of the present invention to overcome the problems with regard to the applicability of rapid immunochromatographic test devices for the detection of hCG, HBsAG, anti-HBs, IgG, e.g. HIV antibodies, in urine, blood, serum or saliva by enhanced sensitivity. Therefore, it is an object to enhance the sensitivity of the rapid immunochromatographic detection system by the improvement of the colloidal gold signal.
It is the object of the present invention to solve the rapid immunochromatographic detection of a target in a sample by a new method through sensitivity enhancement using different colloidal gold conjugates conjugated with a first and a second specific antibody or antigen, respectively. The rapid immunochromatographic detection method is using the double sandwich immunoassay detection to multiply the colloidal gold signal system comprising a detection test strip of two gold conjugate releasing pads with different compositions.
In one embodiment the present invention concerns a method for rapid immunochromatographic detection of a target in a sample comprising the step of forming a double sandwich by contacting
Thus, this is achieved by sensitivity enhancement using different colloidal gold conjugates conjugated with a first and a second specific antibody or antigen, respectively. The rapid immunochromatographic detection method is using the double sandwich immunoassay detection to multiply the colloidal gold signal system comprising a detection test strip of two gold conjugate releasing pads with different compositions.
In one embodiment the present invention further relates to a method, comprising the following steps of
In a further embodiment the present invention concerns a test device for conducting the method for rapid immunochromatographic detection of a target in a sample according to the present invention comprising a housing comprising a test strip comprising a sample application site; a first conjugate releasing site; a second conjugate releasing site; a nitrocellulose membrane; a test zone and a control zone; and a sample absorbent site. The second conjugate releasing site may also be laminated within the upper side of the housing.
In another embodiment the present invention relates to the use of the method for diagnosing and monitoring a disease or a specific condition of a subject by detecting a target in a sample.
In a further embodiment the present invention refers to a kit for rapid immunochromatographic detection of a target in a sample comprising the test device according to the present invention.
In a further embodiment the present invention concerns a method for the manufacture of the test device according to the present invention comprising the following steps of
In another embodiment the present invention relates to a method for the manufacture of the test device according to the present invention comprising the following steps of
In a further embodiment the present invention relates to a method for the manufacture of the test device according to the present invention comprising the following steps of
The first conjugate releasing site of the rapid immunochromatographic detection system contains a colloidal gold conjugate that is conjugated with the first specific antibody or antigen to capture the target from the first site (let us consider it as site A), while the second conjugate releasing site contains a colloidal gold conjugate conjugated with the second specific antibody or antigen to capture the target from the second site (let us consider it as site B). The last mentioned conjugated antibody or antigen is the same antibody or antigen that is immobilized onto the nitrocellulose membrane.
By the sample flow within the rapid immunochromatographic test the antibody on the first conjugate releasing site will capture the first specific antigen or antibody in the sample from site A and carry it to be captured by the second specific antibody or antigen that is immobilized onto the nitrocellulose membrane from site B to form the sandwich detection. Taking in mind that the first conjugate mostly capture more than one target molecule, one ore more targets will be captured from A and will be free from site B.
Then, when the second conjugate releasing site releases its colloidal gold conjugate that is conjugated with the same antibody or antigen that is immobilized onto the nitrocellulose membrane, this conjugate will capture site B of the already captured targets by the first conjugate which is captured by the antibody or antigen onto the membrane.
The new binding of the second conjugate will make new probability choices of capturing the unbound target or captured target (by the first conjugate), similar for the unbound first conjugate, to form more and more branched bonds that propagate the accumulation of colloidal gold particles onto the test zone. This propagation and accumulation of colloidal gold signal will improve the signal and increase the sensitivity.
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. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties.
Preferably, the terms used herein are defined as described in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, Leuenberger, H. G. W., Nagel, B. and Kölbl. H. eds. (1996), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).
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.
As outlined above there is a need in the prior art to provide a new method for rapid immunochromatographic detection of a target in a sample for the detection of a disease or a specific condition such as pregnancy in a subject. There is also a need in the art for methods suitable for rapid and sensitive detection of an antibody and/or antigen having a higher sensitivity than methods from the prior art.
In a first aspect the present invention provides a method for rapid immunochromatographic detection of a target in a sample comprising the step of forming a double sandwich by contacting
The first colloidal gold conjugated with a first antibody or antigen captures the target in the sample and forms a complex “target-first colloidal conjugate”. Preferably this target in the sample is an antigen and/or antibody.
In a further first aspect the present invention provides a method comprising the following steps of
In one embodiment the method comprises further a specific first antibody or antigen which is selected from the group consisting of anti-beta chorionic gonadotropin hormone (anti-βhCG), anti-lipoarabinomannan (LAM), hepatitis virus antibodies against or antigens from hepatitis virus type A, hepatitis virus type B, or hepatitis virus type C or human immunoglobulin G antibodies or antigens.
Other antigens or antibodies that could be used for related antibody detection are: H. Pylori antigen, hepatitis B envelope antigen, hepatitis C NS3 antigen, hepatitis C core antigen, HIV p160, HIV p24. Toxoplasma antigen, anti-Malaria HRP-II, anti-human immunoglobulin G, anti-LH, anti-prostate specific antigen, anti-pneumolysin, anti-leishmania antigen, anti-H. Pylori antigen, and anti-toxoplasma antigen. Monoclonal antibodies are preferred while polyclonal antibodies are applicable.
In one preferred embodiment the hepatitis virus antigen is hepatitis B surface antigen (HbsAg) and the hepatitis virus antibody is anti-HBsAg.
In one embodiment the method comprises a second specific antibody or antigen which is selected from the group consisting of anti-alpha chorionic gonadotropin hormone (anti-αhCG), anti-lipoarabinomannan (LAM), hepatitis virus antibodies against or antigens from hepatitis virus type A, hepatitis virus type B, or hepatitis virus type C or human immunodeficiency virus (HIV) antibodies or antigens from the HIV type HIV-1 and HIV-2 or HIV subtype HIV-1-N, HIV-1-O or HIV-1-M.
In one preferred embodiment the hepatitis virus antigen is hepatitis B surface antigen (HbsAg), the hepatitis virus antibody is anti-HBsAg and the human immunodeficiency virus (HIV) antigen is HIV p160.
Other antigens or antibodies that could be used for related antibody detection are: H. Pylori antigen, hepatitis B envelope antigen, hepatitis C NS3 antigen, hepatitis C core antigen, HIV p160, HIV p24, Toxoplasma antigen, anti-Malaria HRP-II, anti-human immunoglobulin G, anti-LH, anti-prostate specific antigen, anti-pneumolysin, anti-leishmania antigen, anti-H. Pylori antigen, and anti-toxoplasma antigen. Monoclonal antibodies are preferred while polyclonal antibodies are applicable.
In one embodiment of the method the sample comprises a body fluid of a subject.
In one preferred embodiment the body fluid is selected from the group consisting of urine, whole blood, serum plasma and saliva.
In another aspect the present invention concerns a test device for conducting the method for rapid immunochromatographic detection of a target in a sample according to the present invention comprising a housing comprising a test strip 101 comprising a sample application site 102; a first conjugate releasing site 103.1; a second conjugate releasing site 103.2; a nitrocellulose membrane 104; a test zone 108 and a control zone 109; and a sample absorbent site 105.
The first conjugate releasing pad 103.1 is laminated on the test strip between the sample pad and the nitrocellulose membrane while the second 103.2 is above the first pad separated by a divider 110 to be released directly toward the nitrocellulose membrane without flow through the first conjugate pad to avoid interact with the first conjugate before reaching the membrane, see
In one embodiment the test device further comprises the test strip 101 which is attached to a supporting backing 107 by means of an adhesive 106.
In a preferred embodiment the supporting backing 107 of the test device is a plastic backing.
In another embodiment the test zone 108 of the test device comprises the second specific antibody or antigen.
In another preferred embodiment the test zone 108 of the test device comprises the second specific antibody or antigen which is selected from the group consisting of anti-alpha chorionic gonadotropin hormone (anti-αhCG), anti-lipoarabiniomannan (LAM), hepatitis virus antibodies against or antigens from hepatitis virus type A, hepatitis virus type B, or hepatitis virus type C or human immunodeficiency virus (HIV) antibodies or antigens from the HIV type HIV-1 and HIV-2 or HIV subtype HIV-1-N, HIV-1-O or HIV-1-M.
In a more preferred embodiment the hepatitis virus antigen is hepatitis B surface antigen (HBsAg), the hepatitis virus antibody is anti-HBsAg and the human immunodeficiency virus (HIV) antigen is HIV p160.
In another embodiment the second conjugate releasing site 103.2 of the test device is laminated within the upper side of the housing.
In another aspect the invention relates to the use of the method for diagnosing and monitoring a disease or a specific condition of a subject by detecting a target in a sample.
In one embodiment the specific condition is pregnancy.
In a preferred embodiment the target of the specific condition is human chorionic gonadotropin hormone (hCG).
In another embodiment the disease is hepatitis selected of the group consisting of hepatitis type A, hepatitis type B, or hepatitis type C.
In a preferred embodiment the selected hepatitis type is hepatitis type B.
In a more preferred embodiment the target of the disease which is hepatitis type is hepatitis B surface antigen (HbsAg).
In another embodiment the disease is an HIV infection selected from the HIV infection group consisting of HIV type HIV-1 and HIV-2 or HIV subtype HIV-1-N, HIV-1-O or HIV-1-M.
In a more preferred embodiment the target of the HIV infection is selected from an HIV antibody or antigen selected from the group consisting of p41,p120, p160, p18, p24/25, p55, p34, p40, p52, p68.
In a further more preferred embodiment the HIV antigen is p160.
In a further aspect the invention concerns a kit for rapid immunochromatographic detection of a target in a sample comprising the test device comprising the housing according to the invention.
in one embodiment the kit comprises further reagents, wash buffers and a manual.
There is no need for special reagents or buffers except in the case of a whole blood sample, where the used assay buffer is 0.1M Tris buffer (pH 7.5) with sodium azide 0.01M as a preservative.
In another aspect the invention relates to a method for the manufacture of the test device according to the invention.
In one embodiment the method comprises the following steps of
In one embodiment the method further comprises the following steps of
In anther embodiment the method further comprises the following steps of
a:
a shows top and side views of a typical rapid-flow immunochromatographic test device in the form of a test strip 101 including 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:
b shows top and side views of our modified rapid-flow immunochromatographic test device in the form of a test strip 101 including 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, a test zone 108, a control zone 109, and the two conjugates (divider) 110.
By the sample flow within the rapid immunochromatographic test device the target in the sample will be captured by the first specific antibody or antigen 202 of the first colloidal gold 201 to form the complex “target-first colloidal gold”. This complex flows to the test zone, where it will be captured by the second specific antibody or antigen 203 that is immobilized onto the membrane 104 of the test zone 108. Then, the second colloidal gold 211 conjugated with the first specific antibody or antigen 203 will be released and will bind to the target as well as to the first colloidal gold conjugate 201 and enhance the signal by forming a double sandwich.
The following examples illustrate the present invention without, however, limiting the same thereto.
The first gold conjugate is mouse anti-βhCG conjugated with colloidal gold conjugate, and the second gold conjugate is mouse anti-αhCG conjugated with colloidal gold conjugate. The first gold conjugate 103.1 was laminated in the side of the nitrocellulose membrane 104 while the second gold conjugate 103.2 was laminated above the first pad 103.1 separated by a divider 110 that enables the second conjugate to take a part of the sample and release directly onto the nitrocellulose membrane 104.
The first conjugate releasing pad 103.1 is laminated on the test strip between the sample pad and the nitrocellulose membrane while the second 103.2 is above the first pad separated by a divider 110 to be released directly toward the nitrocellulose membrane without flow through the first conjugate pad to avoid interact with the first conjugate before reaching the membrane, see
The test zone 108 is a mouse anti-αhCG immobilized onto the nitrocellulose membrane 104. The control zone 109 is anti-mouse IgG. Test 108 and control 109 zones turn into purple color in case of hCG availability in the sample; only the control zone 109 turns into purple color in case of hCG free sample.
The commercially available rapid tests sensitivity for the pregnancy hormone which is human chorionic gonadotropin hormone (hCG) is around 25 mIU/ml while according to this system it is so simple to detect less than 5 mIU/ml.
The first gold conjugate is made of mouse anti-HBsAg (clone 1) conjugated with colloidal gold conjugate, and the second gold conjugate is mouse anti-HBsAg (clone 2) conjugated with colloidal gold conjugate. The numbering of clones are only for explanation and to recognize that we use always two different clones of monoclonal antibodies; these two monoclonal antibodies capture the target antigen from two different sites, so we call them as: a pair of monoclonal antibodies. The first gold conjugate 103.1 was laminated in the side of the nitrocellulose membrane 104, while the second gold conjugate was laminated above the first pad 103.1 separated by a divider 110 that enables the second conjugate to take a part of the sample and release it directly onto the nitrocellulose membrane 104.
The first conjugate releasing pad 103.1 is laminated on the test strip between the sample pad and the nitrocellulose membrane while the second 103.2 is above the first pad separated by a divider 110 to be released directly toward the nitrocellulose membrane without flow through the first conjugate pad to avoid interact with the first conjugate before reaching the membrane, see
The test zone 108 is mouse anti-HBsAg (clone2) immobilized onto the nitrocellulose membrane 104. The control zone 109 is anti-mouse IgG. Test 108 and control zones 109 turn into purple color in case of HBsAg availability in the sample; only the control zone 109 turns into purple color in case of HBsAg free sample, see
The commercially available rapid tests sensitivity for hepatitis B surface antigen is within the range 500-1000 pg/ml while according to this system it is so simple to detect less than 50 pg/ml.
The first gold conjugate is mouse anti-human Immunoglobulin G (anti-hIgG) conjugated with colloidal gold conjugate, and the second gold conjugate is HIV p160 conjugated with colloidal gold conjugate. The first gold conjugate 103.1 was laminated in the side of nitrocellulose membrane 104, while the second gold conjugate was laminated above the first pad 103.1 separated by a divider 110 that enables the second conjugate to take a part of the sample and release directly onto the nitrocellulose membrane 104.
The first conjugate releasing pad 103.1 is laminated on the test strip between the sample pad and the nitrocellulose membrane while the second 103.2 is above the first pad separated by a divider 110 to be released directly toward the nitrocellulose membrane without flow through the first conjugate pad to avoid interact with the first conjugate before reaching the membrane, see
The test zone 108 is HIV p160 antigen immobilized onto the nitrocellulose membrane 104. The control zone 109 is anti-mouse IgG. Sample 108 and control 109 zones turn into purple color in case of HIV antibodies availability in the sample; only the control zone 109 turns into purple color in case of HIV antibodies free sample, see
According to this system it is so simple to detect very low titers of HIV antibodies.
| Number | Date | Country | Kind |
|---|---|---|---|
| 06025526.2 | Dec 2006 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2007/010624 | 12/6/2007 | WO | 00 | 10/29/2009 |
