Patent Publication Number: US-2010120017-A1

Title: Rapid immune chromatographic detection by amplification of the colloidal gold signal

Description:
The present invention relates in general to the field of diagnostics, namely to device for the detection of at least one target in a sample, preferably in urine. More precisely, the present invention relates to a high sensitive rapid immunochromatographic test device preferably providing a unique system for the detection of more than one target in a single assay at the same time. The present invention further refers to a method for the production of the test device, to the uses of the test device for the detection of more than one disease infection such as Human Immunodeficiency virus (HIV) and Tuberculosis Lipoarabinomannan (LAM) at the same time in urine, as well as to a kit comprising the test device. 
     BACKGROUND OF THE INVENTION 
     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 ( FIG. 1   a ). 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 zones) 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 . 
     Rapid immunochromatographic test devices for diagnostic purposes are easy to operate and thus do not only contribute to the comfort of professional users, e.g. medical stuff, but also allow the operation by non-professionals users, e.g. most patients. 
     Using rapid immunochromatographic tests, a physician can instantly not only examine a sample by himself, but also diagnose a patient on the basis of the obtained results. The main disadvantage of the one rapid immunochromatographic test is that it only detects one parameter at a time. Thus, many different rapid immunochromatographic test devices are needed to check the suspected parameters, resulting in the requirement of enormous sample volume obtained from a patient as well as cumbersome test procedures. 
     In addition, the use of urine samples may be attractive due to the ease of sample collection, possible cost savings, better safety (against needle-stick injuries) and higher compliance rates. Assays for this type of specimens might be a useful alternative when it is for instance difficult or impossible to perform HIV-tests in blood samples. Further, it might be possible that blood cannot be drawn for religious reasons or that difficulties may be experienced in collecting blood samples. Therefore, urinary samples are the more safety type of specimens in most of infection cases. 
     Thus, there is a need to develop a multi-parameter rapid immunochromatographic detection test device suitable to detect more than two types of antigens and/or antibodies in a single assay at the same time. Further there is a need in the prior art to develop a multi-parameter rapid immunochromatographic detection test device sensitive enough to detect more than two types of antigens and/or antibodies in a sample such as urine. 
     Thus, it is an object of the present invention to provide a rapid immunochromatographic detection test overcoming the drawbacks of the prior art, being preferably suitable for ultra sensitive detection of at least one antibody and at least one antigen, preferably in urine at the same time. 
     SUMMARY OF THE INVENTION 
     In one embodiment the present invention concerns a rapid immunochromatographic test device for the detection of at least one target in a sample, comprising
         a) a first gold conjugate releasing pad, comprising at least two colloidal gold conjugates I and II, wherein gold I is conjugated with a first antibody and at least one oligonucleotide, and wherein gold II is conjugated with a first antigen or second antibody and at least one oligonucleotide, wherein the oligonucleotide of gold II differs from the oligonucleotide of gold I, and   b) a second gold conjugate releasing pad, comprising at least two colloidal gold conjugates III and IV, wherein gold III is conjugated with a third antibody and at least one oligonucleotide, wherein the oligonucleotide of gold III is complementary to the oligonucleotide of gold I, and wherein gold IV is conjugated with a second antigen or fourth antibody and at least one oligonucleotide, wherein the oligonucleotide of gold IV is complementary to the oligonucleotide of gold II;
           wherein both releasing pads are located at different positions within the test device.   
               

     In another embodiment the present invention relates to a rapid immunochromatographic test device for the detection of at least one target in a sample, comprising
         a) a first gold conjugate releasing pad, comprising at least two colloidal gold conjugates I and II, wherein gold I is conjugated with a first antibody and at least one further antibody or antigen (A- 1 ), and wherein gold II is conjugated with a first antigen or second antibody and at least one further antibody or antigen (A- 2 ), wherein said antibodies or antigens (A- 1 ) and (A- 2 ) differ from each other and differ from the first and second antigens and antibodies, and   b) a second gold conjugate releasing pad, comprising at least two colloidal gold conjugates III and IV, wherein gold III is conjugated with a third antibody and at least one further antibody or antigen (A- 3 ), wherein said antibody or antigen (A- 3 ) is complementary to said antibody or antigen (A- 1 ), and wherein gold IV is conjugated with a second antigen or fourth antibody and at least one further antibody or antigen (A- 4 ), wherein said antibody or antigen (A- 4 ) is complementary to said antibody or antigen (A- 2 ),
           wherein both releasing pads are located at different positions within the test device.   
               

     In a further embodiment the present invention concerns a method for the production of a device according to the present invention comprising the steps of
         a) preparing a colloidal gold solution;   b) preparing a conjugation buffer;   c) partitioning the conjugation buffer by dividing it into a first, second, third and fourth flask;   d) adding an antibody according to the present invention to the conjugation buffer in the first flask;   e) adding an antigen according to the present invention to the conjugation buffer in the second flask;   f) adding an antibody according to the present invention to the conjugation buffer in the third flask, wherein said antibody differs from the antibody used in step d);   g) adding an antigen according to the present invention to the conjugation buffer in the fourth flask, wherein said antigen differs from the antigen used in step f);   h) adding colloidal gold solution into each flask;   i) adding stabilizing buffer to each flask;   j) concentrating each conjugate;   k) mixing conjugates of the first and second flask; adding a surfactant and soaking a glass fibre sheet conjugate pad into the conjugate;   l) mixing conjugates of the third and fourth flask; soaking another glass fibre sheet conjugate pad into the conjugate;   m) printing sample and control lines onto the membrane;   n) laminating cards; and   o) cutting cards into strips.       

     In another embodiment the present invention relates to the use of a device according to the present invention for the detection of at least two diseases in at least one sample. 
     In a further embodiment the present invention refers to a kit for detection of a disease comprising the device according to the present invention and a manual 
     DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION 
     Before the present invention is described in more detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. 
     Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. 
     Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer&#39;s specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. 
     As outlined above there is a need in the prior art to provide a new test device suitable for the detection of at least one antibody and one, preferably in urine at the same time. 
     In a first aspect the present invention provides a rapid immunochromatographic test device for the detection of at least one target in a sample, comprising
         a) a first gold conjugate releasing pad, comprising at least two colloidal gold conjugates I and II, wherein gold I is conjugated with a first antibody and at least one oligonucleotide, and wherein gold II is conjugated with a first antigen or second antibody and at least one oligonucleotide, wherein the oligonucleotide of gold II differs from the oligonucleotide of gold I, and   b) a second gold conjugate releasing pad, comprising at least two colloidal gold conjugates III and IV, wherein gold III is conjugated with a third antibody and at least one oligonucleotide, wherein the oligonucleotide of gold III is complementary to the oligonucleotide of gold I, and wherein gold IV is conjugated with a second antigen or fourth antibody and at least one oligonucleotide, wherein the oligonucleotide of gold IV is complementary to the oligonucleotide of gold II;
           wherein both releasing pads are located at different positions within the test device.   
               

     The first and second colloidal (gold I and II) will capture the antigen/antibody in the sample and form the complexes “target-gold conjugate I” and “target-gold conjugate II”. Preferably the targets in the sample, preferably urine, are an antigen and/or antibody. 
     In one preferred embodiment of the device according to the present invention the first antibody of gold I will capture the target antigen from a first site (let us consider this site as site A), the first antigen of gold II will be captured by the target antibody, the third antibody of gold III will capture the target antigen from a second site (let us consider this site as site B), and the second antigen of gold IV will be captured by the same target antibody that capture the first antigen as mentioned above. 
     In another preferred embodiment of the device according to the present invention each gold conjugate comprises between 1 and 6 different oligonucleotides. Preferably each gold conjugate comprises between 2 and 4 different oligonucleotides. These oligonucleotides usually have a length of about 15 to 25 nucleotides, preferably of about 20 nucleotides. Further these oligonucleotides have an amino group at the 5′ terminus, preferably conjugated with bovine serum albumin. The bonds formed between the gold and oligonucleotides are the same bonds as those usually formed between gold and antibodies/antigens, and which are known to a person skilled in the art. Preferably such bonds are driven by hydrophobic, hydrophilic and dative binding forces. 
     In one preferred embodiment of the device according to the present invention the first gold conjugate releasing pad contains the gold conjugates I and II. Gold conjugate I is specific for an antigen, preferably the antigen is a target in a sample. Therefore gold I is conjugated with “clone one”. Please note, that the numbering of clones are only for explanation and to recognize that always two different clones of monoclonal antibodies were used. Preferably, the two monoclonal antibodies capture the target antigen from two different sites, so they were called as a pair of monoclonal antibodies. In addition, “clone one” is specific for one site (A) of the antigen. At the same time gold 1 is conjugated with some oligonucleotides (O- 1 ). Gold conjugate II is specific for an antibody, preferably the antibody is a target in a sample, and is conjugated with an antigen or a specific antibody for the antigen-specific antibody and is at the same time conjugated with some oligonucleotides (O- 2 ) which are different from the oligonucleotides (O- 1 ). 
     In a further preferred embodiment the second conjugate releasing pad contains the two gold conjugates III and IV. Gold conjugate III is specific for an antigen, preferably the antigen is a target in a sample, and therefore is conjugated with “clone two”, whereas “clone two” is specific for the second site (B) of the antigen. At the same time gold III is conjugated with the complementary oligonucleotides (O- 3 ) of that in the related conjugate I in the first conjugate pad. Gold conjugate IV is specific for an antibody, preferably the antibody is a target in a sample, and conjugated with the antigen or specific antibody for the antigen-specific antibody and at the same time conjugated with the complementary oligonucleotides (O- 3 ) of that in the related conjugate II in the first conjugate pad ( FIG. 2   a  and  FIG. 2   b ). All used oligonucleotides should be tested for nonspecific interaction theoretically and experimentally. 
     In one preferred embodiment of the device according to the present invention the rapid immunochromatographic test device for the detection of at least one target in a sample, comprises
         c) a first gold conjugate releasing pad, comprising at least two colloidal gold conjugates I and II, wherein gold I is conjugated with a first antibody  205  and two different oligonucleotides  209 ,  210 , and wherein gold II is conjugated with a first antigen  206  and two different oligonucleotides  211 ,  212 , wherein the oligonucleotides  211 ,  212  of gold II differs from the oligonucleotides  209 ,  210  of gold I, and   d) a second gold conjugate releasing pad, comprising at least two colloidal gold conjugates III and IV, wherein gold III is conjugated with a third antibody  208  and two different oligonucleotides  209 ′,  210 ′, wherein the oligonucleotides  209 ′,  210 ′ of gold III are complementary to the oligonucleotides  209 ,  210  of gold I, and wherein gold IV is conjugated with a second antigen  207  and two oligonucleotides  211 ′,  212 ′, wherein the oligonucleotides  211 ′,  212 ′ of gold IV are complementary to the oligonucleotides  211 ,  212  of gold II;
           wherein both releasing pads are located at different positions within the test device ( FIG. 2   a ).   
               

     In one preferred embodiment of the device according to the present invention the first antibody  205  of gold I will capture the target antigen from a first site  205 ′, the a first antigen  206  of gold II will be captured by the target antibody  206 ′, the third antibody  208  of gold III will capture the target antigen from a second site  208 ′, and the second antigen  207  of gold IV will be captured by the same target antibody  207 ′ that capture the first antigen as mentioned above, ( FIGS. 2   a  and  2   b ). 
     In a second aspect the present invention provides a rapid immunochromatographic test device for the detection of at least one target in a sample, comprising
         a) a first gold conjugate releasing pad, comprising at least two colloidal gold conjugates I and II, wherein gold I is conjugated with a first antibody and at least one further antibody or antigen (A- 1 ), and wherein gold II is conjugated with a first: antigen or second antibody and at least one further antibody or antigen (A- 2 ), wherein said antibodies or antigens (A- 1 ) and (A- 2 ) differ from each other and differ from the first and second antigens and antibodies, and   b) a second gold conjugate releasing pad, comprising at least two colloidal gold conjugates III and IV, wherein gold III is conjugated with a third antibody and at least one further antibody or antigen (A- 3 ), wherein said antibody or antigen (A- 3 ) is complementary to said antibody or antigen (A- 1 ), and wherein gold IV is conjugated with a second antigen or fourth antibody and at least one further antibody or antigen (A- 4 ), wherein said antibody or antigen (A- 4 ) is complementary to said antibody or antigen (A- 2 ),
           wherein both releasing pads are located at different positions within the test device.   
               

     Preferably, the antibodies or antigens (A- 1  to A- 4 ) differs from said first antibody or antigen. The first antibody or antigen captures the target from the sample, whereas the other antibodies (A- 1  to A- 4 ) only functions to amplify the signal by recognizing the specifically-related antibodies or antigens (A- 1  to A- 4 ). In addition, the antibodies or antigens (A- 1  and A- 2 ) conjugated with gold conjugates I and II are specifically-related to the antibodies or antigens (A- 3  and A- 4 ) of the gold conjugates III and IV, respectively. 
     In one preferred embodiment of the device according to the present invention the device is suitable to detect the targets in urine. 
     In one embodiment of the device according to the present invention the device comprises a test strip comprising
         a) a sample pad,   b) a conjugate pad comprising the first gold conjugate pad,   c) a conjugate pad comprising the second gold conjugate pad,   d) a membrane comprising two capture test zones and a negative control zone, and   e) an absorbent pad.       

     In a preferred embodiment of the device according to the present invention one capture test zone suitable to detect an antigen in a sample comprises a third antibody specific for one site of the antigen, and the other capture test zone suitable to detect an antibody in a sample comprises a second antigen or a fourth antibody. 
     In another preferred embodiment the antibodies or antigens within the test zones are immobilized. 
     The complexes “target-gold conjugate I” and “target-gold conjugate II will be captured by the specific antibodies or antigens within the test zones and therefore be kept within these test zones to form the sandwich detection ( FIG. 4 ). Then, the second gold conjugate releasing pad will release its gold conjugated with the other specific antibodies or antigens to capture the target analyte from the second site (let us consider it as site B) and at the same time with the complementary oligonucleotides that are conjugated with the colloidal gold conjugate. The last mentioned conjugate would bind with the first conjugate from different sites, this binding could be happened between any of the conjugated oligonucleotides with its complementary oligonucleotide on the other gold conjugate or between any free site B of the analyte with its specific antibody or antigen on the conjugate ( FIG. 5 ). Nevertheless, the other oligonucleotides will be able to link with their complementary oligonucleotides beside the probability of capturing the first conjugate that will capture the second conjugate to form more and more branched bonds that propagate the accumulation of colloidal gold particles onto the capturing/sample line ( FIG. 5 ). 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. 
     In one embodiment of the device according to the present invention the membrane is attached by means of an adhesive to a supporting backing. Preferably an acrylic pressure sensitive adhesive as known in the art is used. 
     In another embodiment of the device according to the present invention the first and second gold conjugate pad are laminated between the sample pad and the membrane, wherein the two gold conjugates are separated by a divider. 
     In a preferred embodiment of the device according to the present invention the first  103 . 1  and second gold conjugate pad  103 . 2  are laminated between the sample pad  102  and the membrane  104 , wherein the two gold conjugates are separated by a divider  110  ( FIG. 1   b ). Preferably, the divider is an inert divider, more preferably the divider is a plastic divider 
     In another embodiment, the device according to the present invention the first gold conjugate pad is attached between the sample pad and the membrane while the second gold conjugate pad is within the upper part of the plastic housing to be released after sample application onto the nitrocellulose membrane directly. 
     In one preferred embodiment of the device according to the present invention the supporting backing is a plastic backing. 
     In another preferred embodiment of the device according to the present invention the membrane is nitrocellulose membrane. 
     In one embodiment of the device according to the present invention the first or second antibody is selected from the group comprising mouse anti-HIV p24, mouse anti-HBsAg, anti-hlgG, anti-Lipoarabinomannan, anti- H. Pylori  antigen, anti- Leishmania  antigen, anti-Pneumonia antigen, anti-Malaria antigen, anti-Chlamydia antigen, anti- Toxoplasma  antigen, anti- Schistosoma  antigen, HIV 1 antibody, and HIV 2 antibody. 
     In a preferred embodiment of the device according to the present the first or second antibody is a monoclonal or polyclonal antibody, preferably a monoclonal antibody. 
     In another embodiment of the device according to the present invention the first antigen is selected from the group comprising conjugate of HIV antigen, conjugate of hepatitis C antigen, HIV 1 antigen, HIV 2 antigen, Lipoarabinomannan,  H. Pylori  antigen,  Toxoplasma  antigen. 
     In a further embodiment of the device according to the present invention the control zone comprises a non-specific capturing antibody and/or a non-specific antibody capturing protein. 
     In one preferred embodiment of the device according to the present invention the non-specific antibody is selected from the group consisting of anti-mouse IgG, anti-rabbit IgG, anti-goat IgG, anti-donkey IgG, Anti-sheep IgG, anti-HIV p24, anti-Lipoarabinomannan, anti- H. Pylori  antigen, anti- Leishmania  antigen, anti-Pneumonia antigen, anti-Malaria antigen, anti-Chlamydia antigen, anti- Toxoplasma  antigen, anti- Schistosoma  antigen, HIV 1 antibody, and HIV 2 antibody. 
     In another preferred embodiment of the device according to the present invention the non-specific capturing protein is either Protein A or Protein G. 
     In a preferred embodiment of the device according to the present invention the device comprises a housing comprising at least one test strip according to the present invention. 
     In another preferred embodiment of the device according to the present invention the housing comprises two, three, four, five, six, seven, eight, nine, or ten test strips. Preferably the housing comprises two, three, four, or five test strips, more preferably the housing comprises two or three test strips. 
     In one preferred embodiment of the device according to the present invention each test strip comprises at least four antibodies or antigens, wherein two of these antibodies or antigens are immobilized onto the membrane and at the same time are conjugated with the two colloidal gold conjugates and wherein the other two antibodies or antigens are conjugated with the other two gold conjugates. 
     In another aspect the present invention concerns a method for the production of a device according to the present invention, comprising the steps of
         a) preparing a colloidal gold solution;   b) preparing a conjugation buffer;   c) partitioning the conjugation buffer by dividing it into a first, second, third and fourth flask;   d) adding an antibody according to the present invention to the conjugation buffer in the first flask;   e) adding an antigen according to the present invention to the conjugation buffer in the second flask;   f) adding an antibody according to the present invention to the conjugation buffer in the third flask, wherein said antibody differs from the antibody used in step d);   g) adding an antigen according to the present invention to the conjugation buffer in the fourth flask, wherein said antigen differs from the antigen used in step f);   h) adding colloidal gold solution into each flask;   i) adding stabilizing buffer to each flask;   j) concentrating each conjugate;   k) mixing conjugates of the first and second flask; adding a surfactant and soaking a glass fibre sheet conjugate pad into the conjugate;   l) mixing conjugates of the third and fourth flask; soaking another glass fibre sheet conjugate pad into the conjugate;   m) printing sample and control lines onto the membrane;   n) laminating cards; and   o) cutting cards into strips.       

     In a preferred embodiment of the method according to the present invention the method further comprises the steps of
         a) preparing and adding oligonucleotides labelled BSA aqueous solution to the first flask;   b) preparing and adding oligonucleotides labelled BSA aqueous solution to the second flask, wherein said oligonucleotides differ from the oligonucleotides used in step a);   c) preparing and adding oligonucleotides labelled BSA aqueous solution to the third flask, wherein said oligonucleotides are complementary to the oligonucleotides used in step a);   d) preparing and adding oligonucleotides labelled BSA aqueous solution to the fourth flask, wherein said oligonucleotides are complementary to the oligonucleotides used in step b); and       

     wherein the solutions are added before step h) according to the method of the present invention. 
     In another preferred embodiment of the method according to the present invention the method further comprises the steps of
         a) preparing and adding aqueous solution comprising antibodies or antigens to the first flask, wherein said antibodies or antigens are different from the antibodies or antigens used in step d), e), g) and f) of the method of the present invention;   b) preparing and adding aqueous solution comprising antibodies or antigens to the second flask, wherein said antibodies or antigens are different from the antibodies or antigens used in step d), e), g) and f) of the method of the present invention and of the antibodies used in step a);   c) preparing and adding aqueous solution comprising antibodies or antigens to the third flask, wherein said antibodies or antigens are complementary to the antibodies or antigens used in step a);   d) preparing and adding aqueous solution comprising antibodies or antigens to the fourth flask, wherein said antibodies or antigens are complementary to the antibodies or antigens used in step b); and   wherein the solutions are added before step h) according to the method of the present invention.       

     In another aspect the present invention relates to the use of a device according to the present invention for the detection of at least one disease in at least one sample. 
     In one preferred embodiment of the use of the device according to the present invention preferably two diseases are detected at the same time in the same sample. 
     In one preferred embodiment of the use according to the present invention the antibody in one sample (e.g. specimen) and the antigen in another sample (e.g. specimen) is detected. For example, in the case two test according to the present invention are used, Lipoarabinomannan-antigen can be detected in urine, while anti-lipoarabinomannan is detected in serum ( FIGS. 6   a  and  b ). 
     In another preferred embodiment of the use according to the present invention the antibody and antigen are detected in the same sample (specimen). For example, HIV antibodies and the HIV p24 antigen are detected in the same serum sample (specimen) using a device of two different strips ( FIGS. 7   a  and  b ). 
     In another embodiment of the use of the device according to the present invention the sample is obtained from a human. 
     In one preferred embodiment of the use of the device according to the present invention the sample is selected from the group comprising of whole blood, serum, plasma, saliva, and urine. Preferably the sample is urine. 
     In another preferred embodiment of the use of the device according to the present invention the disease detected in said sample is selected from the group consisting of HIV, Hepatitis A, Hepatitis B, Hepatitis C,  H. Pylori, Leishmania,  Schistosomiasis, Malaria, Pneumonia, Toxoplasmosis, Tubercolosis and Chlamydial infection. 
     In a further aspect the present invention refers to a kit for detection of a disease comprising the device according to the present invention and a manual. 
     In one preferred embodiment of the kit according to the present invention the kit further comprises an assay buffer. The assay buffer can be any buffer known in the art suitable for the use of whole blood samples. Preferably in the case of whole blood samples Tris buffer is used, more preferably 0.1M Tris buffer having a pH of 7.5 and comprising a preservative. Any preservative known by a person skilled in the art can be used, preferably sodium azide and even more preferably 0.01M sodium azide is used. 
     The following example illustrate the present invention without, however, limiting the same thereto. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWING 
         FIG. 1   a:  shows top and side views of a typical rapid-flow immunochromatographic test device known in the art in the form of a test strip  101  comprising a sample pad  102 , a conjugate pad  103 , a membrane  104 , an absorbent pad  105 , an adhesive  106 , a supporting backing  107 , a test zone  108 , and a control zone  109 . 
         FIG. 1   b:  shows top and side views of a preferred embodiment of a rapid-flow immunochromatographic test device according to the present invention in the form of a test strip  101  comprising a sample pad  102 , a first conjugate pad  103 . 1 , a second conjugate pad  103 . 2 , a membrane  104 , an absorbent pad  105 , an adhesive  106 , a supporting backing  107 , two test zones  108   a  and  108   b,  a control zone  109 , and the conjugates divider  110 . 
         FIG. 2   a : shows the schematically view of a preferred embodiment of the colloidal gold conjugates I to IV according to the present invention, wherein gold I is conjugated with an specific antibody  205  suitable to detect the target from one side (A) and two different oligonucleotides  209  and  210 , gold II is conjugated with a specific antigen  206  and two different oligonucleotides  211  and  212 , gold III is conjugated with another specific antibody  208  suitable to detect the antigen from the other side (B) and two different oligonucleotides  209 ′ and  210 ′, and gold IV is conjugated with another antigen  207  and two different oligonucleotides. The oligonucleotides of gold I and II are complementary to the oligonucleotides of gold III and IV, respectively. 
         FIG. 2   b : shows the schematically view of the sample  213  that contains the two targets; two distinguish sites of the antibody  206 ′ and  207 ′ and that of the antigen&#39;s  205 ′ and  208 ′. 
         FIG. 3 : shows a simplified scheme of a preferred embodiment of the test device according to the present invention. It shows the enlarged top view of the two test zones  108   a  and  108   b  of a test strip, whereas the second specific antibody  208  and the second specific antigen  207  are immobilized on the membrane  104 . 
         FIG. 4 : shows the main principle of a preferred embodiment of the signal development according to the present invention. By the sample flow within the rapid immunochromatographic test the targets in the sample will be captured by the antibody or antigen of the colloidal gold I or II to form the complexes “target-gold conjugate I” and “target-gold conjugate II”. These complexes flow to the two test zones  108   a  and  108   b,  where they will be captured by a specific antibody or antigen that is immobilized onto the membrane  104  of both test zones to form a sandwich detection. 
         FIG. 5 : shows the main principle of a preferred embodiment of the signal amplification and multiplication according to the present invention. After the complexes “target-gold conjugate I” and “target-gold conjugate II” were captured within the two test zones  108   a  and  108   b,  the second gold conjugate releasing pad will release the gold conjugates III and IV with the second specific antibody or antigen to capture the target analyte from the second site (let us consider it as site B) and at the same time with the complementary oligonucleotides (O- 3  and O- 4 ) that are conjugated with the first colloidal gold conjugate. The last mentioned conjugate would bind with the first conjugate from different sites, this binding could be happened between any of the conjugated oligonucleotides with its complementary oligonucleotide on the other gold conjugate or between any free site B of the analyte with its specific antibody or antigen on the conjugate. 
         FIG. 6   a : shows an internal view of a preferred embodiment of a test device according to the present invention suitable to detect two different targets within the same sample (specimen) from the same person (patient). In this preferred embodiment the device comprises two test strips  101 . a  and  101 . b,  wherein both test strips share the same sample pad  102 , the same humidity indicator  110  and the same absorbent pad  105 . Further each test strip  101 . a  and  101 . b  comprises its own gold conjugate  103 . a  and  103 . b,  sample line  108 . a  and  103 . b  and control line  109 . a  and  109 . b.    
         FIG. 6   b : shows an internal view of a preferred embodiment of a test device according to the present invention suitable to detect two different targets within two different samples (specimens) from the same person (patient). In this preferred embodiment the device comprises two test strips  101 . a  and  101 . b,  wherein each of these two strips comprises its own sample pad  102 . a  and  102 . b,  gold conjugate  103 . a  and  103 . b,  sample line  108 . a  and  103 . b,  control line  109 . a  and  109 . b,  humidity indicator  110 . a  and  110 . b,  and absorbent pad  105 . a  and  105 . b.    
         FIG. 7   a : shows an external view of a preferred embodiment of a test device  501  according to the present invention suitable to detect two targets within the same sample (specimen) from the same person (patient). In this preferred embodiment the device  501  comprises a sample application window  502 , two test result windows  503 . a  and  503 . b,  two control result windows  504 . a  and  504 . b,  humidity indication window  505  and a patient ID area  506 . 
         FIG. 7   b : shows an external view of a preferred embodiment of a test device  501  according to the present invention suitable to detect two targets within two different samples (specimens) from the same person (patient). In this preferred embodiment the device  501  comprises two sample application windows  502 . a  and  502 . b,  two test result windows  503 . a  and  503 . b,  two control result windows  504 . a  and  504 . b,  two humidity indication windows  505 . a  and  505 . b  and a patient ID area  506 . 
     
    
    
     EXAMPLES 
     Example 1  
     Preparation of Oligonucleotide- and Complementary Oligonucleotide Labeled Bovine Serum Albumin 
     5 mg of bovine serum albumin (BSA) was linked to each oligonucleotide and another 5 mg to the complementary oligonucleotide. Every oligonucleotide had a length of about 20 nucleotides having an amino group at the 5′ terminus. The procedure was performed according to the method described by Duncan et al. 1983 20  comprising the following steps: 
     
       
         
         
             
             
         
       
     
     Example 2  
     Preparation of an Preferred Embodiment of a Test Device According to the Present Invention 
     The oligonucleotide and complementary oligonucleotide linked BSA prepared as described in Example 1 are further processed according to a procedure comprising the following steps:
         a) prepare oligonucleotide linked BSA solution, according to example 1 (solution 1);   b) prepare oligonucleotide linked BSA solution, according to example 1 (solution 2), wherein the oligonucleotides of solution 2 differs from the oligonucleotides of solution 1;   c) prepare oligonucleotide linked BSA solution, according to example 1 (solution 3), wherein the oligonucleotides of solution 3 are complementary to the oligonucleotides of solution 1;   d) prepare oligonucleotide linked BSA solution, according to example 1 (solution 4), wherein the oligonucleotides of solution 4 are complementary to the oligonucleotides of solution 2;   e) prepare 1% aqueous solution of tetrachloroauric acid at room temperature;   f) prepare 4% trisodium citrate aqueous solution at room temperature;   g) prepare 0.05 M Potassium Carbonate aqueous solution at room temperature;   h) prepare 1000 ml of phosphate stabilizing buffer of pH 7.4, containing BSA, Tween 20, Sucrose, polyvinylpurrolidone and a preservative, e.g. sodium azide, at room temperature;   i) prepare colloidal gold solution by reduction of 3.4 ml boiling tetrachloroauric acid solution (after dilution into 200 ml) using 2 ml trisodium citrate solution and let it takes the room temperature;   j) dilute the colloidal gold solution as 1:1 using distilled water. Adjust the pH to 7.4 using potassium carbonate solution at room temperature;   k) prepare 400 ml of phosphate conjugation buffer of pH 7.4 at room temperature;   l) partition the 400 ml conjugation buffer by dividing it into four flasks (100 ml of each);   m) add 0.5 mg of aqueous antibody (e.g. anti-Lipoarabinomannan 1 st  clone) to the conjugation buffer in the first flask with stirring at room temperature;   n) add 0.5 mg of oligonucleotides labelled BSA aqueous solution (solution 1) to the first flask at room temperature;   o) add 0.5 mg of aqueous antigen (e.g. HIV p160) to the conjugation buffer in the second flask with stirring at room temperature;   p) add 0.5 mg of oligonucleotides labelled BSA aqueous solution (solution 2) to the second flask with stirring at room temperature;   q) add 0.5 mg of aqueous antibody (e.g. anti-Lipoarabinomannan 2 nd  clone) to the conjugation buffer in the third flask with stirring at room temperature;   r) add 0.5 mg of oligonucleotides labelled BSA aqueous solution (solution 3) to the third flask at room temperature;   s) add 0.5 mg of aqueous antigen (e.g. HIV p160) to the conjugation buffer in the fourth flask with stirring at room temperature;   t) add 0.5 mg of oligonucleotides labelled BSA aqueous solution (solution 4) to the second flask with stirring at room temperature;   u) add 100 ml colloidal gold solution into each flask with stirring at room temperature;)   v) after about 45 minutes; add 200 ml of stabilizing buffer to each flask;   w) after about 20 minutes; concentrate each conjugate by cooled (temperature around 15° C.) high speed centrifugation (10,000 rpm for one hour);   x) discard the supernatant and re-suspend the concentrated conjugates using the stabilising buffer at room temperature;   y) adjust the concentration for each of the two conjugates to O.D. 520 =2.0;   z) mix conjugates of flasks I and II, add 0.2 ml of Tween 20 and soak glass fibre sheet conjugate pad into the conjugate, then heat dry at temperature around 50° C. (first gold conjugate).   aa) mix conjugates of flasks III and IV, soak another glass fiber sheet conjugate pad into the conjugate, then heat dry at temperature around 50° C. (second gold conjugate).       

     (* In case of antibodies/antigens and their specific antigens/antibodies there is no need for these steps of bovine serum albumin or any other protein labelling. ** Other proteins or peptides could be used other than bovine serum albumin). 
     Additionally, print first sample line (e.g. anti-HIV p24, 2 nd  clone), second sample line (e.g. HIV p160) and control line (e.g. mixture of goat anti-p160 and goat anti-mouse IgG) onto nitrocellulose membrane, then heat dry at temperature around 50° C. 
     Finally, laminate cards according to the following procedure: 
     A. In case of Conjugate Releasing Site Laminated Within the Upper Side of the Device Plastic Housing 
     Lamination of cards using the first gold conjugate. Laminate card components onto the backing material with the sequence:
         1. laminate the nitrocellulose membrane nearly in the middle of the card;   2. laminate the absorbent pad in the end of the card (overlaps from the nitrocellulose membrane side);   3. laminate the first conjugate pad in the other side of the nitrocellulose membrane; and   4. laminate the sample pad.       

     B. In Case of Conjugate Releasing Site Laminated Onto the Test Strip Itself Separated From the First Conjugate By a Divider 
     Laminate card components onto the backing material with the sequence (see  FIG. 1   b ):
         1. laminate the nitrocellulose membrane nearly in the middle of the card;   2. laminate the absorbent pad in the end of the card (overlaps from the nitrocellulose membrane side);   3. laminate the first conjugate pad in the other side of the nitrocellulose membrane;   4. laminate the plastic divider onto the first conjugate (overlaps from the nitrocellulose membrane side);   5. laminate the second conjugate pad onto the divider (overlaps from the nitrocellulose membrane side);   6. laminate the sample pad onto the other end of the card, the sample pad will overlaps with the two conjugate pads; and   7. then cut cards into strips.       

     C. Alternatively 
     Lamination of the second gold conjugate could be applied within the plastic housing itself to ensure that the two conjugates will not propagate before release from the releasing pad and so stick within the releasing pad. 
     Example 3  
     Ultra-Sensitive Urinary HIV Antibody and Tuberculosis Antigen Detection Test 
     The 1st gold conjugate pad contains:
         Gold labeled mouse anti-LAM clone  1  and at the same time the same colloidal gold is conjugated with oligonucleotides O- 1 .   Gold labeled mouse anti-hlgG and at the same time the colloidal gold is conjugated with oligonucleotides O- 2 .       

     The 2nd gold conjugate pad contains:
         Gold labeled anti-LAM clone  2  and at the same time the colloidal gold is conjugated with oligonucleotides O- 3  complementary to oligonucleotides O- 1 .   Gold labeled recombinant envelop protein gp160 and at the same time the colloidal gold is conjugated with oligonucleotides O- 4  complementary to oligonucleotides O- 2 .       

     The 1st sample line  108   a  (tuberculosis specific detection line) comprises anti-LAM clone  2  immobilized onto the nitrocellulose membrane. The second sample line  108   b  (HIV specific detection line) comprises of recombinant envelop protein gp160 immobilized onto the nitrocellulose membrane. The control line  109  is anti-mouse IgG. Sample line  108   a  and control line  109  turn into purple color in case of LAM availability in the sample, sample line  108   b  and control line  109  turn into purple color in case of HIV gp160 antibody availability in the sample, and only the control line  109  turns into purple color in case of HIV antibody and LAM antigen free sample. 
     There aren&#39;t any commercially available rapid tests for urinary HIV/Tuberculosis detection in urine. 
     Example 4  
     Ultra-Sensitive Urinary Hepatitis C (HCV) Antibody and Tuberculosis Antigen Detection Test 
     The 1st gold conjugate pad contains:
         Gold labeled mouse anti-LAM clone  1  and at the same time the same colloidal gold is conjugated with oligonucleotides O- 1 .   Gold labeled mouse anti-hlgG and at the same time the colloidal gold is conjugated with oligonucleotides O- 2 .       

     The 2nd gold conjugate pad contains:
         Gold labeled anti-LAM clone  2  and at the same time the colloidal gold is conjugated with oligonucleotides O- 3  complementary to the oligonucleotides O- 1 .   Gold labeled recombinant HCV antigen &amp; at the same time the colloidal gold is conjugated with oligonucleotides O- 4  complementary to the oligonucleotides O- 2 .       

     The 1st sample line  108   a  (tuberculosis specific detection line) comprises of anti-LAM clone  2  immobilized onto the nitrocellulose membrane. The second sample line  108   b  (HCV specific detection line) comprises of recombinant HCV antigen immobilized onto the nitrocellulose membrane. The control line  109  is anti-mouse IgG. Sample line  108   a  and control line  109  turn into purple color in case of LAM availability in the sample, sample line  108   b  and control line  109  turn into purple color in case of HCV antibody availability in the sample, and only the control line  109  turns into purple color in case of HCV antibody and LAM antigen free sample. 
     There aren&#39;t any commercially available rapid tests for urinary HCV/Tuberculosis detection in urine. 
     Example 5  
     Ultra-Sensitive Urinary  H. Pylori  Antibody and Tuberculosis Antigen Detection Test 
     The 1st gold conjugate pad contains:
         Gold labeled mouse anti-LAM clone  1  and at the same time the same colloidal gold is conjugated with oligonucleotides O- 1 .   Gold labeled mouse anti-hlgG and at the same time the colloidal gold is conjugated with oligonucleotides O- 2 .       

     The 2nd gold conjugate pad contains:
         Gold labeled anti-LAM clone  2  and at the same time the colloidal gold is conjugated with oligonucleotides O- 3  complementary to the oligonucleotides O- 1 .   Gold labeled recombinant  H. Pylori  antigen and at the same time the colloidal gold is conjugated with oligonucleotides O- 4  complementary to the oligonucleotides O- 2 .       

     The 1st sample line  108   a  (tuberculosis specific detection line) comprises of anti-LAM clone  2  immobilized onto the nitrocellulose membrane. The second sample line  108   b  ( H. Pylori  specific detection line) comprises of recombinant  H. Pylori  antigen immobilized onto the nitrocellulose membrane. The control line  109  is anti-mouse IgG. Sample line  108   a  and control line  109  turn into purple color in case of LAM availability in the sample, sample line  108   b  and control line  109  turn into purple color in case of  H. Pylori  antibody availability in the sample, and only the control line  109  turns into purple color in case of  H. Pylori  antibody and LAM antigen free sample. 
     There aren&#39;t any commercially available rapid tests for urinary  H. Pylori/ Tuberculosis detection in urine. 
     Example 6  
     Other Combinations are Applicable Using the Same Method Like 
     
         
         
           
             a. Urinary HIV/HCV/Tuberculosis Detection test.
           The 1st gold conjugate pad contains:
               Gold labeled mouse anti-LAM clone  1  and at the same time the same colloidal gold is conjugated with oligonucleotides O- 1 .   Gold labeled mouse anti-hlgG and at the same time the colloidal gold is conjugated with oligonucleotides O- 2 .   
               The 2nd gold conjugate pad contains:
               Gold labeled anti-LAM clone  2  and at the same time the colloidal gold is conjugated with oligonucleotides O- 3  complementary to the oligonucleotides O- 1 .   Gold labeled recombinant Hepatitis C antigen and at the same time the colloidal gold is conjugated with oligonucleotides O- 4  complementary to the oligonucleotides O- 2 .   Gold labeled recombinant envelop protein gp160 and at the same time the colloidal gold is conjugated with oligonucleotides O- 4  complementary to the oligonucleotides O- 2 .   
               The 1st sample line  108   a  (tuberculosis specific detection line) comprises of anti-LAM clone  2  immobilized onto the nitrocellulose membrane. The second sample line  108   b  (HCV specific detection line) comprises of recombinant HCV antigen immobilized onto the nitrocellulose membrane. The third sample line  108   c  (HIV specific detection line) comprises of recombinant envelop protein gp160 immobilized onto the nitrocellulose membrane. The control line  109  is anti-mouse IgG. Sample line  108   a  and control line  109  turn into purple color in case of LAM availability in the sample, sample line  108   b  and control line  109  turn into purple color in case of HCV antibody availability in the sample, sample line  108   c  and control line  109  turn into purple color in case of HIV antibody availability in the sample and only the control line  109  turns into purple color in case of HCV antibodies, HIV antibodies and LAM antigen free sample.   
         
             b. Urinary Tuberculosis antigen, Pneumonia antigen and  H. Pylori  antibody Detection test.
           The 1st gold conjugate pad contains:
               Gold labeled mouse anti-LAM clone  1  and at the same time the same colloidal gold is conjugated with oligonucleotides O- 1 .   Gold labeled mouse anti-hlgG and at the same time the colloidal gold is conjugated with oligonucleotides O- 2 .   
               The 2nd gold conjugate pad contains:
               Gold labeled anti-LAM clone  2  and at the same time the colloidal gold is conjugated with oligonucleotides O- 3  complementary to the oligonucleotides O- 1 .   Gold labeled recombinant Hepatitis C antigen and at the same time the colloidal gold is conjugated with oligonucleotides O- 4  complementary to the oligonucleotides O- 2 .   Gold labeled recombinant envelop protein gp160 and at the same time the colloidal gold is conjugated with oligonucleotides O- 4  complementary to the oligonucleotides O- 2 .   
               The 1st sample line  108   a  (tuberculosis specific detection line) comprises of anti-LAM clone  2  immobilized onto the nitrocellulose membrane. The second sample line  108   b  (Pneumonia specific detection line) comprises of anti-pneumolysin (PLY) clone  2  immobilized onto the nitrocellulose membrane. The third sample line  108   c  ( H. Pylori  specific detection line) comprises of recombinant  H. Pylori  antigen immobilized onto the nitrocellulose membrane. The control line  109  is anti-mouse IgG. Sample line  108   a  and control line  109  turn into purple color in case of LAM availability in the sample, sample line  108   b  and control line  109  turn into purple color in case of PLY antibody availability in the sample, sample line  108   c  and control line  109  turn into purple color in case of  H. Pylori  antibodies availability in the sample and only the control line  109  turns into purple color in case of  H. Pylori  antibodies, PLY and LAM antigen free sample.   
         
             c. Urinary HIV/HCV/Malaria Detection test.
           The 1st gold conjugate pad contains:
               Gold labeled mouse anti-HRP-II clone  1  and at the same time the same colloidal gold is conjugated with oligonucleotides O- 1 .   Gold labeled mouse anti-hlgG and at the same time the colloidal gold is conjugated with oligonucleotides O- 2 .   
               The 2nd gold conjugate pad contains:
               Gold labeled anti-HRP-II clone  2  and at the same time the colloidal gold is conjugated with oligonucleotides O- 3  complementary to the oligonucleotides O- 1 .   Gold labeled recombinant Hepatitis C antigen and at the same time the colloidal gold is conjugated with oligonucleotides O- 4  complementary to the oligonucleotides O- 2 .   Gold labeled recombinant envelop protein gp160 and at the same time the colloidal gold is conjugated with oligonucleotides O- 4  complementary to the oligonucleotides O- 2 .   
               The 1st sample line  108   a  (malaria specific detection line) comprises of anti-HRP-II clone  2  immobilized onto the nitrocellulose membrane. The second sample line  108   b  (HCV specific detection line) comprises of recombinant HCV antigen immobilized onto the nitrocellulose membrane. The third sample line  108   c  (HIV specific detection line) comprises of recombinant envelop protein gp160 immobilized onto the nitrocellulose membrane. The control line  109  is anti-mouse IgG. Sample line  108   a  and control line  109  turn into purple color in case of HRP-II availability in the sample, sample line  108   b  and control line  109  turn into purple color in case of HCV antibody availability in the sample, sample line  108   c  and control line  109  turn into purple color in case of HIV antibody availability in the sample and only the control line  109  turns into purple color in case of HCV antibodies, HIV antibodies and HRP-II antigen free sample.   
         
             d. Urinary HIV/HCV/Malaria/Tuberculosis Detection test. 
             e. Urinary HIV antibody and Malaria antigen Detection test. 
             f. Urinary HIV antibody, Malaria antigen and Tuberculosis Antigen Detection test. 
             g. Urinary HCV antibody and Malaria antigen Detection test. 
           
         
       
    
     The features disclosed in the foregoing description, in the claims and/or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in divers forms thereof. 
     REFERENCES  
     (1) 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. 
     (2) J Chandler, 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 
     (3) 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. 
     (4) Zaaijer, H. L., Exel-Oehlers, P. V., Kraaijeveld, T., Altena, E., Lelie, P. N. (1992) Early detection of antibodies to HIV-1 by third-generation assays.  Lancet  340, 770-772. 
     (5) Constantine, N. T., van der Groen, G., Belsey, E. M., Tamashiro, H. (1994) Sensitivity of HIV-antibody assays determined by seroconversion panels.  AIDS  8, 1715-1720. 
     (6) Satten, G. A., Busch, M. P., et al. (1997) Effect of transmission route on window period estimates. Fourth Conference on Retroviruses and Opportunistic Infections, Washington D.C., Abstract 122. 
     (7) WHO. Rapid HIV tests: Guidelines for use in HIV testing and counseling services in resource-constrained settings. Geneva 2004. http://www.who.int/hiv/pub/vct/rapidhivtests/en/ 
     (8) Holodniy M, et al. (1991) Reduction in plasma human immunodeficiency virus ribonucleic acid following dideoxynucleoside therapy as determined by the polymerase chain reaction.  J. Clin. Invest  88, 1755-1759. 
     (9) Katzenstein D. A., et al. (1994) Quantitation of human immunodeficiency virus by culture and polymerase chain reaction in response to didanosine after long-term therapy with zidovudine.  J. Infect. Dis.  169, 416-419. 
     (10) Jackson J B, et al. (1998) Practical diagnostic testing for human immunodeficiency virus.  Clin. Microb. Rev.  1, 124-138. 
     (11) Goudsmit J, et al. (1986) Expression of human Immunodeficiency virus antigen (HIV-Ag) in serum and cerebrospinal fluid during acute and chronic infection.  Lancet  2, 177-180. 
     (12) Aubuchon, J. P., Birkmeyer, J. D., Busch, M. P. (1997) Cost-effectiveness of expanded human immunodeficiency virus-testing protocols for donated blood.  Transfusion  45, 45-51. 
     (13) Ward, J. M., Holmberg, S. D., Allen, J. R., Cohn, D. L., et al. (1988) Transmission of human immunodeficiency virus (HIV) by blood transfusion screened as negative for HIV antibody.  N. Engl. J. Med.  8, 473-478. 
     (14) Alter, H. J., et al. (1990) Prevalence of human immunodeficiency virus type 1 p24 antigen in U.S. blood donors—an assessment of the efficacy of testing in donor screening.  N. Engl. J. Med.  323, 1312-1317. 
     (15) Mayers. D. L. (1998) Drug-resistant HIV-1. The virus strikes back.  JAMA  279, 2000-2002. 
     (16) Stephenson, J. (2002) Cheaper HIV drugs for poor nations bring a new challenge: monitoring treatment.  JAMA  288, 2. 
     (17) WHO. HIV assays: Operational characteristics ( Phase  1). Report 15/antigen/antibody ELISAs. Geneva 2004. http://www.who.int/diagnostics_laboratory/evaluations/hiv/en/ 
     (18) WHO. HIV assays: Operational characteristics ( Phase  1). Report 14/simple/rapid tests. Geneva 2003. http://www.who.int/diagnostics_laboratory/evaluations/hiv/en/ 
     (19) Meier, T, et al. (2001) Evidence for a diagnostic window in fourth generation assays for HIV.  J. Clin. Virol.  23, 113-116. 
     (20) 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.