Abstract:
The present invention refers to an immuno-chromatographic detection cup and its use for simultaneously detecting at least one antigen, e.g. tuberculosis antigen, malaria antigen and pneumonia antigen, and at least one antibody, e.g. HIV antibody, HCV antibody and  H. pylori  antibody, in a urine sample. The urinary detection cup comprises (a) a sample-collecting container; (b) a conjugate releasing pad comprising a gold labelled antibody X and an oligo-nucleotide linked antibody X′, wherein both antibodies are directed against the same antigen, and a gold labelled antigen Y and an oligo-nucleotide linked antigen Y′, wherein both antigens are recognized by the same antibody; (c) a test means inside the container separated from the conjugate releasing pad, which test means comprises a region comprising an oligo-nucleotide complementary to the oligo-nucleotide linked to antibody X′, a region comprising an oligo-nucleotide complementary to the oligo-nucleotide linked antigen Y′, and at least one region comprising a control antibody and/or a control antigen; and (d) at least two sample absorbent pads linked to the test device at different positions.

Description:
[0001]    The present invention refers to an immuno-chromatographic detection cup and its use for simultaneously detecting at least one antigen, e.g. tuberculosis antigen, malaria antigen and pneumonia antigen, and at least one antibody, e.g. HIV antibody, HCV antibody and  H. pylori  antibody, in a urine sample. The detection makes use of gold-labelled antibodies and antigens, oligo-nucleotide linked antibodies and antigens, and complementary oligo-nucleotides for capturing the linked antibodies. 
       BACKGROUND OF THE INVENTION 
       [0002]    Testing 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 should be as accurate as possible, given the serious ethical, legal and social issues that accompany HIV infection 1 . 
         [0003]    The use of body fluids other than blood as specimens for detecting antibodies to HIV has been reported to have potential as an alternative medium for HIV testing. 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 these types of specimens can be a useful alternative when it is difficult or impossible to test for HIV in blood samples. It may be that blood can not be drawn for religious reasons or difficulties may be experienced in collecting blood samples in hard to reach places where it is, nevertheless, important to have epidemiological surveillance 1 . Moreover, urinary samples are the more safety type of specimens in case of HIV infection. 
         [0004]    Today, new HIV tests have been developed for use with urine specimens. Urine contains very low levels of IgG, frequently around 1 mg/l, and therefore very sensitive techniques are required to detect specific antibody. Thus, to date, the tests for urine specimens have been based on ELISA and Western blot techniques, however, efforts are being made to develop simple and/or rapid tests for the detection of antibody to HIV in urine specimens 1 . It would be of particular advance to have rapid immuno-chromatographic test devices available for detecting HIV antibodies in urine. 
         [0005]    In addition to HIV infection, tuberculosis (TB) is an important 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. 
         [0006]    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  Mycobacteriurm 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. 
         [0007]    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. 
         [0008]    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 needed most 
         [0009]    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 reliable method for LAM detection and quantification was reported using an enzyme-linked immunosorbent assay (ELISA) in AFB positive sputa from TB patients 2 . However, it would be of particular advantage to have rapid immuno-chromatographic test devices available for detecting LAM in urine. 
         [0010]    In recent years, the in vitro diagnostics (IVD) industry has made enormous efforts to develop immuno-chromatographic tests. Such tests have found applications in both clinical and non-clinical fields 3 . 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 4 . The wide range of applications for such devices has been reviewed 3, 5 . 
         [0011]    Rapid immuno-chromatographic test devices, e.g. in the form of a test strip, are made up of a number of components (see  FIG. 1 ). Such a test strip  101  commonly includes a sample pad  102 , a conjugate pad  103 , a membrane  104 , e.g. a nitrocellulose membrane, and an absorbent pad  105 . The membrane  104  is usually attached by means of an adhesive  106  to a supporting backing  107 , e.g. made of plastic. In practice, the user dispense a patient sample (usually urine or whole blood) onto the sample pad  102 . The sample then flows through the sample pad  102  into the conjugate pad  103 , where it mixes with and releases the detector reagent. This mixture then flows across the membrane  104 , where it binds with the test and control reagents located in the capture test zone  108  (sample zone) and negative control zone  109 , respectively. When the mixture binds to the reagent that forms the test line, a positive result is indicated. The colour intensity of the test line is proportional to the concentration of analyte in the sample. Excess sample that flows beyond the test and control zones  108 ,  109  is taken up in the absorbent pad  105 . 
         [0012]    Using rapid immuno-chromatographic test devices, a physician can instantly not only examine a sample by himself, but also diagnose a patient on the basis of the obtained results. Such 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. 
         [0013]    However, a main demerit is that one immuno-chromatographic test device only detects one parameter at a time with the consequence that many different rapid immuno-chromatographic test devices are needed to check the suspected parameters, resulting in the requirement of enormous sample volume to be obtained from a patient as well as cumbersome test procedures. 
         [0014]    Therefore, it is an object of the present invention to provide a rapid immuno-chromatographic test device for detecting different analytes (antibodies and antigens) in urine in a single assay at the same time. 
       SUMMARY OF THE INVENTION 
       [0015]    The object of the present invention is solved by a urinary immuno-chromatographic detection cup comprising:
       (a) a sample-collecting container;   (b) at least one conjugate releasing pad, which conjugate releasing pad comprises at least one gold labelled antibody X and at least one oligo-nucleotide linked antibody X′, wherein both antibodies are directed against the same antigen, and at least one gold labelled antigen Y and at least one oligo-nucleotide linked antigen Y′, wherein both antigens are recognized by the same antibody;   (c) at least one test means inside the container separated from the conjugate releasing pad, which test means comprises at least one region comprising an oligo-nucleotide complementary to the oligo-nucleotide linked to the antibody X′, at least one region comprising an oligo-nucleotide complementary to the oligo-nucleotide linked antigen Y′, and at least one region comprising a control antibody and/or a control antigen;   (d) at least two sample absorbent pads linked to the test device at different positions.       
 
         [0020]    Antibodies X and X′ may be identical or different, as long as they are directed against the same antigen. Antigens Y and Y′ may be identical or different as long as they are recognized by the same antibody; 
         [0021]    In one embodiment, the antibodies X and X′ are directed against an antigen selected from tuberculosis antigen, malaria antigen and pneumonia antigen. 
         [0022]    In a preferred embodiment, the tuberculosis antigen is LAM, the malaria antigen is HRP-II, and the pneumonia antigen is PLY. 
         [0023]    In one embodiment, the control antibody is anti-mouse IgG. 
         [0024]    In one embodiment, the antigens Y and Y′ are selected from envelop protein gp 160 from HIV, NS3 antigen from HCV, and  H. pylori  antigen. 
         [0025]    In one embodiment, the test device further comprises a nitrocellulose membrane on which the complementary oligo-nucleotides are immobilized. 
         [0026]    In one embodiment, the conjugate releasing pad is located at the internal surface of the container wall. 
         [0027]    In one embodiment, a first sample absorbent pad is located at the bottom, and a second sample pad is located at the top of the container. 
         [0028]    In a preferred embodiment, the sample absorbent pad at the top is fitted into a container cap. 
         [0029]    In a further preferred embodiment, the sample absorbent pad at the top has a capacity of absorbing at least 15 ml of sample. 
         [0030]    In one embodiment, the conjugate releasing pad further comprises optionally modified chitosan. 
         [0031]    The object of the present invention is further solved by a use of the urinary immuno-chromatographic detection cup according to the present invention for detecting at least one antigen selected from tuberculosis antigen, malaria antigen and pneumonia antigen in a sample of urine of a subject, preferably human, and at least one antibody selected from HIV antibody, HCV antibody and  H. pylori  antibody. 
         [0032]    In one embodiment of the use, the volume of the urine sample is approximately 15 ml. 
         [0033]    The object of the present invention is further solved by a method for preparing the urinary fluid immuno-chromatographic detection cup according to the present invention, wherein optionally modified water-soluble chitosan is added during the preparation of gold labelled antibody X or gold labelled antigen Y prior to conjugation of colloid gold with the antibody or antigen. 
         [0034]    In one embodiment of the method, the colloidal gold is prepared by reduction of 1% aqueous solution of tetra-chloroauric acid using tri-sodium citrate aqueous solution to produce spheroidal gold particles. 
         [0035]    The object of the present invention is also solved by a urinary immuno-chromatographic detection cup comprising:
       (a) a sample-collecting container;   (b) at least one conjugate releasing pad, which conjugate releasing pad comprises at least two different gold labelled antibodies (consider as anti-A, anti-B) and at least two different oligo-nucleotide (consider as 1 and 2) linked antibodies (consider as anti-A˜1 and anti-B˜2);   (c) at least one test strip inside the container separated from the conjugate releasing pad, the nitrocellulose membrane of this test strip printed with at least two lines of different oligo-nucleotides (consider as 1′ and 2′) complementary to each of the oligo-nucleotides linked to the antibodies, and at least a third line of one control antibody (non-specific binding of anti-A and anti-B);   (d) at least two sample absorbent pads linked to the test device at different positions.       
 
         [0040]    The object of the present invention is also solved by a urinary immuno-chromatographic detection cup comprising:
       (a) a sample-collecting container;   (b) at least one conjugate releasing pad, which conjugate releasing pad comprises at least two different gold labelled antigens (consider as C, D) and at least two different oligo-nucleotide (consider as 3 and 4) linked antigens (consider as C˜3 and D˜4);   (c) at least one test strip inside the container separated from the conjugate releasing pad, the nitrocellulose membrane of this test strip printed with at least two lines of different oligo-nucleotides (consider as 3′ and 4′) complementary to each of the oligo-nucleotides linked to the antigens, and at least a third line of one control antigen (non-specific binding of C and D);   (d) at least two sample absorbent pads linked to the test device at different positions.       
 
         [0045]    The object of the present invention is also solved by a urinary immuno-chromatographic detection cup comprising:
       (a) a sample-collecting container;   (b) at least one conjugate releasing pad, which conjugate releasing pad comprises at least one gold labelled antibody (consider as anti-E), at least one oligo-nucleotide (consider as 5) linked antibody (consider as anti-E˜5), at least one gold labelled antigen (consider as F) and at least one oligo-nucleotide (consider as 6) linked antigen (consider as F˜6);   (c) at least one test strip inside the container separated from the conjugate releasing pad, the nitrocellulose membrane of this test strip printed with at least one line of oligo-nucleotide (consider as 5′) complementary to the oligo-nucleotide linked to the antibody, at least one oligo-nucleotide (consider as 6′) complementary to the oligo-nucleotide linked antigen, at least a third line of one control antibody and antigen (non-specific binding of F and anti-E);   (d) at least two sample absorbent pads linked to the test device at different positions.       
 
         [0050]    In one embodiment, the two antibodies are directed against two different antigens selected from tuberculosis antigen, malaria antigen and pneumonia antigen. 
         [0051]    In a preferred embodiment, the tuberculosis antigen is LAM, the malaria antigen is HRP-II, and the pneumonia antigen is PLY. 
         [0052]    In one embodiment, the control antibody is anti-mouse IgG. 
         [0053]    In one embodiment, the two different antigens are selected from envelop protein gp 160 from HIV, NS3 antigen from HCV, envelop antigen from HCV, and  H. pylori  antigen, 
         [0054]    In one embodiment, the test device further comprises a nitrocellulose membrane on which the complementary oligo-nucleotides are immobilized. 
         [0055]    In one embodiment, the conjugate releasing pad is located at the internal surface of the container wall. 
         [0056]    In one embodiment, a first sample absorbent pad is located at the bottom and a second sample pad is located at the top of the container. 
         [0057]    In a preferred embodiment, the sample absorbent pad at the top is fitted into a container cap. 
         [0058]    In a particularly preferred embodiment, the sample absorbent pad at the top has a capacity of absorbing at least 15 ml of sample. 
         [0059]    In one embodiment, the conjugate releasing pad further comprises optionally modified chitosan. 
         [0060]    The object of the present invention is further solved by a use of the urinary immuno-chromatographic detection cup according to the present invention for detecting at least two different antigens selected from tuberculosis antigen, malaria antigen and pneumonia antigen in a sample of urine of a subject, preferably human. 
         [0061]    In a preferred embodiment, the tuberculosis antigen is LAM, the malaria antigen is HRP-II, and the pneumonia antigen is PLY. 
         [0062]    The object of the present invention is further solved by a use of the urinary immuno-chromatographic detection cup according to the present invention for detecting at least two different antibodies selected from HIV, HCV and  H. pylori  antibody in a sample of urine of a subject, preferably human. 
         [0063]    The object of the present invention is further solved by a use of the urinary immuno-chromatographic detection cup according to the present invention for detecting at least one antigen selected from tuberculosis antigen, malaria antigen and pneumonia antigen in a sample of urine of a subject, preferably human, and at least one antibody selected from HIV, HCV and  H. pylori  antibody 
         [0064]    In one embodiment, the volume of the urine sample is approximately 15 ml. 
         [0065]    The object of the present invention is further solved by a method for preparing the urinary fluid immuno-chromatographic detection cup according to the present invention, wherein optionally modified water-soluble chitosan is added during the preparation of gold labelled antibody or antigen prior to conjugation of colloid gold with the antibody or antigen. 
         [0066]    In one embodiment, the colloidal gold is prepared by reduction of 1% aqueous solution of tetra-chloroauric acid using tri-sodium citrate aqueous solution to produce spheroidal gold particles. 
         [0067]    Thus, the present invention provides a urinary multi-parameter rapid immuno-chromatographic detection cup using a unique system for the detection of at least one antigen and/or at least one antibody in a single assay at the same time. This is achieved by using (1) colloidal gold-labelled antibodies and oligo-nucleotide-linked antibodies, and (2) colloidal gold-labelled antigens and oligo-nucleotide-linked antigens. The oligo-nucleotide-linked anti-bodies and/or antigens are captured by complementary oligo-nucleotides. 
         [0068]    One of the advantages of the test device of the present invention is that there is no requirement for the preparation of different rapid immuno-chromatographic test devices to check more than one parameter. Another advantage is that the patient&#39;s sample is readily obtainable by non-invasive procedures, namely urine. 
         [0069]    High sensitivity of measurements carried out in urine by means of the test device of the present invention is achieved by using a large volume of urine as a sample (about 15 ml urine instead of 17 μl of serum; concentration of IgG in urine is about 1:6000; factor reduction to 1:7) and by using water-soluble chitosan (or modified water-soluble chitosan) as a colour intensity modification agent. 
     
    
     
       DETAILED DESCRIPTION OF THE INVENTION 
       Brief Description of the Figures 
         [0070]      FIG. 1  shows top and side views of a typical rapid-flow immuno-chromatographic 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 . 
           [0071]      FIG. 2   a  shows an assembly comprising a test strip  201 , an absorbent sample pad  202 , and an absorbent pad  205 . 
           [0072]      FIG. 2   b  shows an immuno-chromatographic detection cup comprising a sample-collecting container  210 , a cap  211 , and the assembly shown in  FIG. 2   a.    
           [0073]      FIG. 3   a  shows an assembly comprising a test strip  101 , an absorbent sample pad  102 , a nitrocellulose membrane  104 , a conjugate pad  103 , an adhesive  106 , a supporting backing  107 , a control zone  109 , and an absorbent pad  105 . The conjugate pad  103  contains the gold-labelled antibody  302 , oligonucleotide 1 labelled antibody  304 , gold labelled antigen  303 , oligonucleotide 2 labelled antigen  301 . Capturing complementary oligo-nucleotide 1′ and 2′ are immobilized within sample zone(s)  305  and  306 , respectively, onto the nitrocellulose membrane  104 . 
           [0074]      FIG. 3   b  shows the detection test strip of  FIG. 3   a  after the application of a sample  307  that contains a target antibody and a target antigen. 
       
    
    
     EXAMPLES 
     Example 1 
     Urinary Immuno-chromatographic Detection Cup (Multi-parameter) 
       [0075]    One embodiment of the rapid immuno-chromatographic detection system of the present invention is shown in  FIGS. 2   a, b . The immuno-chromatographic detection cup comprises a sample-collecting container  210 , actually the “cup”, a cap  211  for closing the container, and a detection test strip  201  inserted into the container  210 . The test strip  201  may be placed inside the transparent container wall, and the test result can be read on the outer surface. The test strip  201  comprises a nitrocellulose membrane  204  and is linked via an absorbent pad  202  on the test strip  201  to an absorbent sample pad  202  placed on the bottom of the container  210 . The absorbent sample pad  202  is designed to cover the inner surface of the container bottom. At the opposite end, the test strip  201  is linked via an absorbent pad  205  on the test strip to an absorbent pad  205  which is fitted into the cap  211  of the container and is designed to be thick enough to absorb more than 15 ml of sample. 
         [0076]    A gold conjugate releasing pad  203  is fixed to the internal surface of the container wall which pad  203  comprises (1a) colloidal gold-labelled antibodies and (1b) oligo-nucleotide-linked antibodies, and (2a) colloidal gold-labelled antigens and (2b) oligo-nucleotide-linked antigens. The gold conjugates will begin releasing from the pad  203  during the urine sample is streaming into the container  210 . Release into the sample will increase the possibility of interaction between the conjugates and the analytes, e.g. antibodies and/or antigens. 
         [0077]    Capturing complementary oligo-nucleotides are immobilized within one or more sample zones  208  onto the nitrocellulose membrane  204  (see also  FIG. 3   a  and  FIG. 3   b  where it is referred to as  305 ,  306 , i.e. “capturing complementary oligo-nucleotide 1 or 2 sample zones”). If the target antigen is present in the sample, the colloidal gold-labelled antibody and oligo-nucleotide-linked antibody will form a complex with the antigen. If the target antibody is present in the sample, the colloidal gold-labelled antigen and oligo-nucleotide-linked antigen will form a complex with the antibody. Subsequently, the formed complexes migrate to the fixed complementary oligo-nucleotides located at the sample zones  208  and are bound to the solid phase via nucleotide-nucleotide interaction. As a result, at least two types of reactions can be detected on a single test device by the combination of (1) colloidal gold-labelled and oligo-nucleotide-linked antibodies and capturing complementary oligo-nucleotides for detecting an antigen, and (2) colloidal gold-labelled and oligo-nucleotide-linked antigens and capturing complementary oligo-nucleotides for detecting antibodies, wherein antibodies, antigens and complementary oligo-nucleotides are immobilized on a nitrocellulose membrane. 
         [0078]    Non-specific antibody and/or antigen is immobilized as a control zone  209  onto the nitrocellulose membrane  204  for non-specific capturing of the gold conjugates (see also  FIG. 3 ). The complementary oligo-nucleotides within the sample zones  208  are for specific capturing of the specific colloidal gold-labelled antibody or antigen-linked oligo-nucleotides. 
         [0079]    The antigens used may be synthetic or recombinant. The antibodies used may be polyclonal or monoclonal. In the embodiment shown in  FIGS. 2   a, b  the sample zone  208  is realized by a sample line, and the control zone  209  is realized by a control line. Preferably, one sample zone is provided for each parameter. More than one sample zone  208  for each parameter and/or more than one control zone  209  are also contemplated. 
         [0080]    Detection of the following diseases is taken into consideration: 
       (1) Tuberculosis 
       [0000]    
       
         
           
             Target: LAM (antigen) 
             gold-labelled anti-LAM 
             oligonucleotide 1-linked anti-LAM 
             complementary oligonucleotide 1′ 
           
         
       
     
       (2) HIV Infection 
       [0000]    
       
         
           
             Target: antibody against envelop protein gp160 
             gold-labelled recombinant envelop protein gp 160 
             oligonucleotide 2-linked recombinant envelop protein gp160 
             complementary oligonucleotide 2′ 
           
         
       
     
       (3) Malaria 
       [0000]    
       
         
           
             Target: HRP-II (antigen) 
             gold-labelled anti-HRP-II 
             oligonucleotide 3-linked anti-HRP-II 
             complementary oligonucleotide 3′ 
           
         
       
     
       (4) HCV Infection 
       [0000]    
       
         
           
             Target: antibody against HCV antigen NS3 
             gold-labelled recombinant HCV NS3 antigen 
             oligonucleotide 4-linked HCV NS3 antigen 
             complementary oligonucleotide 4′ 
           
         
       
     
       (5) Pneumonia 
       [0000]    
       
         
           
             Target: pneumolysin PLY (antigen) 
             gold-labelled anti-pneumolysin (PLY) 
             oligonucleotide 5-linked anti-pneumolysin (PLY) 
             complementary oligonucleotide 5′
 
(6)  H. pylori  Infection
 
             Target: antibody against  H. pylori  antigen 
             gold-labelled  H. pylori  antigen 
             oligonucleotide 6-linked  H. pylori  antigen 
             complementary oligonucleotide 6′ 
           
         
       
     
         [0105]    The preparation of oligo-nucleotide-labelled antibody Fab′ comprises the following steps 6 : 
         [0000]    
       
                 
         
             
             
         
       
     
         [0106]    The preparation of oligo-nucleotide-labelled protein comprises the following steps 7 : 
         [0000]    
       
                 
         
             
             
         
       
     
       Example 2 
     Urinary HIV Antibody and Tuberculosis Antigen Detection Cup (Two-Parameters) 
       [0107]    The conjugate releasing pad  203  immobilized onto the internal surface of the sample-collecting container  210  comprises gold labelled anti-LAM, oligo-nucleotide 1 linked anti-LAM, gold labelled recombinant envelop protein gp160, oligo-nucleotide 2 linked recombinant envelop protein gp160. A first sample zone  208  (tuberculosis antigen=LAM specific detection zone) comprises complementary oligo-nucleotide 1′ immobilized onto the nitrocellulose membrane  204 . A second sample zone  208  (HIV antibody specific detection zone) comprises complementary oligo-nucleotide 2′ immobilized onto the nitrocellulose membrane. The control zone  209  comprises a mixture of anti-mouse IgG and goat anti-gp160. The first sample zone  208  and control zone  209  turn into purple colour in case that tuberculosis antigen (LAM) is present in the sample, and the second sample zone  208  and control zone  209  turn into purple colour in case that HIV antibody (anti-gp160) is present in the sample; only the control zone turns into purple colour in case of HIV antibody and tuberculosis antigen free sample. 
       Example 3 
     Urinary HIV Antibody and Malaria Antigen Detection Cup (Two-Parameters) 
       [0108]    The conjugate releasing pad  203  immobilized onto the internal surface of the sample-collecting container  210  comprises gold labelled anti-HRP-II, oligo-nucleotide 3 linked anti-HRP-II, gold labelled recombinant envelop protein gp160, oligo-nucleotide 2 linked recombinant envelop protein gp160. A first sample zone  208  (malaria antigen=HRP-II specific detection zone) comprises complementary oligo-nucleotide 3′ immobilized onto the nitrocellulose membrane  204 . A second sample zone  208  (HIV antibody specific detection zone) comprises complementary oligo-nucleotide 2′ immobilized onto the nitrocellulose membrane. The control zone  209  comprises a mixture of anti-mouse IgG and goat anti-gp160. The first sample zone  208  and control zone  209  turn into purple colour in case that malaria antigen (HRP-II) is present in the sample, and the second sample zone  208  and control zone  209  turn into purple colour in case that HIV antibody (anti-gp160) is present in the sample; only the control zone turns into purple colour in case of HIV antibody and malaria antigen free sample. 
       Example 4 
     Urinary HIV Antibody, Malaria Antigen and Tuberculosis Detection Cup (Three-Parameters) 
       [0109]    The conjugate releasing pad  203  immobilized onto the internal surface of the sample-collecting container  210  comprises gold labelled anti-LAM, oligo-nucleotide 1 linked anti-LAM, gold labelled anti-HRP-II, oligo-nucleotide 3 linked anti-HRP-II, gold labelled recombinant envelop protein gp160, and oligo-nucleotide 2 linked recombinant envelop protein gp160. A first sample zone  208  (tuberculosis antigen=LAM specific detection zone) comprises complementary oligo-nucleotide 5′ immobilized onto the nitrocellulose membrane  204 . A second sample zone  208  (malaria antigen=HRP-II specific detection zone) comprises complementary oligo-nucleotide 3′ immobilized onto the nitrocellulose membrane. A third sample zone  208  (HIV antibody specific detection zone) comprises complementary oligo-nucleotide 2′ immobilized onto the nitrocellulose membrane. The control zone  209  comprises a mixture of anti-mouse IgG and goat anti-gp160. The first sample zone  208  and control zone  209  turn into purple colour in case that tuberculosis antigen (LAM) is present in the sample, the second sample zone  208  and control zone  209  turn into purple colour in case that malaria antigen (HRP-II) is present in the sample, and the third sample zone  208  and control zone  209  turn into purple colour in case that HIV antibody (anti-gp160) is present in the sample; only the control zone turns into purple colour in case of HIV antibody, tuberculosis antigen and malaria antigen free sample. 
       Example 5 
     Urinary HCV Antibody and Malaria Antigen Detection Cup (Two-Parameters) 
       [0110]    The conjugate releasing pad  203  immobilized onto the internal surface of the sample-collecting container  210  comprises gold labelled anti-HRP-II, oligo-nucleotide 3 linked anti-HRP-II, gold labelled recombinant HCV NS3 antigen, oligo-nucleotide 4 linked HCV NS3 antigen. A first sample zone  208  (malaria antigen=HPR-II specific detection zone) comprises complementary oligo-nucleotide 3′ immobilized onto the nitrocellulose membrane  204 . A second sample zone  208  (HCV NS3 antibody specific detection zone) comprises complementary oligo-nucleotide 4′ immobilized onto the nitrocellulose membrane. The control zone  209  comprises a mixture of anti-mouse IgG and goat anti-HCV NS3 antigen. The first sample zone  208  and control zone  209  turn into purple colour in case that malaria antigen (HRP-II) is present in the sample, and the second sample zone  208  and control zone  209  turn into purple colour in case that HCV antibody (anti-NS3) is present in the sample; only the control zone turns into purple colour in case of HCV NS3 antibody and malaria antigen free sample. 
       Example 6 
     Urinary HCV Antibody and Tuberculosis Antigen Detection Cup (Two-Parameters) 
       [0111]    The conjugate releasing pad  203  immobilized onto the internal surface of the sample-collecting container  210  comprises gold labelled anti-LAM, oligo-nucleotide 1 linked anti-LAM, gold labelled recombinant HCV NS3 antigen, oligo-nucleotide 4 linked HCV NS3 antigen. A first sample zone  208  (tuberculosis antigen=LAM specific detection zone) comprises complementary oligo-nucleotide 1′ immobilized onto the nitrocellulose membrane  204 . A second sample zone  208  (HCV NS3 antibody specific detection zone) comprises complementary oligo-nucleotide 4′ immobilized onto the nitrocellulose membrane. The control zone  209  comprises a mixture of anti-mouse IgG and goat anti-HCV NS3 antigen. The first sample zone  208  and control zone  209  turn into purple colour in case that tuberculosis antigen (LAM) is present in the sample, and the second sample zone  208  and control zone  209  turn into purple colour in case that HCV NS3 antibody is present in the sample; only the control zone turns into purple colour in case of HCV NS3 antibody and tuberculosis antigen free sample. 
       Example 7 
     Urinary HIV Antibody, HCV Antibody and Malaria Antigen Detection Cup (Three-Parameters) 
       [0112]    The conjugate releasing pad  203  immobilized onto the internal surface of the sample-collecting container  210  comprises gold labelled anti-HRP-II, oligo-nucleotide 3 linked anti-HRP-II, gold labelled recombinant HCV NS3 antigen, oligo-nucleotide 4 linked HCV NS3 antigen, gold labelled recombinant envelop protein gp160, and oligo-nucleotide 2 linked recombinant envelop protein gp160. A first sample zone  208  (malaria antigen=HRP-II specific detection zone) comprises complementary oligo-nucleotide 3′ immobilized onto the nitrocellulose membrane  204 . A second sample zone  208  (HCV NS3 antibody specific detection zone) comprises complementary oligo-nucleotide 4′ immobilized onto the nitrocellulose membrane. A third sample zone  208  (HIV gp160 antibody specific detection zone) comprises complementary oligo-nucleotide 2′ immobilized onto the nitrocellulose membrane. The control zone  209  comprises a mixture of anti-mouse IgG, goat anti-HCV NS3 antigen and goat anti-gp160. The first sample zone  208  and control zone  209  turn into purple colour in case that malaria antigen (HPR-II) is present in the sample, the second sample zone  208  and control zone  209  turn into purple colour in case that HCV antibody (anti-NS3) is present in the sample, and the third sample zone  208  and control zone  209  turn into purple colour in case that HIV antibody (anti-gp160) is present in the sample; only the control zone turns into purple colour in case of HCV NS3 antibody, HIV gp160 antibody and malaria antigen (HRP-II) free sample. 
       Example 8 
     Urinary HIV Antibody, HCV Antibody and Tuberculosis Antigen Detection Cup (Three-Parameters) 
       [0113]    The conjugate releasing pad  203  immobilized onto the internal surface of the sample-collecting container  210  comprises gold labelled anti-LAM, oligo-nucleotide 1 linked anti-LAM, gold labelled recombinant HCV NS3 antigen, oligo-nucleotide 4 linked HCV NS3 antigen gold labelled recombinant envelop protein gp160, and oligo-nucleotide 2 linked recombinant envelop protein gp160. A first sample zone  208  (tuberculosis antigen=LAM specific detection zone) comprises complementary oligo-nucleotide 1′ immobilized onto the nitrocellulose membrane  204 . A second sample zone  208  (HCV NS3 antibody specific detection zone) comprises complementary oligo-nucleotide 4′ immobilized onto the nitrocellulose membrane. A third sample zone  208  (HIV gp160 antibody specific detection zone) comprises complementary oligo-nucleotide 2′ immobilized onto the nitrocellulose membrane. The control zone  209  comprises a mixture of anti-mouse IgG, goat anti-HCV NS3 antigen and goat anti-gp160. The first sample zone  208  and control zone  209  turn into purple colour in case that tuberculosis antigen (LAM) is present in the sample, the second sample zone  208  and control zone  209  turn into purple colour in case that HCV antibody (anti-NS3) is present in the sample, and the third sample zone  208  and control zone  209  turn into purple colour in case that HIV antibody (anti-gp160) is present in the sample; only the control zone turns into purple colour in case of HCV antibody (anti-NS3), HIV antibody (anti-gp160) and tuberculosis antigen (LAM) free sample. 
       Example 9 
     Urinary HIV Antibody, HCV Antibody, Malaria Antigen and Tuberculosis Antigen Detection Cup (Four-Parameters) 
       [0114]    The conjugate releasing pad  203  immobilized onto the internal surface of the sample-collecting container  210  comprises gold labelled anti-LAM, oligo-nucleotide 1 linked anti-LAM, gold labelled anti-HRP-II, oligo-nucleotide 3 linked anti-HRP-II, gold labelled recombinant HCV NS3 antigen, oligo-nucleotide 4 linked HCV NS3 antigen, gold labelled recombinant envelop protein gp160, and oligo-nucleotide 2 linked recombinant envelop protein gp160. A first sample zone  208  (tuberculosis antigen=LAM specific detection zone) comprises complementary oligo-nucleotide 1′ immobilized onto the nitrocellulose membrane  204 . A second sample zone  208  (malaria antigen=HRP-II specific detection zone) comprises complementary oligo-nucleotide 3′ immobilized onto the nitrocellulose membrane  204 . A third sample zone  208  (HCV NS3 antibody specific detection zone) comprises complementary oligo-nucleotide 4′ immobilized onto the nitrocellulose membrane. A fourth sample zone  208  (HIV gp160 antibody specific detection zone) comprises complementary oligo-nucleotide 2′ immobilized onto the nitrocellulose membrane. The control zone  209  comprises a mixture of anti-mouse IgG, goat anti-HCV NS3 antigen and goat anti-gp160. The first sample zone  208  and control zone  209  turn into purple colour in case that tuberculosis antigen (LAM) is present in the sample, the second sample zone  208  and control zone  209  turn into purple colour in case that malaria antigen (HRP-II) is present in the sample, the third sample zone  208  and control zone  209  turn into purple colour in case that HCV antibody (anti-NS3) is present in the sample, and the fourth sample zone  208  and control zone  209  turn into purple colour in case that HIV antibody (anti-gp160) is present in the sample; only the control zone turns into purple colour in case of HCV antibody, HIV antibody, malaria antigen and tuberculosis antigen free sample. 
       Example 10 
     Urinary Tuberculosis Antigen, Pneumonia Antigen and  H. pylori  Antibody Detection Cup (Three-Parameters) 
       [0115]    The conjugate releasing pad  203  immobilized onto the internal surface of the sample-collecting container  210  comprises gold labelled anti-LAM, oligo-nucleotide 1 linked anti-LAM, gold labelled anti-pneumolysin (PLY), oligo-nucleotide 5 linked anti-pneumolysin (PLY), and gold labelled  H. pylori  antigen, oligo-nucleotide 6 linked  H. pylori  antigen. A first sample zone  208  (tuberculosis antigen=LAM specific detection zone) comprises complementary oligo-nucleotide 1′ immobilized onto the nitrocellulose membrane  204 . A second sample zone  208  (pneumonia antigen=PLY specific detection zone) comprises complementary oligo-nucleotide 5′ immobilized onto the nitrocellulose membrane. A third sample zone  208  ( H. pylori  antibody specific detection zone) comprises complementary oligonucleotide 6′ immobilized onto the nitrocellulose membrane. The control zone  209  comprises a mixture of anti-mouse IgG, and goat anti- H. pylori  antigen. The first sample zone  208  and control zone  209  turn into purple colour in case that tuberculosis antigen (LAM) is present in the sample, the second sample zone  208  and control zone  209  turn into purple colour in case that Pneumonia antigen (PLY) is present in the sample, and the third sample zone  208  and control zone  209  turn into purple colour in case that  H. pylori  antibody is present in the sample; only the control zone turns into purple colour in case of  H. pylori  antibody, tuberculosis antigen and Pneumonia antigen free sample. 
       Example 11 
     Amplification of Gold Conjugate Colour Intensity 
       [0116]    The colloidal gold conjugate colour intensity was amplified using water-soluble chitosan (or modified water-soluble chitosan) as a colour intensity modification agent. The action of water-soluble chitosan (or modified water-soluble chitosan) in connection with the antibodies or antigens is on the colour intensity of the colloidal gold. Chitosan is added during the preparation of colloidal gold, but prior to the conjugation of colloidal gold with the antibodies or the antigens. Water-soluble chitosan (or modified water-soluble chitosan) affect the colour intensity of colloidal gold and so increases the ability of the human eye to identify the colour, and, thus, enables to detect very low concentrations of the analyte. Signal amplification lies in the range of up to 10 folds. Colloidal gold could be prepared by the reduction of 1% aqueous solution of tetra-chloroauric acid (HAuCl 4 ) using tri-sodium citrate aqueous solution to produce spheroidal gold particles. After colloidal gold preparation, water-soluble chitosan (or modified water-soluble chitosan) aqueous solution was added with a suitable volume and concentration to convert colour from purple to violet pending on the volume and concentration of the added modification solution. 
       REFERENCES 
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         (3) J Chandler, N Robinson, and K Whiting, “Handling False Signals in Gold-Based Rapid Tests”, IVD Technology 7, no. 2 (2001): 34-45; http://www.devicelink.com/ivdt/archive/01/03/002.html. 
         (4) T C Tisone et al., “ Image Analysis for Rapid - Flow Diagnostics” , IVD Technology 5, no. 5 (1999): 52-58; http://www.devicelink.com/ivdt/archive/99/09/010.html. 
         (5) J Chandler, T Gurmin, and N Robinson, “The Place of Gold in Rapid Tests”, IVD Technology 6, no. 2 (2000): 37-49; http://www.devicelink.com/ivdt/archive/00/03/004.html 
         (6) Oku, Y., Uesaka, Y., Hirayama, T., Takeda, Y., 1988. Development of a highly sensitive Bead-ELISA to detect bacterial protein toxins. Microbiol. Immunol. 32, 807. 
         (7) Duncan, R. J. S., Weston, P. D., Wrigglesworth, R., 1983. A new reagent which may be used to introduce sulfhydryl groups into proteins, and its use in the preparation of conjugates for immunoassay. Anal. Biochem. 132, 68.