Patent Application
20100075441
The present invention relates to a 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 different colloidal gold conjugates for sensitivity enhancement by signal amplification. The present invention further refers to rapid immunochromatographic detection devices, to the uses of the method for detecting diseases or specific conditions, and to a method for the manufacture of the devices as well as to a kit which comprises the devices.
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 targets 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, requires 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 8-11 weeks after last menstrual period.
3. <5 mIU/ml 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 human immunodeficiency virus (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 HIV 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 times 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).
Tuberculosis (TB) is a major and increasing public health problem in both industrialized and developing countries. Hence, the development of new inexpensive, rapid and field adapted methods for its diagnosis is urgently needed. Sputum culture, which is still the reference method for the diagnosis of pulmonary TB, is cumbersome and time-consuming, and requires access to expensive biosafety level 3 (BSL3) laboratories. Microscopy of direct smears for acid-fast bacilli (AFB) as recommended by WHO for developing countries is the most commonly used method for diagnosis of TB. A major disadvantage with this method is its low sensitivity, even after concentration of the sputum samples.
The availability of new field adapted, low-cost, and rapid diagnostic tests to supplement AFB microscopy, and especially methods improving the diagnosis in AFB-negative disease, would be of great benefit for TB control programs, in particular in areas lacking appropriate safety laboratories. Among the newly developed methods for rapid diagnosis of TB, nucleic acid amplification methods such as PCR seem most promising, but the technology is still too complex to be feasible for TB control programs in developing countries. Antibodies against a number of mycobacterial antigens have been identified in patients using a variety of immunological techniques, but no antibody test has so far reached sufficient sensitivity and/or specificity for routine diagnostic purposes. Detection of circulating or secreted Mycobacterium tuberculosis antigens seems attractive and has been explored in a number of studies. However, no satisfactory commercial test for mycobacterial antigens in serum or sputum is currently available.
The idea of identifying mycobacterial antigens in urine of TB patients is attractive for several reasons: urine is more readily obtainable than serum samples and urinary specimens do not carry the risks inherent to needles and blood-based laboratory work. Furthermore, if the urine specimens are boiled before handling, there is no need for BSL3 facilities.
In 1920s, mycobacterial antigens were detected in the urine of TB patients, and the diagnostic potential of such antigens was subsequently discussed by other scientists. More recently, the diagnostic value of mycobacterial antigens in the urine of leprosy patients has been assessed. Unfortunately, the techniques involved turned out to be insufficiently sensitive in paucibacillary patients, the patient group where improved diagnostic tests are most needed.
Lipoarabinomannan (LAM) is a major and structurally important glycolipid component of the outer cell wall of all mycobacteria and may account for up to 15% of the total bacterial weight. LAM is a carbohydrate antigen with glycosidic linkages for which no human degrading glycosidases are known. Hence, we assumed that in active mycobacterial disease LAM may be cleared through the kidneys and occur in urine in antigenically intact form. Furthermore, since LAM is a carbohydrate antigen and thus inherently heat-stable, LAM may be detectable by sensitive immunological techniques, even after boiling of the urine. At least theoretically, the amount of LAM in the urine should reflect the bacterial load, metabolic activity and/or rate of degradation of the bacteria, and hence permit a semi-quantitative assessment of the infectious status. A high sensitive, simple, fast and method for LAM detection and quantification was reported using an enzyme-linked immunosorbent assay (ELISA) in AFB positive sputa from TB patients (7).
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, anti-lipoarabinomannan (LAM), HBsAG, anti-HBs, IgG, e.g. HIV antibodies, in urine, blood, serum or saliva by enhanced sensitivity.
It is an object of the present invention to solve the rapid immunochromatographic detection of a target in a sample by a new method especially through sensitivity enhancement and to overcome the disadvantages of the prior art.
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 sandwich by contacting
Surprisingly, the object is achieved by the inventive method through signal amplification using different colloidal gold conjugates conjugated with a specific antibody and some oligonucleotides and their complementary oligonucleotides and/or antibodies and their related antigens. The rapid immunochromatographic detection method is using the oligonucleotides to multiply the colloidal gold signal by branched links between both colloidal gold conjugates.
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, see
In another embodiment the present invention concerns another test device for conducting the method for rapid immunochromatographic detection of a target in a sample comprising a detection cup, see
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 devices according to the present invention, reagents, wash buffers and a manual.
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
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
The rapid immunochromatographic detection system comprises a detection test strip of two gold conjugate releasing sites or pads with different compositions. The first conjugate releasing site or pad 103.1 is laminated on the test strip between the sample application site or sample pad and the nitrocellulose membrane, while the second conjugate releasing site or pad 103.2 is laminated above the first conjugate releasing site separated by a divider 110 to be released directly toward the nitrocellulose membrane without flow through the first conjugate releasing site to avoid interaction with the first conjugate before reaching the membrane, see
By the sample flow within the rapid immunochromatographic test the antibody on the first pad which contains the oligonucleotides will capture the antigen/antibody in the sample and carry it to be captured by the other antibody/antigen that is immobilized on the nitrocellulose membrane to form the sandwich detection. Then, the second conjugate releasing pad will release its gold that is conjugated with the complementary oligonucleotides. The last mentioned conjugate would bind with the first conjugate from the oligonucleotide(s) side. This binding could be happened by any of the conjugated oligonucleotides with its complementary oligonucleotide on the other gold conjugate. At the same time, the other oligonucleotides will be able to link with their complementary oligonucleotides besides the probability of capturing the first conjugate that will capture the second conjugate to form more and more branched bonds which propagates the accumulation of colloidal gold particles onto the capturing/sample line. This propagation and accumulation of colloidal gold signal will amplify the signal and highly increase the sensitivity. This will enable us to detect very low concentrations that are not detectable using the same technique without signal amplification.
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 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 preferred embodiment of the method according to the present invention each gold conjugate comprises between 1 and 15 different oligonucleotides, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, preferably between 2 and 10 different oligonucleotides, more preferably between 3 and 6 different oligonucleotides. In addition, the oligonucleotides conjugated with the second gold conjugate are complementary to the oligonucleotides of the first colloidal gold conjugate.
Mostly 2-4 oligonucleotides per gold conjugate are used. These oligonucleotides are of about 20-nucleotides in length. These oligonucleotides have an amino group at the 5′ terminus which is conjugated with bovine serum albumin. The bond between the gold and the oligonucleotides is the same as the one between the gold and the antibodies or antigens.
In a further first aspect the present invention provides a method comprising the following steps of
In one embodiment the method comprises further a first specific 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 antibodies and antigens which can be used are HIV specific antibodies or antigens, tuberculosis specific antibodies or antigens, malaria specific antibodies, toxoplasmosis specific antibodies or antigens, rubella specific antibodies, Leishmania specific antibodies or Pneumonia specific antibodies. Monoclonal antibodies are preferred, whereas 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 further another specific antibody or antigen which is selected from the group consisting of anti-alpha chorionic gonadotropin hormone (anti-ahCG), 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.
Moreover, antigen detection antibodies are often pairs of monoclonal antibodies for example of anti-hepatitis B surface antigen (anti-HBsAg), anti-HIV p160 or anti-HIV p24.
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 site or pad 103.1 is laminated on the test strip between the sample pad and the nitrocellulose membrane, while the second conjugate releasing site or pad 103.2 is located above the first conjugate releasing pad separated by a divider 110, in order 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 another specific antibody or antigen.
In another preferred embodiment the test zone 108 of the test device comprises another specific antibody or antigen which is selected from the group consisting of anti-alpha chorionic gonadotropin hormone (anti-ahCG), 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 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.
Moreover, antigen detection antibodies are often pairs of monoclonal antibodies for example of anti-hepatitis B surface antigen (anti-HBsAg), anti-HIV p160 or anti-HIV p24.
In another embodiment of the present invention the second conjugate releasing site 103.2 is laminated within the upper side of the housing.
In a further 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 detection cup 510.
In one embodiment the detection cup 510 of the test device comprises
In one embodiment the test zones of the detection cup further comprise another specific antibody or antigen.
In a preferred embodiment another specific antibody or antigen is selected from the group consisting of anti-alpha chorionic gonadotropin hormone (anti-ahCG), 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.
Moreover, antigen detection antibodies are often pairs of monoclonal antibodies for example of anti-hepatitis B surface antigen (anti-HBsAg), anti-HIV p160 or anti-HIV p24.
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.
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
In a further embodiment the method comprises the following steps of
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 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 the target in the sample 220 will be captured by the 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 another antibody or antigen 108 that is immobilized onto the membrane 104 of the test zone to form a sandwich detection. Then, the second colloidal gold 211 conjugated with the complementary oligonucleotides 203′, 204′, 205′, 206′ to the oligonucleotides 203, 204, 205, 206 of the first colloidal gold will be released and will bind to the first conjugate from the oligonucleotide(s) side and enhance the signal.
The following examples illustrate the present invention without, however, limiting the same thereto.
5 mg of bovine serum albumin (BSA) was linked to each oligonucleotide (about 20 nucleotide having an amino group at 5′ terminus) and another 5 mg to complementary oligonucleotide (about 20 nucleotide having an amino group at 5′ terminus), according to a procedure comprising the following steps, according to the method described by Duncan et al. 1983 (7):
The oligonucleotide and complementary oligonucleotide labeled bovine serum albumin (BSA) prepared as described in Example 2 are further processed according to a procedure comprising the following steps:
Lamination of card components onto the backing material with the sequence (in case of conjugate releasing site laminated onto the test strip itself separated from the first conjugate by a divider), see
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
Finally the lamination of the second gold conjugate will 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.
* 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 labeling.
** Other proteins or peptides could be used other than bovine serum albumin
The first gold conjugate is made of mouse anti-βhCG and four oligonucleotides conjugated with colloidal gold conjugate, and the second gold conjugate is the conjugate of the four complementary oligonucleotides. The first gold conjugate 103.1 was laminated in the side of the nitrocellulose membrane, while the second gold conjugate 103.2 is 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.
The plastic housing is the plastic design where we insert the test strip. 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 sample line is a mouse anti-αhCG immobilized onto the nitrocellulose membrane. The control line is anti-mouse IgG. Sample and control lines turn into purple color in case of hCG availability in the sample; only the control line 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 1 mIU/ml.
The first gold conjugate is made of mouse anti-HBsAg (clone 1) and four oligonucleotides conjugated with colloidal gold conjugate, and the second gold conjugate is the conjugate of the four complementary oligonucleotides. 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 a pair of monoclonal antibodies. The first gold conjugate 103.1 was laminated in the side of 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 it directly onto the nitrocellulose membrane.
The plastic housing is the plastic design, where we insert the test strip. 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 sample line 108 is mouse anti-HBsAg (clone 2) immobilized onto the nitrocellulose membrane 104. The control line 109 is anti-mouse IgG. Sample 108 and control lines 109 turn into purple color in case of HBsAg availability in the sample; only the control line 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 10 pg/ml.
The first gold conjugate is mouse anti-human Immunoglobulin G (anti-hIgG) and four oligonucleotides conjugated with colloidal gold conjugate, and the second gold conjugate is the conjugate of the four complementary oligonucleotides. The first gold conjugate 103.1 was laminated in the side of nitrocellulose membrane 104, while the second gold conjugate 103.2 was laminated above the first pad 103.1 separated by a divider that enables the second conjugate to take a part of the sample and release it directly onto the nitrocellulose membrane 104.
The plastic housing is the plastic design where we insert the test strip. 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 sample line 108 is a combination of synthetic or recombinant HIV antigen immobilized onto the nitrocellulose membrane 104. The control line 109 is anti-mouse IgG. Sample 108 and control 109 lines turn into purple color in case of HIV antibodies availability in the sample; only the control line 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 in serum.
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 diverse forms thereof.
| Number | Date | Country | Kind |
|---|---|---|---|
| 06025530.4 | Dec 2006 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2007/010620 | 12/6/2007 | WO | 00 | 10/29/2009 |
