Title of Invention	"DEVICE FOR EARLY DETECTION OF INFECTION WITH DENGUE FLAVIVIRUS IN HUMAN SERUM, PLASMA OR WHOLE BLOOD"
Abstract	The present invention relates to devices for the early detection of infection with dengue flavivirus in human serum or plasma or whole blood. More specifically, the present invention relates to devices for the simultaneous and differential detection of dengue NS1 antigen and dengue antibodies present in human serum or plasma or whole blood. Further more the subject invention allows one to detect dengue antigens in early acute stage of infection even prior to the development of antibodies thereby allowing early detection of the disease. Also the present invention relates to the kit for simultaneous and differential detection of presence of antibodies and antigens to dengue in human serum or plasma or whole

Title of Invention

"DEVICE FOR EARLY DETECTION OF INFECTION WITH DENGUE FLAVIVIRUS IN HUMAN SERUM, PLASMA OR WHOLE BLOOD"

Abstract

The present invention relates to devices for the early detection of infection with dengue flavivirus in human serum or plasma or whole blood. More specifically, the present invention relates to devices for the simultaneous and differential detection of dengue NS1 antigen and dengue antibodies present in human serum or plasma or whole blood. Further more the subject invention allows one to detect dengue antigens in early acute stage of infection even prior to the development of antibodies thereby allowing early detection of the disease. Also the present invention relates to the kit for simultaneous and differential detection of presence of antibodies and antigens to dengue in human serum or plasma or whole

Full Text	Field of the Invention The present invention relates to a device for the early detection of infection with dengue flavivirus in human serum or plasma or whole blood. It is an improved version of the patent "2484/DEL/2007: A device and a kit for analyzing the presence of anti dengue IgM and anti dengue IgG in a sample and process thereof which is pending in Indian Patent office. More specifically, the present invention relates to the simultaneous and differential detection of dengue NSl antigen and dengue antibodies present in human serum or plasma or whole blood. Further more the subject invention allows one to detect dengue antigens in early acute stage of infection even prior to the development of antibodies thereby allowing early detection of the disease. More specifically, the subject invention relates to a rapid diagnostic kit for the detection of dengue NSl antigen and dengue antibodies present in human serum or plasma or whole blood. It is a visual, rapid, sensitive and accurate immunoassay for the rapid and differential detection of dengue antigen and dengue antibodies in human serum or plasma or whole blood. Background of Invention Dengue fever is a viral disease transmitted by mosquitoes and caused by four serotypes of dengue virus (DEN): DEN: 1, DEN: 2, DEN: 3 and DEN: 4. Dengue fever is the most important arthropod-borne viral disease affecting humans and according to the World Health Organisation estimates, its incidence has increased by a factor of 30 over the last 50 years. Dengue viruses are transmitted to humans by Stegomyia aegypti (formerly Aedes aegypti) mosquitoes and cause a wide range of symptoms from unapparent or mild disease to severe haemorrhagic form. The infection agent, dengue virus has a simple stranded RNA with positive polarity which comprises of nucleotides and poly protein amino acids. It is separated into structural and non structural proteins. The structural proteins are C, prM and E and the nonstructural proteins are NSl, NS2a, NS2b, NS3, NS4a, NS4b and NS5. NSl a non structural protein was identified for the first time in 1970. NSl is a highly conserved glycoprotein which appears essential for the virus viability although no precise function has yet been assigned to it. During the cell infection, NSl is found associated with intracellular organelles or is alternatively transported through the secretory pathway to the cell surface. A soluble hexameric form may be released in a glycosylation-dependent fashion from infected mammalian cells but not from vector derived mosquito cells. Dengue virus infection is currently detected by means of several biological tests. Virus isolation on mosquito cells, viral RNA detection by reverse transcription - PCR (RT-PCR) or serological tests such as immunoglobulin M (IgM) capture enzyme linked immunosorbent assay (MAC-ELISA). RT-PCR remains expensive and its routine use in the clinical diagnostic laboratories is difficult. Commercial MAC-ELISA kits are available but they cannot be used for early diagnosis, because IgM does not become detectable until 5 to 10 days after the onset of illness in secondary infection. In primary infection, IgM and IgG arise approximately 5 and 14 days respectively after symptoms onset. In secondary infection, IgM levels are low or undetectable while IgG arise 1-2 days after symptoms onset with higher levels than in primary infection. More recently, detection in patient's sera of circulating dengue virus in nonstructural protein NSl has been described as an alternative method for early diagnosis. NSl antigens were found circulating from the first day and up to 9 days after the onset of fever with comparable levels observe in primary and secondary infection. Dengue virus specific IgM first appeared at about day 3 and was prominent from day 5 onward. IgM did not seem to interfere with NS1 detection during the clinical phase. There is no commercially available vaccine against the dengue virus. In the absence of vaccine, it is necessary to monitor epidemics. To do this, active monitoring programs have in particular been set up by the World Health organization. It essentially comprises the monitoring of cases of fever and of vector insects and the serological and virological screening of individuals having a fever and suspected of being infected with the dengue virus. The present invention is a device for early and rapid detection of dengue in human serum or plasma or whole blood. Dengue NS1 antigen can be detected during the acute and the symptomatic phase of dengue. Antibodies to dengue can be detected during the entire period of infection. Prior Art US 2005186562 (Al) The invention concerns a method for early detection of a flavivirus-induced infection, comprising the detection of the flavivirus non-structural glycoprotein NS1 in a biological sample during the clinical phase of the infection, by an immumological method using at least two identical or different antibodies, the first antibody consisting of polyclonal or monoclonal antibodies pre-selected for their high affinity for said NS 1 protein hexameric in shape. US 5824506: Peptide antigens derived from the dengue virus type-2 glycoprotein NS1 are provided. The peptide antigens are specifically immunoreactive with sera from individuals infected with the dengue virus. The antigens are useful as diagnostic tools in determining whether an individual has been or is infected with dengue virus, and for discriminating between infection with dengue virus and infection with related flaviviruses. The antigens are also useful in vaccine compositions for immunizing individuals against infection with the dengue virus. WO 2003/048184 A3: The present invention relates to NS1 proteins or parts thereof of Flaviviruses, in particular of Dengue viruses useful for vaccination against said Flavivirus and against one or more other Flaviviruses. The invention further concerns the NS1 protein or parts thereof of one Dengue virus serotype, in particular serotype 2, useful for vaccination against Dengue viruses from all serotypes. The invention further concerns DNA comprising an expression cassette coding for a Flavivirus NS1 or parts thereof, vectors, comprising said DNA and vaccines containing or expressing a Flavivirus NS1. These patents do not disclose a fast, cost effective, simple and rapid method for dengue diagnosis that combines sensitivity and specificity. It does not provide a method of detection of dengue for a man of ordinary skill to perform. The present invention is a rapid, qualitative, solid phase immunochromatographic assay for differential detection of dengue IgM antibodies, dengue IgG antibodies and dengue NS1 antigen present in human serum or plasma or whole blood. The object of the present invention is to provide a novel, inexpensive, simple, rapid diagnostic device and a kit which differentially detects the dengue NS1 antigen, dengue IgM antibodies and dengue IgG antibodies present in human serum or plasma or whole blood. It is another object of the present invention to provide a safe diagnostic assay for the detection of dengue NS1 antigen, anti dengue IgM antibodies and anti dengue IgG antibodies present in human serum or plasma or whole blood. Summary of the Invention The object of the present invention is to provide a rapid test device and a test kit which detects the dengue NS1 antigen, dengue IgM antibodies and dengue IgG antibodies present in human serum or plasma or whole blood. In the present device control line is used to test the workability of the reagent. The present invention provides an effective device having sensitivity and specificity of 99% and 99% respectively. The device for the test can be in two formats, i.e. lateral flow immunochromatographic test device or flow through test device. The lateral flow immunochromatographic test device comprises of top cover and a bottom receptacle adapted to be pressed with each other. The top cover is provided with well/s, through which sample is added on to the sample pad/s. The bottom receptacle is provided with track/s for holding strip/s having nitrocellulose membrane. The sample pad is provided at one end of strip and absorbant pad on other end. The membrane is having lines meant for detection of dengue NS1 antigen, anti dengue IgM antibodies and anti dengue IgG antibodies. The line for detection of,dengue NS1 antigen is immobilized with anti dengue NS1 antibodies, and the line for detection of anti dengue IgM antibodies is immobilized with anti human IgM antibodies and the line for detection of anti dengue IgG antibody is immobilized with anti human IgG antibody. Control line is also immobilized on membrane to test the working of reagents. The conjugate is impregnated on to the conjugate pad of strip. The second form of device is flow through test device. The device consists of top cover and a bottom receptacle. The top cover is provided with a hole in the centre through which membrane is exposed. The bottom receptacle is provided with an absorbant pad over which nitrocellulose membrane is mounted, exposing through centre of top cover. The conjugate is in solution form/lyophilized form. The membrane is coated with dots meant for detection of dengue NS1 antigen, anti dengue IgM antibodies and anti dengue IgG antibodies. The dot for detection of dengue NS1 antigen is immobilized with anti dengue NS1 antibodies, and the dot for detection of anti dengue IgM antibody is immobilized with antihuman IgM antibodies, and the dot for detection of anti dengue IgG antibody is immobilized with antihuman IgG antibody. The control dot is also provided to test the working of reagents simultaneously. Brief Description with figures Figure 1 shows the dengue detection device in format 1. Figure 1(a) shows only control line in colour. Figure 1(b) shows the control line and line for the detection of dengue IgM antibodies in colour. Figure 1(c) shows the control line and the line for detection of dengue IgG antibodies in colour. Figure 1(d) shows the control line and the line for the detection of dengue NSl antigen in colour. Figure 1(e) shows the control line as well as the line for the detection of dengue IgM antibodies and dengue IgG antibodies in colour. Figure 1(f) shows the control line and the line for the detection of dengue IgM antibodies and the line for the detection of dengue NSl antigen in colour. Figure 1(g) shows the control line and line for the detection of the dengue IgG antibodies and the line for the detection of dengue NSl antigen in colour. Figure 1(h) shows the control line and the line for the detection of dengue IgM antibodies and dengue IgG antibodies and dengue NSl antigen detection line in colour. Figure 2 shows the device in format 2. Figure 2(a) shows only the control line in colour. Figure 2(b) shows the control line and the line for detection of dengue IgM antibodies in colour. Figure 2(c) the control line and line for detection of dengue IgG antibodies in colour. Figure 2(d) shows the control line and the line for detection of dengue NSl antigen in colour. Figure 2(e) shows the control line and the line for the detection of anti dengue IgG antibodies and anti dengue IgM antibodies in colour. Figure 2(f) shows the control line and the line for detection of dengue IgM antibodies and dengue NSl antigen in colour. Figure 2(g) shows the control line and the line for the detection of dengue IgG antibodies and dengue NS1 antigen in colour. Figure 2(h) shows the control line and the line for the detection of dengue IgM antibodies and dengue IgG antibodies and dengue NSl antigen detection line in colour. Figure 3 shows the device in Format 3. Figure 3(a) shows the device with only control line in colour. Figure 3(b) shows the control line and the line for detection of dengue IgM antibodies in colour. Figure 3(c) shows the control line and the line for the detection of the dengue IgG antibodies in colour. Figure 3(d) shows the control line and the line for the detection of dengue NSl antigen in colour. Figure 3(e) shows the control line and the line for the detection of dengue IgM antibodies and the line for detection of dengue IgG antibodies in colour. Figure 3(f) shows the control line and the line for the detection of dengue IgM antibodies and the line for the detection of dengue NSl antigen in colour. Figure 3(g) shows the control line and the line for the detection of dengue IgG antibodies and the line for detection of dengue NSl antigen in colour. Figure 3(h) shows the control line and the line for the detection of anti dengue IgM antibodies, anti dengue IgG antibodies and dengue NS 1 antigen detection line in colour. Figure 4 shows the flow through device in format 4. Figure 4(a) shows the device with only control dot in colour. Figure 4(b) shows the control dot and the dot for the detection of dengue IgG antibodies in colour. Figure 4(c) shows the control dot and the dot for the detection of dengue IgM antibodies in colour. Figure 4(d) shows the control dot and the dot for the detection of dengue NS 1 antigen in colour. Figure 4(e) shows the control dot and the dot for the detection of dengue IgM antibodies and dengue IgG antibodies in colour. Figure 4(f) shows the control dot and the dot for detection of dengue IgM antibodies and the dot for the detection of dengue NS 1 antigen in colour. Figure 4(g) shows the control dot and the dot for the detection of dengue IgG antibodies and dot for detection of dengue NS 1 antigen in colour. Figure 4(h) shows the control dot and the dot for the detection of dengue IgM antibodies and dengue IgG antibodies and dengue NS 1 antigen in colour. Figure 5 shows-the device in format 5. Figure 5(a) shows the device with only control dot in colour. Figure 5(b) shows the control dot and the dot for the detection of dengue IgM antibodies in colour. Figure 5(c) shows the control dot and the dot for the detection of dengue IgG antibodies in colour. Figure 5(d) shows the control dot and the dot for detection of dengue NSl antigen in colour. Figure 5(e) shows the control dot and the dot for the detection of dengue IgM antibodies and dengue IgG antibodies in colour. Figure 5(f) shows the control dot and the dot for detection of the dengue IgM antibodies and dengue NS1 antigen in colour. Figure 5(g) shows the control dot and the dot for the detection of the dengue IgG antibodies and the dengue NSl antigen detection dot in colour. Figure 5(h) shows the control dot and the dot for the detection of the dengue IgM antibodies and dengue IgG antibodies and dengue NS 1 antigen in colour. On addition of sample, if only the control line/dot appears in colour, then the result is interpreted as negative and the sample is not reactive for Dengue. If the line/dot for detection of anti dengue IgM and the control line/dot are in colour, then the result is interpreted as positive and sample is reactive for dengue IgM antibody i.e. it is a case of primary dengue-infection. If the control line/dot and the line/dot for the detection of anti dengue IgG antibodies are in colour, then the result is interpreted as positive and sample is reactive for dengue IgG antibody and it is a case of secondary infection. If the control line/dot and the line/dot for the detection of dengue NS 1 antigen are in colour, then the result is interpreted as positive and sample is reactive for dengue. If the control line/dot, line/dot for detection of dengue IgM antibodies and line/dot for detection of dengue IgG antibodies are in colour, then the result is interpreted as positive and the sample is reactive for dengue and it is a case of secondary infection. If the control line/dot, the line/dot for the detection of dengue IgM antibodies and the line/dot for the detection of dengue NS1 antigen are in colour, then the result is interpreted as positive and sample is reactive for dengue. If the control dot/line, the line/dot for detection of dengue IgG antibodies and the line/dot for detection of dengue NS1 antigen are in colour, then the sample is interpreted as positive and the sample is reactive for dengue. If the control line/dot, the anti dengue IgM antibody detection line/dot, anti dengue IgG detection line/dot and the dot/line for the detection of dengue NS1 antigen are in colour, then the result is interpreted as positive and the sample is reactive for dengue. Detailed Description The object of the present invention is to provide a device for early detection of dengue flaviviral infection. It comprises of a device for the simultaneous and differential detection of non-structural glycoprotein NS1 of dengue flavivirus, anti dengue IgM antibodies and anti dengue IgG antibodies in human serum or plasma or whole blood. The device can be based on lateral flow immunochromatographic test device or in form of flow through assay format. The device comprises of top cover and bottom receptacles adapted to be fit into each other. The bottom receptacle is having nitrocellulose membrane. A mean for pouring sample is provided in the test device. An absorbant pad is provided to absorb excess buffer. The membrane is coated with line/dot for detection of dengue NS1 antigen, anti dengue IgM antibodies and anti dengue IgG antibodies. In an embodiment of present invention, the device can be prepared in different formats. The process for preparation of device for the detection of presence of dengue NS1 antigen, anti dengue IgM antibodies and anti dengue IgG antibodies is as follows. Format 1 In this format, the device (as shown in figure 1) is based on principle of lateral flow immunochromatography. The casing comprises of a top cover and a bottom receptacle adapted to be pressed with each other. The bottom receptacle has a base for holding the strip. The strip is mounted with a nitrocellulose membrane, and provided with a sample pad at one end of the strip and an absorbant pad on the other end of the strip. The strip is impregnated with conjugate pad. A sample well is provided at one end on the top cover of device through which the sample is dropped onto sample pad. The membrane is provided with lines for the detection of dengue NS1 antigen, anti dengue IgM antibodies and anti dengue IgG antibodies. The line for the detection of dengue NS1 antigen is immobilized with anti dengue NS1 antibodies, the line for the detection of anti dengue IgM antibodies is immobilized with anti human IgM antibodies and the line for the detection of anti dengue IgG antibodies is immobilized with antihuman IgG antibodies. The sample is dropped on the sample pad through sample cup; it will migrate laterally through membrane. Analytes if present in sample will bind to signal reagent (conjugate) forming an analyte- conjugate complex. This complex will further move towards the membrane and bind to the corresponding capture reagent immobilized on membrane, resulting in formation of coloured line. The test lines for detection of anti dengue IgG antibodies, anti dengue IgM antibodies and dengue NS1 antigen are coated as follows. 1 mg/ml to 2.5 mg/ml of monoclonal anti human IgG and/or polyclonal anti human IgG antibodies are added to 10 mM to 50 mM sodium citrate buffer, pH 5.0 to 7.0 containing 150 mM Sodium chloride and mixed. The mixture is then dispensed at the position of test line meant for detection of anti dengue IgG antibodies. The antibodies can be biotinylated using method well known in art and these biotinylated antibodies can then immobilized at respective position on membrane. 0.5 mg/ml to 1.5 mg/ml of monoclonal anti human IgM and/or polyclonal anti human IgM antibodies are added to 10 mM to 50 mM Sodium citrate buffer; pH 5.0 to 7.0 containing 150 mM sodium chloride and mixed. The mixture is then dispensed at the position of test -line meant for detection of anti dengue IgM antibodies. The antibodies can be biotinylated using method well known in art and these biotinylated antibodies can then immobilized at respective position on membrane. 1 mg/ml to 3 mg/ml of monoclonal anti dengue NS1 antibody and/or polyclonal anti dengue NS1 antibody are added to 10 mM to 50 mM MOPS buffer; pH 7.0 to 8.0 containing 150 mM sodium chloride and mixed. The mixture is then dispensed at position of test line meant for detection of dengue NS1 antigen. The antibodies can also be biotinylated using method well known in art and these biotinylated antibodies can then be immobilized at respective position on membrane. The control line is immobilized with anti species IgG in phosphate buffer saline having pH 7.5. The strip is finally dried at 37°C for 1 to 4 hours. The conjugate used for detection of anti dengue IgG antibody, anti dengue IgM antibodies and dengue NS1 antigen in human serum or plasma or whole blood is Dengue viral lysate conjugated to gold colloid or modified microsphere and/or dengue recombinant antigen conjugated to gold colloid or modified microsphere; And monoclonal anti dengue NS1 antibodies conjugated to gold colloid or modified microsphere and/or polyclonal anti dengue NS1 antibody conjugated to gold colloid or modified microsphere. The assay buffer used for performing the test consists of lOmM to 50 mM Disodium hydrogen phosphate, lOmM to 50 mM Sodium dihydrogen phosphate, 20mM TRIS, 0.5 M to 2 M Potassium chloride, 0.5%-3% Goat albumin or sheep albumin, 0.2% preservative like sodium Azide and l%-3% zwitterionic detergent such as CHAPSO (3-[(3 -cholamidopropyl)dimethylammonio] -2-hydroxy-1 -propanesulfonate); the assay buffer has pH 7 to 8. Format 2 In this format, the device is a bi-directional device (as shown in fig 2) and is based on principle of lateral flow immunochromatography. The casing comprises of top cover and bottom receptacles adapted to be pressed with each other. The bottom receptacle has a base for holding the strip. The strip is provided with a sample pad in the centre, and absorbant pads are provided on both ends of the strip. The strip is mounted with nitrocellulose membrane on both sides of sample pad. The top cover is having means for adding sample in form of sample well in the centre of top cover. The corresponding conjugates are impregnated on both sides of the strip. The membrane on one side of sample pad is meant for detection of dengue NS 1 antigen and is immobilized with anti dengue NS1 antibodies. The membrane on other side of sample pad is meant for detection of anti dengue IgG and anti dengue IgM antibodies and is immobilized with anti human IgG antibodies and anti human IgM antibodies. Both the membranes are provided with control lines to test the workability of reagents. The sample when added on to the sample pad through sample cup will migrate laterally on both sides, such that analytes if present in the sample will bind to the signal reagents (conjugate) forming a complex. The complex will further move, and bind to the corresponding capture reagent immobilized on the membrane, resulting in formation of coloured line. The test lines on the membrane present on one side of sample pad meant for detection of anti dengue IgG and anti dengue IgM antibodies is coated as follows. 1 mg/ml to 2.5 mg/ml of monoclonal anti human IgG and/or polyclonal anti human IgG antibodies are added to 10 mM to 50 mM sodium citrate buffer; pH 5.0 to 7.0 containing 150 mM Sodium chloride and mixed. The mixture is then dispensed at position of test line meant for detection of anti dengue IgG antibodies. The antibodies can be biotinylated using method well known in art and these biotinylated antibodies can then be immobilized at respective position on membrane. 0.5 mg/ml to 1.5 mg/ml of monoclonal anti human IgM and/or polyclonal anti human IgM antibodies are added to 10 mM to 50 mM Sodium citrate buffer; pH 5.0 to 7.0 containing 150 mM sodium chloride and mixed. The mixture is then dispensed at position of test line meant for detection of anti dengue IgM antibodies. The antibodies can be biotinylated using method well known in art and these biotinylated antibodies can then be immobilized at respective position on membrane. The control line is immobilized with anti species IgG in phosphate buffer saline having pH 7.5. The Strip is then dried at 37°C for 1 to 4 hours. The test line on the membrane present on other side of sample pad meant for detection of dengue NS1 antigen is coated as follows. 1 mg/ml to 3 mg/ml of monoclonal anti dengue NS1 antibody and/or polyclonal anti dengue NS1 antibody are added to 10 mM to 50 mM MOPS buffer; pH 7.0 to 8.0 containing 150 mM sodium chloride and mixed. The mixture is then dispensed at position of test line meant for detection of dengue NS1 antigen. The antibodies can be biotinylated using method well known in art and these biotinylated antibodies can then be immobilized at respective position on membrane. The control line is immobilized with anti species IgG in phosphate buffer saline having pH 7.5. The strip is then dried at 37°C for 1 to 4 hours. The conjugate used for detection of anti dengue IgG antibody and anti dengue IgM antibodies in human serum or plasma or whole blood is Dengue viral lysate conjugated to gold cold colloid or modified microsphere and/or dengue recombinant antigen conjugated to gold colloid or modified microsphere. The conjugate used for detection of dengue NS1 antigen consist of monoclonal anti dengue NS1 antibodies conjugated to gold colloid or modified microsphere and/or polyclonal anti dengue NS1 antibody conjugated to gold colloid or modified microsphere. The assay buffer used for performing the test consist of lOmM to 50 mM Disodium hydrogen phosphate, lOmM to 50 mM Sodium dihydrogen phosphate, 20mM TRIS, 0.5 M to 2 M Potassium chloride, 0.5%-3% Goat albumin or sheep albumin, 0.2% preservative like sodium Azide and l%-3% zwitterionic detergent such as CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1 -propanesulfonate); the assay buffer has pH 7 to 8. Format 3 In this format, the device is as shown in Figure 3. The device is based on principle of lateral flow immunochromatography. The device comprises top cover and bottom receptacle adapted to be pressed with each other. Two sample wells, adjacent to each other are provided at one end of the top cover of device, through which sample is added onto the sample pad. The bottom receptacle is provided with two base, adjacent to each other for holding strips. Two strips are provided, each with an absorbant pad on one end and a sample pad on other end, such that sample pad faces the sample well when mounted on the base. The strips are mounted with a nitrocellulose membrane, and are impregnated with corresponding conjugate pad. One strip is coated with control line and a test line for detection of dengue NS1 antigen, whereby test line is immobilized with anti dengue NS1 antibodies. The other strip is coated with control line and two test lines, one for detection of anti dengue IgM antibodies and the other for the detection of anti dengue IgG antibodies. The test line on second strip meant for detection of anti dengue IgM antibodies is immobilized with anti human IgM antibodies and test line meant for detection of dengue IgG antibodies is immobilized with anti human IgG antibodies. When samples are added through sample well on sample pad, it will migrate laterally through strips. The analytes if present in sample will bind to the corresponding signal reagent (conjugate) forming a complex. The complex will further move on strip and will bind with corresponding capture reagent immobilized on the strip, leading to formation of coloured line. The first strip having control line and two test lines meant for detection of anti dengue IgG and anti dengue IgM antibodies is coated as follows. 1 mg/ml to 2.5 mg/ml of monoclonal anti human IgG and/or polyclonal anti human IgG antibodies are added to 10 mM to 50 mM sodium citrate buffer; pH 5.0 to 7.0 containing 150 mM Sodium chloride and mixed. The mixture is then dispensed at position of test line meant for detection of anti dengue IgG antibodies. The antibodies can be biotinylated using method well known in art and these biotinylated antibodies can then be immobilized at respective position on membrane. 0.5 mg/ml to 1.5 mg/ml of monoclonal anti human IgM and/or polyclonal anti human IgM antibodies are added to 10 mM to 50 mM Sodium citrate buffer; pH 5.0 to 7.0 containing 150 mM sodium chloride and mixed. The mixture is then dispensed at position of test line meant for detection of anti dengue IgM antibodies. The antibodies can be biotinylated using method well known in art and these biotinylated antibodies can then immobilized at respective position on membrane. The control line is immobilized with anti species IgG in phosphate buffer saline having pH 7.5. The strip is then dried at 37°C for 1 to 4 hours. The second strip having line for dengue NSl antigen detection and control line is coated as follows. 1 mg/ml to 3 mg/ml of monoclonal anti dengue NSl antibody and/or polyclonal anti dengue NSl antibody are added to 10 mM to 50 mM MOPS buffer; pH 7.0 to 8.0 containing 150 mM sodium chloride and mixed. The mixture is then dispensed at position of test line meant for detection of dengue NSl antigen. The antibodies can be biotinylated using method well known in art and these biotinylated antibodies can then immobilized at respective position on membrane. The control line is immobilized with anti species IgG in phosphate buffer saline having pH 7.5. The strip is then dried at 37°C for 1 to 4 hours. The conjugate used for detection of anti dengue IgG antibody and anti dengue IgM antibodies in human serum or plasma or whole blood is Dengue viral lysate conjugated to gold cold colloid or modified microsphere and/or dengue recombinant antigen conjugated to gold colloid or modified microsphere. The conjugate used for detection of dengue NSl antigen consist of monoclonal anti dengue NSl antibodies conjugated to gold colloid or modified microsphere and/or polyclonal anti dengue NSl antibody conjugated to gold colloid or modified microsphere. The assay buffer used for performing the test consist of lOmM to 50 mM Disodium hydrogen phosphate, lOmM to 50 mM Sodium dihydrogen phosphate, 20mM TRIS, 0.5 M to 2 M Potassium chloride, 0.5%-3% Goat albumin or sheep albumin, 0.2% preservative like sodium Azide and l%-3% zwitterionic detergent such as CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1 -propanesulfonate); the assay buffer has pH 7 to 8. Format 4 In this format, the device is as shown in figure 4. The casing comprises of rectangular top cover and bottom cover, tightly pressed with each other. A circular hole is provided in the top cover at the centre of the device. The bottom receptacle is provided with absorbant pad. The membrane is cut from the cellulose material and is mounted on an absorbant pad housed in casing, exposed through the hole in centre of top cover The device is a flow through test device whereby membrane is coated with four dots. The first dot is immobilized with anti dengue NSl antibodies and is meant for detection of dengue NSl antigen in the sample. The second dot is immobilized with anti human IgM antibodies and is meant for detection of anti dengue IgM antibodies in the sample. The third dot is immobilized with anti human IgG antibodies and is meant for detection of dengue IgG antibodies in sample. The fourth dot is control dot and is meant for testing the workability of reagent. The sample is added to the membrane through circular hole provided on top cover. The analytes if present in the sample will bind to the corresponding capture reagent immobilized as dots on membrane forming a complex. When conjugate solution is added to the device, the conjugate will bind to the corresponding complex leading to formation of coloured dot. The membrane with test dots for detection of anti dengue IgG antibodies, anti dengue IgM antibodies and dengue NSl antigen is coated as follows. Each spot/dot for detection of anti dengue IgG antibody is coated with 250 nanogram to 5 ug, preferably 1 jig to 2 (j,g of monoclonal antihuman IgG antibody and/or polyclonal anti human IgG antibody. For coating the dot for detection of anti dengue IgG antibodies, 250 nanogram to 5ug, preferably 1 jxg to 2 u£ monoclonal anti human IgG and/or polyclonal antihuman IgG antibodies are added to 10 mM to 50 mM Sodium citrate buffer; pH 5.0 to 7.0 containing 150 mM Sodium chloride and 100 nanogram to 500 nanogram Bovine Serum albumin and mixed. The mixture is then dispensed at position of test dot meant for detection of anti dengue IgG antibodies. Each spot/dot for detection of anti dengue IgM antibody is coated with 250 nanogram to 10 ug, preferably 3 ug to 5 ug monoclonal anti human IgM antibody and/or polyclonal anti human IgM antibody. For coating the dot for detection of anti dengue IgM antibodies, 250 nanogram to 10 ug, preferably 3 ug to 5 ug monoclonal anti human IgM antibody and/or polyclonal anti human IgM antibody are added to l0mM to 50 mM sodium citrate buffer; pH 5.0 to 7.0 containing 150 mM Sodium chloride and 100 nanogram to 500 nanogram Bovine serum albumin and mixed. The mixture is then dispensed at position of dot for detection of anti dengue IgM antibodies. Each spot/dot for detection of dengue NSl antigen is coated with 500 nanogram to 5 ug, preferably 2 ug to 3 ug monoclonal anti dengue NSl antibody and/or polyclonal anti dengue NS1 antibody. For coating the dot for detection of dengue NSl antigen, 500 nanogram to 5 ug, preferably 2 ug to 3 ug monoclonal anti dengue NSl antibody and/or polyclonal anti dengue NSl antibody are added to 10 mM to 50 mM MOPS buffer; pH 7.0 to 8.0 containing 150 mM Sodium chloride and 100 nanogram to 500 nanogram Bovine Serum albumin and mixed. The mixture is then dispensed at position of dot meant for detection of dengue NS 1 antigen. The control dot is immobilized with anti species IgG in phosphate buffer saline having pH 7.5. The membrane is then dried at 37°C for at least 1 hour. The conjugate used for detection of anti dengue IgG antibodies, anti dengue IgM antibodies and dengue NSl antigen comprises dengue viral lysate conjugated to gold colloid or modified microsphere and/or dengue recombinant antigen conjugated to gold colloid or modified microsphere; And monoclonal anti dengue NSl antibody conjugated to gold colloid or modified microsphere and/or polyclonal anti dengue NSl antibody conjugated to gold colloid or modified microsphere. Biotinylated dengue recombinant antigen and/or biotinylated dengue viral lysate and biotinylated monoclonal anti dengue NS1 antibodies and/or biotinylated polyclonal anti dengue NS1 antibodies can also be used for detection of anti dengue IgG and anti dengue IgM antibodies dengue NS1 antigen followed by addition of conjugate. The conjugate then comprises streptavidin conjugated to gold colloid or modified microsphere. The sample diluent used for the test consist of 10 mM to 50 mM Tris buffer, pH 7.0 to 8.5 containing 1 M to 3 M Sodium Chloride, 0.02% to 0.2% Casein, 0.2% to 0.5% polyoxyethylene sorbitan monolaurate, 0.05% to 0.2% polyoxyethylene-23-lauryl ether, 0.2 % Sodium Azide and 0.01% to 0.05 %Merthiolate. Format 5 In this format, the device used is as shown in figure 5. The casing comprises of rectangular top cover and bottom cover, tightly pressed with each other. Two circular holes are provided in the top cover of the casing. The bottom receptacle is provided with absorbant pad. Two circular membranes are cut from the cellulose material and are mounted on the absorbant pad housed in bottom part of casing, which are exposed through the holes of top cover. The device is a flow through test device whereby two membranes are used. The first membrane is immobilized with dot for detection of dengue NS1 antigen and a control dot. The second membrane is immobilized with dots for detection of anti dengue IgM antibodies and anti dengue IgG antibodies; and a control dot. The dot on first membrane meant for detection of dengue NS1 antigen is immobilized with anti dengue NS1 antibodies. The dot on second membrane meant for detection of dengue IgG antibodies is immobilized with anti human IgG antibodies and the dot meant for detection of dengue IgM antibodies is immobilized with antihuman IgM antibodies. The samples are added to the membrane through holes in the top cover. The analytes if present in the sample will bind to the corresponding dot of immobilized capture reagent forming a complex. When conjugate solution is added to the device, the conjugate will bind to the corresponding complex leading to formation of coloured dot. The membrane with test dot for detection of dengue NSl antigen and control dot is coated as follows. Each spot/dot for detection of dengue NSl antigen is coated with 500 nanogram to 5 µg, preferably 2 µg to 3 µg monoclonal anti dengue NSl antibody and/or polyclonal anti dengue NS1 antibody. For coating the dot for detection of dengue NSl antigen, 500 nanogram to 5 µg, preferably 2 µg to 3µg monoclonal anti dengue NSl antibody and/or polyclonal anti dengue NSl antibody are added to 10 mM to 50 mM MOPS buffer; pH 7.0 to 8.0 containing 150 mM Sodium chloride and 100 nanogram to 500 nanogram Bovine Serum albumin and mixed. The mixture is then dispensed at position of dot meant for detection of dengue NSl antigen. The control dot is immobilized with anti species IgG in phosphate buffer saline having pH 7.5. The membrane is then dried at 37°C for at least 1 hour. The membrane with test dot for detection of anti dengue IgG antibodies, anti dengue IgM antibodies and a control dot is coated as follows. Each spot/dot for detection of anti dengue IgG antibody is coated with 250 nanogram to 5 µg, preferably lµg to 2 µg of monoclonal antihuman IgG antibody and/or polyclonal anti human IgG antibody. For coating the dot for detection of anti dengue IgG antibodies, 250 nanogram to 5µg, preferably 1µg to 2µg monoclonal anti human IgG and/or polyclonal antihuman IgG antibodies are added to 10 mM to 50 mM Sodium citrate buffer; pH 5.0 to 7.0 containing 150 mM Sodium chloride and 100 nanogram to 500 nanogram Bovine Serum albumin and mixed. The mixture is then dispensed at position of test dot meant for detection of anti dengue IgG antibodies. Each spot/dot for detection of anti dengue IgM antibody is coated with 250 nanogram to 10 µg, preferably 3 µg to 5 µg monoclonal anti human IgM antibody and/or polyclonal anti human IgM antibody. For coating the dot for detection of anti dengue IgM antibodies, 250 nanogram to 10 ug, preferably 3 µg to 5 µg monoclonal anti human IgM antibody and/or polyclonal anti human IgM antibody are added to l0mM to 50 mM sodium citrate buffer; pH 5.0 to 7.0 containing 150 mM Sodium chloride and 100 nanogram to 500 nanogram Bovine serum albumin and mixed. The mixture is then dispensed at position of dot for detection of anti dengue IgM antibodies. The control dot is immobilized with anti species IgG in phosphate buffer saline having pH 7.5. The membrane is then dried at 37°C for at least 1 hour. The conjugate used for detection of dengue NSl antigen consist of monoclonal anti dengue NSl antibody conjugated to gold colloid or modified microsphere and/or polyclonal anti dengue NSl antibody conjugated to gold colloid or modified microsphere. Biotinylated monoclonal anti dengue NSl antibodies and/or biotinylated polyclonal anti dengue NSl antibodies can also be used, followed by addition of conjugate. The conjugate then comprises streptavidin conjugated to gold colloid or modified microsphere. The conjugate used for detection of anti dengue IgG antibodies and anti dengue IgM antibodies is dengue viral lysate conjugated to gold colloid or modified microsphere and/or dengue recombinant antigen conjugated to gold colloid or modified microsphere. Biotinylated dengue viral lysate and/or biotinylated dengue recombinant antigens can also be used, followed by addition of conjugate. The conjugate then comprises streptavidin conjugated to gold colloid or modified microsphere. The sample diluent used for the test consist of 10 mM to 50 mM Tris buffer, pH 7.0 to 8.5 containing 1 M to 3 M Sodium Chloride, 0.02% to 0.2% Casein, 0.2% to 0.5% polyoxyethylene sorbitan monolaurate, 0.05% to 0.2% polyoxyethylene-23-lauryl ether, 0.2 % Sodium Azide and 0.01% to 0.05 %Merthiolate. The invention can be described more clearly with reference to following examples but these are for illustrative purpose only and same should not be construed to restrict the scope of invention. Example 1 For lateral flow immunochromatographic test device, 1 mg/ml of monoclonal antihuman IgG is added to 20 mM Sodium citrate buffer, pH 5.5, containing 150 mM Sodium Chloride, and mixed. The mixture is then dispensed at position of test line meant for detection of anti dengue IgG antibodies. 1.2 mg/ml of biotinylated monoclonal antihuman IgM antibody is added to 50 mM Sodium citrate buffer, pH 7.0, containing 150 mM Sodium Chloride, and mixed. The mixture is then dispensed at position of test line meant for detection of anti dengue IgM antibodies. 2.5 mg/ml of polyclonal anti dengue NS1 antibody is added to 10 mM MOPS buffer, pH 8.0, containing 150 mM Sodium Chloride and mixed. The mixture is then dispensed at position of test line meant for detection of dengue NS1 antigen. The strip is then dried at 37°C for 1 to 4 hours. The conjugate used for detection of anti dengue IgG antibody, anti dengue IgM antibody and dengue NS1 antigen is dengue viral lysate and dengue recombinant antigen conjugated to modified microsphere and monoclonal anti dengue NS1 antibody conjugated to modified microsphere (conjugation of antibody to modified microsphere is well known in art). The conjugate is dried on conjugate release pads and impregnated on strip adjacent to sample pad. Example 2 For bidirectional and two strip lateral flow immunochromatographic device as described in format 2 and 3, the strip for detection of anti dengue IgG and anti dengue IgM is coated as follows. For coating line for detection of anti dengue IgG antibodies, 1.5 mg/ml of biotinylated polyclonal anti human IgG antibody is added to 25 mM sodium citrate buffer, pH 7.0, containing 150 mM Sodium chloride and mixed. The mixture is then dispensed at position of test line meant for detection of dengue IgG line. For coating test line meant for detection of anti dengue IgM antibodies, 1 mg/ml of monoclonal anti human IgM is added to 10 mM Sodium citrate buffer, pH 6.0 containing 150 mM Sodium chloride and mixed. And 1.5 mg/ml of polyclonal anti human IgM is added to 20 mM Sodium citrate buffer, pH 5.0, containing 150 mM Sodium chloride. Mix both the solution together. Dispense the mixture at position of test line meant for detection of anti dengue IgM antibodies. The strip is then dried at 37°C for 1 to 4 hours. The conjugate used for detection of anti dengue IgG antibodies and anti dengue IgM antibodies is dengue viral lysate and dengue recombinant antigen conjugated to gold colloid. The conjugate is dried on conjugate release pad and is impregnated on strip adjacent to sample pad. The strip for detection of dengue NS1 antigen is coated as follows. 1.5 mg/ml of monoclonal anti dengue NS1 antibody is added to 25 mM MOPS buffer, pH 7.5, containing 150 mM Sodium Chloride. And 1 mg/ml of biotinylated polyclonal anti dengue NS1 antibody is added to 10 mM MOPS buffer, pH 7.0, containing 150 mM Sodium Chloride. Mix both the solution together and dispensed the mixture at position of test line meant for detection of dengue NS1 antigen. The strip is then dried at 37°C for 1 to 4 hours. The conjugate used is monoclonal anti dengue NS1 antibody conjugated to gold colloid. The conjugate is dried on conjugate release pad and impregnated on the strip. Example 3 For two holes flow through device as described in format 5, the membrane for detection of anti dengue IgG antibody and anti dengue IgG antibody is coated as follows. For each spot to be immobilized for detection of anti dengue IgG antibody, prepare 10 mM Sodium Citrate buffer, pH 7.0, containing 150 mM Sodium chloride and 200 nanogram Bovine Serum Albumin. To this add 1.3µgof monoclonal anti human IgG antibody. The mixture is then dispensed at position of test dot meant for detection of anti dengue IgG antibodies. For each spot to be immobilized for detection of anti dengue IgM antibody, prepare 25 mM Sodium citrate buffer of pH 6.5 containing 150 mM Sodium chloride and add 100 nanogram of BSA. To this buffer add 3µg of polyclonal anti human IgM antibodies. The mixture is then dispensed at position of test dot meant for detection of dengue IgM antibodies. The conjugate used for detection of anti dengue IgG and anti dengue IgM antibody is dengue recombinant antigen conjugated to gold colloid. The membrane for detection of dengue NSl antigen is coated as follows. For each spot to be immobilized for detection of dengue NS1 antigen, prepare 25 mM MOPS buffer, pH 7.0, containing 150 mM Sodium chloride and add 300 nanogram BSA. To this add 2.4 µg of monoclonal anti dengue NSl antibody. The mixture is then dispensed at position of test dot meant for detection of dengue NSl antigen. The conjugate used for detection of dengue NSl antigen is polyclonal anti dengue NSl antigen conjugated to gold colloid. I claim: 1. A kit for the simultaneous and differential detection of the presence of dengue NS1 antigen, dengue IgM antibodies and dengue IgG antibodies in human serum or plasma or whole blood comprising of assay buffer, sample diluent and a device either in immunochromatography or in flow through assay; having nitrocellulose membrane coated with plurality of lines/dots; such that line/dot for detection of anti dengue IgM antibodies is coated with anti human IgM, line/dot for detection of anti dengue IgG antibodies is coated with anti human IgG and line/dot for detection of dengue NS1 antigen is coated with anti dengue NS1 antibody as herein described; such that in lateral flow immunochromatographic format, the membrane is mounted on a strip having sample pad, absorbant pad and conjugate pad; and in flow through assay device, conjugate is in liquid form or in lyophilized form and membrane is mounted onto absorbant pad and is exposed through hole/s in the top cover; and the kit further comprising of assay buffer or sample diluent. 2. A kit as claimed in claim 1, wherein the lateral immunochromatography, the line for detection of dengue NS1 antigen is coated by adding 1 mg/ml to 3 mg/ml monoclonal anti dengue NS1 antibody or polyclonal anti dengue NS1 antibody or a mixture thereof to 10 mM to 50 mM MOPS buffer; pH 7.0 to 8.0 containing 150 mM Sodium chloride; mixing the solution; dispensing the mixture at position of test line meant for detection of dengue NS1 antigen. 3. A kit as claimed in claim 1, wherein the lateral flow immunochromatography, the line meant for detection of anti dengue IgM antibody is coated by adding 0.5 mg/ml to 1.5 mg/ml of monoclonal anti human IgM or polyclonal anti human IgM antibodies or a mixture thereof to 10 mM to 50 mM Sodium citrate buffer, pH 5.0 to 7.0 containing 150 mM sodium chloride; mixing the solution; dispensing the mixture at position of test line meant for detection of anti dengue IgM antibodies. 4. A kit as claimed in claim 1, wherein the lateral flow immunochromatography, the test line meant for detection of anti dengue IgG antibody is coated by adding 1 mg/ml to 2.5 mg/ml of monoclonal anti human IgG or polyclonal anti human IgG antibodies or a mixture thereof to 10 mM to 50 mM sodium citrate buffer; pH 5.0 to 7.0 containing 150 mM Sodium chloride; mixing the solution; dispensing the mixture at position of test line meant for detection of anti dengue IgG antibodies. 5. A kit as claimed in claims 1 and 2, wherein the lateral flow immunochromatography, the conjugate used for detection of dengue NS1 antigen consist of monoclonal anti dengue NS1 antibodies conjugated to gold colloid or modified microsphere or polyclonal anti dengue NS1 antibody conjugated to gold colloid or modified microsphere or a combination thereof. 6. A kit as claimed in claim 1, 3 and 4, wherein the lateral flow immunochromatography, the conjugate used for detection of anti dengue IgG antibodies and anti dengue IgM antibodies consist of Dengue viral lysate conjugated to gold cold colloid or modified microsphere and/or dengue recombinant antigen conjugated to gold colloid or modified microsphere. 7. A kit as claimed in claims 1, 2, 3 and 4, wherein the assay buffer consisting of lOmM to 50 mM Disodium hydrogen phosphate, lOmM to 50 mM Sodium dihydrogen phosphate, 20mM TRIS, 0.5 M to 2 M Potassium chloride, 0.5%-3% Goat albumin or sheep albumin, 0.2% preservative like sodium Azide and l%-3% zwitterionic detergent such as CHAPSO (3-[(3-cholamidopropyl)dimethyl ammonio] -2-hydroxy-l-propanesulfonate);assay buffer has pH 7 to 8. 8. A kit as claimed in claim 1, wherein flow through assay, the test dot for detection of dengue NS1 antigen is coated by adding 500 nanogram to 5 jag, preferably 2 ug to 3 ug monoclonal anti dengue NS1 antibody or polyclonal anti dengue NS1 antibody or a mixture thereof to buffer comprising 10 mM to 50 mM MOPS; pH 7.0 to 8.0, 150 mM Sodium chloride and 100 nanogram to 500 nanogram Bovine Serum albumin; mixing the solution; dispensing the mixture at the position of dot meant for detection of dengue NS1 antigen. 9. A kit as claimed in claims 1, wherein flow through assay, the test dot for detection of anti dengue IgG is coated by adding 250 nanogram to 5ug, preferably 1 ug to 2 ug monoclonal anti human IgG or polyclonal antihuman IgG antibodies or a mixture thereof to buffer comprising 10 mM to 50 mM Sodium citrate; pH 5.0 to 7.0, containing 150 mM Sodium chloride and 100 nanogram to 500 nanogram Bovine Serum albumin; mixing the solution; dispensing the mixture at the position of test dot meant for detection of anti dengue IgG antibodies. 10. A kit as claimed in claims 1, wherein flow through assay device, the test dot for detection of anti dengue IgM is coated by adding 250 nanogram to 10 ug, preferably 3 ug to 5 ug monoclonal anti human IgM antibody or polyclonal anti human IgM antibody or a mixture thereof to buffer comprising lOmM to 50 mM sodium citrate; pH 5.0 to 7.0, containing 150 mM Sodium chloride and 100 nanogram to 500 nanogram Bovine serum albumin; mixing the solution; dispensing the mixture at position of dot meant for detection of anti dengue IgM antibodies. 11. A kit as claimed in claims 1 and 8, wherein the conjugate used for the detection of dengue NS1 antigen consisting of monoclonal anti dengue NS1 antibody conjugated to gold colloid or modified microsphere or polyclonal anti dengue NS1 antibody conjugated to gold colloid or modified microsphere or a mixture thereof; OR streptavidin conjugated to gold colloid or modified microsphere, such that biotinylated monoclonal anti dengue NS1 antibody or biotinylated polyclonal anti dengue NS1 antibody or a mixture of two are added prior to the addition of streptavidin conjugate. 12. A kit as claimed in claim 1, 9 and 10, wherein the conjugate used for detection of anti dengue IgG and anti dengue IgM consisting of dengue viral lysate conjugated to gold colloid or modified microsphere or dengue recombinant antigen conjugated to gold colloid or modified microsphere or a combination thereof; Or streptavidin conjugated to gold colloid or modified microsphere, such that biotinylated dengue viral lysate or biotinylated dengue recombinant antigens or a mixture of two are added prior to addition of streptavidin conjugate. 13. A kit as claimed in claims 1, 8, 9 and 10, wherein the sample diluent consisting of 10 mM to 50 mM Tris buffer, pH 7.0 to 8.5 containing 1 M to 3 M Sodium Chloride, 0.02% to 0.2% Casein, 0.2% to 0.5% polyoxyethylene sorbitan monolaurate, 0.05% to 0.2% polyoxyethylene-23-lauryl ether, 0.2 % Sodium Azide and 0.01% to 0.05 % Merthiolate. 14. A kit for detection of dengue NSl antigen, anti dengue IgG antibodies, anti dengue IgM antibodies substantially described herein with reference to and as illustrated in accompanying drawing.

Full Text

Field of the Invention
The present invention relates to a device for the early detection of infection with dengue flavivirus in human serum or plasma or whole blood.
It is an improved version of the patent "2484/DEL/2007: A device and a kit for analyzing the presence of anti dengue IgM and anti dengue IgG in a sample and process thereof which is pending in Indian Patent office.
More specifically, the present invention relates to the simultaneous and differential detection of dengue NSl antigen and dengue antibodies present in human serum or plasma or whole blood. Further more the subject invention allows one to detect dengue antigens in early acute stage of infection even prior to the development of antibodies thereby allowing early detection of the disease.
More specifically, the subject invention relates to a rapid diagnostic kit for the detection of dengue NSl antigen and dengue antibodies present in human serum or plasma or whole blood.
It is a visual, rapid, sensitive and accurate immunoassay for the rapid and differential detection of dengue antigen and dengue antibodies in human serum or plasma or whole blood.
Background of Invention
Dengue fever is a viral disease transmitted by mosquitoes and caused by four serotypes of dengue virus (DEN): DEN: 1, DEN: 2, DEN: 3 and DEN: 4. Dengue fever is the most important arthropod-borne viral disease affecting humans and according to the World Health Organisation estimates, its incidence has increased by a factor of 30 over the last 50 years.

Dengue viruses are transmitted to humans by Stegomyia aegypti (formerly Aedes aegypti) mosquitoes and cause a wide range of symptoms from unapparent or mild disease to severe haemorrhagic form.
The infection agent, dengue virus has a simple stranded RNA with positive polarity which comprises of nucleotides and poly protein amino acids. It is separated into structural and non structural proteins. The structural proteins are C, prM and E and the nonstructural proteins are NSl, NS2a, NS2b, NS3, NS4a, NS4b and NS5. NSl a non structural protein was identified for the first time in 1970.
NSl is a highly conserved glycoprotein which appears essential for the virus viability although no precise function has yet been assigned to it. During the cell infection, NSl is found associated with intracellular organelles or is alternatively transported through the secretory pathway to the cell surface. A soluble hexameric form may be released in a glycosylation-dependent fashion from infected mammalian cells but not from vector derived mosquito cells.
Dengue virus infection is currently detected by means of several biological tests. Virus isolation on mosquito cells, viral RNA detection by reverse transcription - PCR (RT-PCR) or serological tests such as immunoglobulin M (IgM) capture enzyme linked immunosorbent assay (MAC-ELISA). RT-PCR remains expensive and its routine use in the clinical diagnostic laboratories is difficult. Commercial MAC-ELISA kits are available but they cannot be used for early diagnosis, because IgM does not become detectable until 5 to 10 days after the onset of illness in secondary infection.
In primary infection, IgM and IgG arise approximately 5 and 14 days respectively after symptoms onset. In secondary infection, IgM levels are low or undetectable while IgG arise 1-2 days after symptoms onset with higher levels than in primary infection. More recently, detection in patient's sera of circulating dengue virus in nonstructural protein NSl has been described as an alternative method for early diagnosis. NSl antigens were found circulating from the first day and up to 9 days after the onset of fever with

comparable levels observe in primary and secondary infection. Dengue virus specific IgM first appeared at about day 3 and was prominent from day 5 onward. IgM did not seem to interfere with NS1 detection during the clinical phase.
There is no commercially available vaccine against the dengue virus. In the absence of vaccine, it is necessary to monitor epidemics. To do this, active monitoring programs have in particular been set up by the World Health organization. It essentially comprises the monitoring of cases of fever and of vector insects and the serological and virological screening of individuals having a fever and suspected of being infected with the dengue virus.
The present invention is a device for early and rapid detection of dengue in human serum or plasma or whole blood. Dengue NS1 antigen can be detected during the acute and the symptomatic phase of dengue. Antibodies to dengue can be detected during the entire period of infection.
Prior Art
US 2005186562 (Al) The invention concerns a method for early detection of a flavivirus-induced infection, comprising the detection of the flavivirus non-structural glycoprotein NS1 in a biological sample during the clinical phase of the infection, by an immumological method using at least two identical or different antibodies, the first antibody consisting of polyclonal or monoclonal antibodies pre-selected for their high affinity for said NS 1 protein hexameric in shape.
US 5824506: Peptide antigens derived from the dengue virus type-2 glycoprotein NS1 are provided. The peptide antigens are specifically immunoreactive with sera from individuals infected with the dengue virus. The antigens are useful as diagnostic tools in determining whether an individual has been or is infected with dengue virus, and for discriminating between infection with dengue virus and infection with related flaviviruses. The antigens are also useful in vaccine compositions for immunizing individuals against infection with the dengue virus.

WO 2003/048184 A3: The present invention relates to NS1 proteins or parts thereof of Flaviviruses, in particular of Dengue viruses useful for vaccination against said Flavivirus and against one or more other Flaviviruses. The invention further concerns the NS1 protein or parts thereof of one Dengue virus serotype, in particular serotype 2, useful for vaccination against Dengue viruses from all serotypes. The invention further concerns DNA comprising an expression cassette coding for a Flavivirus NS1 or parts thereof, vectors, comprising said DNA and vaccines containing or expressing a Flavivirus NS1.
These patents do not disclose a fast, cost effective, simple and rapid method for
dengue diagnosis that combines sensitivity and specificity.
It does not provide a method of detection of dengue for a man of ordinary skill to
perform.
The present invention is a rapid, qualitative, solid phase immunochromatographic
assay for differential detection of dengue IgM antibodies, dengue IgG antibodies
and dengue NS1 antigen present in human serum or plasma or whole blood.
The object of the present invention is to provide a novel, inexpensive, simple,
rapid diagnostic device and a kit which differentially detects the dengue NS1
antigen, dengue IgM antibodies and dengue IgG antibodies present in human
serum or plasma or whole blood.
It is another object of the present invention to provide a safe diagnostic assay for
the detection of dengue NS1 antigen, anti dengue IgM antibodies and anti
dengue IgG antibodies present in human serum or plasma or whole blood.
Summary of the Invention
The object of the present invention is to provide a rapid test device and a test kit which detects the dengue NS1 antigen, dengue IgM antibodies and dengue IgG antibodies present in human serum or plasma or whole blood. In the present device control line is used to test the workability of the reagent. The present invention provides an effective device having sensitivity and specificity of 99% and 99% respectively.

The device for the test can be in two formats, i.e. lateral flow immunochromatographic test device or flow through test device. The lateral flow immunochromatographic test device comprises of top cover and a bottom receptacle adapted to be pressed with each other. The top cover is provided with well/s, through which sample is added on to the sample pad/s. The bottom receptacle is provided with track/s for holding strip/s having nitrocellulose membrane. The sample pad is provided at one end of strip and absorbant pad on other end. The membrane is having lines meant for detection of dengue NS1 antigen, anti dengue IgM antibodies and anti dengue IgG antibodies. The line for detection of,dengue NS1 antigen is immobilized with anti dengue NS1 antibodies, and the line for detection of anti dengue IgM antibodies is immobilized with anti human IgM antibodies and the line for detection of anti dengue IgG antibody is immobilized with anti human IgG antibody. Control line is also immobilized on membrane to test the working of reagents. The conjugate is impregnated on to the conjugate pad of strip. The second form of device is flow through test device. The device consists of top cover and a bottom receptacle. The top cover is provided with a hole in the centre through which membrane is exposed. The bottom receptacle is provided with an absorbant pad over which nitrocellulose membrane is mounted, exposing through centre of top cover. The conjugate is in solution form/lyophilized form. The membrane is coated with dots meant for detection of dengue NS1 antigen, anti dengue IgM antibodies and anti dengue IgG antibodies. The dot for detection of dengue NS1 antigen is immobilized with anti dengue NS1 antibodies, and the dot for detection of anti dengue IgM antibody is immobilized with antihuman IgM antibodies, and the dot for detection of anti dengue IgG antibody is immobilized with antihuman IgG antibody. The control dot is also provided to test the working of reagents simultaneously.
Brief Description with figures
Figure 1 shows the dengue detection device in format 1.
Figure 1(a) shows only control line in colour.
Figure 1(b) shows the control line and line for the detection of dengue IgM antibodies in
colour.

Figure 1(c) shows the control line and the line for detection of dengue IgG antibodies in
colour.
Figure 1(d) shows the control line and the line for the detection of dengue NSl antigen in
colour.
Figure 1(e) shows the control line as well as the line for the detection of dengue IgM
antibodies and dengue IgG antibodies in colour.
Figure 1(f) shows the control line and the line for the detection of dengue IgM antibodies
and the line for the detection of dengue NSl antigen in colour.
Figure 1(g) shows the control line and line for the detection of the dengue IgG antibodies
and the line for the detection of dengue NSl antigen in colour.
Figure 1(h) shows the control line and the line for the detection of dengue IgM antibodies
and dengue IgG antibodies and dengue NSl antigen detection line in colour.
Figure 2 shows the device in format 2.
Figure 2(a) shows only the control line in colour.
Figure 2(b) shows the control line and the line for detection of dengue IgM antibodies in
colour.
Figure 2(c) the control line and line for detection of dengue IgG antibodies in colour.
Figure 2(d) shows the control line and the line for detection of dengue NSl antigen in
colour.
Figure 2(e) shows the control line and the line for the detection of anti dengue IgG
antibodies and anti dengue IgM antibodies in colour.
Figure 2(f) shows the control line and the line for detection of dengue IgM antibodies and
dengue NSl antigen in colour.
Figure 2(g) shows the control line and the line for the detection of dengue IgG antibodies
and dengue NS1 antigen in colour.
Figure 2(h) shows the control line and the line for the detection of dengue IgM antibodies
and dengue IgG antibodies and dengue NSl antigen detection line in colour.
Figure 3 shows the device in Format 3.
Figure 3(a) shows the device with only control line in colour.

Figure 3(b) shows the control line and the line for detection of dengue IgM antibodies in
colour.
Figure 3(c) shows the control line and the line for the detection of the dengue IgG
antibodies in colour.
Figure 3(d) shows the control line and the line for the detection of dengue NSl antigen in
colour.
Figure 3(e) shows the control line and the line for the detection of dengue IgM antibodies
and the line for detection of dengue IgG antibodies in colour.
Figure 3(f) shows the control line and the line for the detection of dengue IgM antibodies
and the line for the detection of dengue NSl antigen in colour.
Figure 3(g) shows the control line and the line for the detection of dengue IgG antibodies
and the line for detection of dengue NSl antigen in colour.
Figure 3(h) shows the control line and the line for the detection of anti dengue IgM
antibodies, anti dengue IgG antibodies and dengue NS 1 antigen detection line in colour.
Figure 4 shows the flow through device in format 4.
Figure 4(a) shows the device with only control dot in colour.
Figure 4(b) shows the control dot and the dot for the detection of dengue IgG antibodies
in colour.
Figure 4(c) shows the control dot and the dot for the detection of dengue IgM antibodies
in colour.
Figure 4(d) shows the control dot and the dot for the detection of dengue NS 1 antigen in
colour.
Figure 4(e) shows the control dot and the dot for the detection of dengue IgM antibodies
and dengue IgG antibodies in colour.
Figure 4(f) shows the control dot and the dot for detection of dengue IgM antibodies and
the dot for the detection of dengue NS 1 antigen in colour.
Figure 4(g) shows the control dot and the dot for the detection of dengue IgG antibodies
and dot for detection of dengue NS 1 antigen in colour.
Figure 4(h) shows the control dot and the dot for the detection of dengue IgM antibodies
and dengue IgG antibodies and dengue NS 1 antigen in colour.

Figure 5 shows-the device in format 5.
Figure 5(a) shows the device with only control dot in colour.
Figure 5(b) shows the control dot and the dot for the detection of dengue IgM antibodies
in colour.
Figure 5(c) shows the control dot and the dot for the detection of dengue IgG antibodies
in colour.
Figure 5(d) shows the control dot and the dot for detection of dengue NSl antigen in
colour.
Figure 5(e) shows the control dot and the dot for the detection of dengue IgM antibodies
and dengue IgG antibodies in colour.
Figure 5(f) shows the control dot and the dot for detection of the dengue IgM antibodies
and dengue NS1 antigen in colour.
Figure 5(g) shows the control dot and the dot for the detection of the dengue IgG
antibodies and the dengue NSl antigen detection dot in colour.
Figure 5(h) shows the control dot and the dot for the detection of the dengue IgM
antibodies and dengue IgG antibodies and dengue NS 1 antigen in colour.
On addition of sample, if only the control line/dot appears in colour, then the result is interpreted as negative and the sample is not reactive for Dengue. If the line/dot for detection of anti dengue IgM and the control line/dot are in colour, then the result is interpreted as positive and sample is reactive for dengue IgM antibody i.e. it is a case of primary dengue-infection. If the control line/dot and the line/dot for the detection of anti dengue IgG antibodies are in colour, then the result is interpreted as positive and sample is reactive for dengue IgG antibody and it is a case of secondary infection. If the control line/dot and the line/dot for the detection of dengue NS 1 antigen are in colour, then the result is interpreted as positive and sample is reactive for dengue. If the control line/dot, line/dot for detection of dengue IgM antibodies and line/dot for detection of dengue IgG antibodies are in colour, then the result is interpreted as positive and the sample is reactive for dengue and it is a case of secondary infection. If the control line/dot, the line/dot for the detection of dengue IgM antibodies and the line/dot for the detection of

dengue NS1 antigen are in colour, then the result is interpreted as positive and sample is reactive for dengue. If the control dot/line, the line/dot for detection of dengue IgG antibodies and the line/dot for detection of dengue NS1 antigen are in colour, then the sample is interpreted as positive and the sample is reactive for dengue. If the control line/dot, the anti dengue IgM antibody detection line/dot, anti dengue IgG detection line/dot and the dot/line for the detection of dengue NS1 antigen are in colour, then the result is interpreted as positive and the sample is reactive for dengue.
Detailed Description
The object of the present invention is to provide a device for early detection of dengue flaviviral infection. It comprises of a device for the simultaneous and differential detection of non-structural glycoprotein NS1 of dengue flavivirus, anti dengue IgM antibodies and anti dengue IgG antibodies in human serum or plasma or whole blood.
The device can be based on lateral flow immunochromatographic test device or in form of flow through assay format. The device comprises of top cover and bottom receptacles adapted to be fit into each other. The bottom receptacle is having nitrocellulose membrane. A mean for pouring sample is provided in the test device. An absorbant pad is provided to absorb excess buffer. The membrane is coated with line/dot for detection of dengue NS1 antigen, anti dengue IgM antibodies and anti dengue IgG antibodies. In an embodiment of present invention, the device can be prepared in different formats. The process for preparation of device for the detection of presence of dengue NS1 antigen, anti dengue IgM antibodies and anti dengue IgG antibodies is as follows.
Format 1
In this format, the device (as shown in figure 1) is based on principle of lateral flow immunochromatography. The casing comprises of a top cover and a bottom receptacle adapted to be pressed with each other. The bottom receptacle has a base for holding the strip. The strip is mounted with a nitrocellulose membrane, and provided with a sample pad at one end of the strip and an absorbant pad on the other end of the strip. The strip is impregnated with conjugate pad. A sample well is provided at one end on the top cover

of device through which the sample is dropped onto sample pad. The membrane is provided with lines for the detection of dengue NS1 antigen, anti dengue IgM antibodies and anti dengue IgG antibodies. The line for the detection of dengue NS1 antigen is immobilized with anti dengue NS1 antibodies, the line for the detection of anti dengue IgM antibodies is immobilized with anti human IgM antibodies and the line for the detection of anti dengue IgG antibodies is immobilized with antihuman IgG antibodies. The sample is dropped on the sample pad through sample cup; it will migrate laterally through membrane. Analytes if present in sample will bind to signal reagent (conjugate) forming an analyte- conjugate complex. This complex will further move towards the membrane and bind to the corresponding capture reagent immobilized on membrane, resulting in formation of coloured line.
The test lines for detection of anti dengue IgG antibodies, anti dengue IgM antibodies and dengue NS1 antigen are coated as follows.
1 mg/ml to 2.5 mg/ml of monoclonal anti human IgG and/or polyclonal anti human IgG antibodies are added to 10 mM to 50 mM sodium citrate buffer, pH 5.0 to 7.0 containing 150 mM Sodium chloride and mixed. The mixture is then dispensed at the position of test line meant for detection of anti dengue IgG antibodies. The antibodies can be biotinylated using method well known in art and these biotinylated antibodies can then immobilized at respective position on membrane.
0.5 mg/ml to 1.5 mg/ml of monoclonal anti human IgM and/or polyclonal anti human IgM antibodies are added to 10 mM to 50 mM Sodium citrate buffer; pH 5.0 to 7.0 containing 150 mM sodium chloride and mixed. The mixture is then dispensed at the position of test -line meant for detection of anti dengue IgM antibodies. The antibodies can be biotinylated using method well known in art and these biotinylated antibodies can then immobilized at respective position on membrane.
1 mg/ml to 3 mg/ml of monoclonal anti dengue NS1 antibody and/or polyclonal anti dengue NS1 antibody are added to 10 mM to 50 mM MOPS buffer; pH 7.0 to 8.0 containing 150 mM sodium chloride and mixed. The mixture is then dispensed at position of test line meant for detection of dengue NS1 antigen. The antibodies can also be

biotinylated using method well known in art and these biotinylated antibodies can then be
immobilized at respective position on membrane.
The control line is immobilized with anti species IgG in phosphate buffer saline having
pH 7.5.
The strip is finally dried at 37°C for 1 to 4 hours.
The conjugate used for detection of anti dengue IgG antibody, anti dengue IgM antibodies and dengue NS1 antigen in human serum or plasma or whole blood is Dengue viral lysate conjugated to gold colloid or modified microsphere and/or dengue recombinant antigen conjugated to gold colloid or modified microsphere; And monoclonal anti dengue NS1 antibodies conjugated to gold colloid or modified microsphere and/or polyclonal anti dengue NS1 antibody conjugated to gold colloid or modified microsphere.
The assay buffer used for performing the test consists of lOmM to 50 mM Disodium hydrogen phosphate, lOmM to 50 mM Sodium dihydrogen phosphate, 20mM TRIS, 0.5 M to 2 M Potassium chloride, 0.5%-3% Goat albumin or sheep albumin, 0.2% preservative like sodium Azide and l%-3% zwitterionic detergent such as CHAPSO (3-[(3 -cholamidopropyl)dimethylammonio] -2-hydroxy-1 -propanesulfonate); the assay buffer has pH 7 to 8.
Format 2
In this format, the device is a bi-directional device (as shown in fig 2) and is based on principle of lateral flow immunochromatography. The casing comprises of top cover and bottom receptacles adapted to be pressed with each other. The bottom receptacle has a base for holding the strip. The strip is provided with a sample pad in the centre, and absorbant pads are provided on both ends of the strip. The strip is mounted with nitrocellulose membrane on both sides of sample pad. The top cover is having means for adding sample in form of sample well in the centre of top cover. The corresponding conjugates are impregnated on both sides of the strip. The membrane on one side of sample pad is meant for detection of dengue NS 1 antigen and is immobilized with anti

dengue NS1 antibodies. The membrane on other side of sample pad is meant for detection of anti dengue IgG and anti dengue IgM antibodies and is immobilized with anti human IgG antibodies and anti human IgM antibodies. Both the membranes are provided with control lines to test the workability of reagents.
The sample when added on to the sample pad through sample cup will migrate laterally on both sides, such that analytes if present in the sample will bind to the signal reagents (conjugate) forming a complex. The complex will further move, and bind to the corresponding capture reagent immobilized on the membrane, resulting in formation of coloured line.
The test lines on the membrane present on one side of sample pad meant for detection of
anti dengue IgG and anti dengue IgM antibodies is coated as follows.
1 mg/ml to 2.5 mg/ml of monoclonal anti human IgG and/or polyclonal anti human IgG
antibodies are added to 10 mM to 50 mM sodium citrate buffer; pH 5.0 to 7.0 containing
150 mM Sodium chloride and mixed. The mixture is then dispensed at position of test
line meant for detection of anti dengue IgG antibodies. The antibodies can be biotinylated
using method well known in art and these biotinylated antibodies can then be
immobilized at respective position on membrane.
0.5 mg/ml to 1.5 mg/ml of monoclonal anti human IgM and/or polyclonal anti human
IgM antibodies are added to 10 mM to 50 mM Sodium citrate buffer; pH 5.0 to 7.0
containing 150 mM sodium chloride and mixed. The mixture is then dispensed at position
of test line meant for detection of anti dengue IgM antibodies. The antibodies can be
biotinylated using method well known in art and these biotinylated antibodies can then be
immobilized at respective position on membrane.
The control line is immobilized with anti species IgG in phosphate buffer saline having
pH 7.5.
The Strip is then dried at 37°C for 1 to 4 hours.
The test line on the membrane present on other side of sample pad meant for detection of dengue NS1 antigen is coated as follows.

1 mg/ml to 3 mg/ml of monoclonal anti dengue NS1 antibody and/or polyclonal anti
dengue NS1 antibody are added to 10 mM to 50 mM MOPS buffer; pH 7.0 to 8.0
containing 150 mM sodium chloride and mixed. The mixture is then dispensed at position
of test line meant for detection of dengue NS1 antigen. The antibodies can be biotinylated
using method well known in art and these biotinylated antibodies can then be
immobilized at respective position on membrane.
The control line is immobilized with anti species IgG in phosphate buffer saline having
pH 7.5.
The strip is then dried at 37°C for 1 to 4 hours.
The conjugate used for detection of anti dengue IgG antibody and anti dengue IgM antibodies in human serum or plasma or whole blood is Dengue viral lysate conjugated to gold cold colloid or modified microsphere and/or dengue recombinant antigen conjugated to gold colloid or modified microsphere.
The conjugate used for detection of dengue NS1 antigen consist of monoclonal anti dengue NS1 antibodies conjugated to gold colloid or modified microsphere and/or polyclonal anti dengue NS1 antibody conjugated to gold colloid or modified microsphere.
The assay buffer used for performing the test consist of lOmM to 50 mM Disodium hydrogen phosphate, lOmM to 50 mM Sodium dihydrogen phosphate, 20mM TRIS, 0.5 M to 2 M Potassium chloride, 0.5%-3% Goat albumin or sheep albumin, 0.2% preservative like sodium Azide and l%-3% zwitterionic detergent such as CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1 -propanesulfonate); the assay buffer has pH 7 to 8.
Format 3
In this format, the device is as shown in Figure 3. The device is based on principle of lateral flow immunochromatography. The device comprises top cover and bottom receptacle adapted to be pressed with each other. Two sample wells, adjacent to each other are provided at one end of the top cover of device, through which sample is added onto the sample pad. The bottom receptacle is provided with two base, adjacent to each

other for holding strips. Two strips are provided, each with an absorbant pad on one end and a sample pad on other end, such that sample pad faces the sample well when mounted on the base. The strips are mounted with a nitrocellulose membrane, and are impregnated with corresponding conjugate pad. One strip is coated with control line and a test line for detection of dengue NS1 antigen, whereby test line is immobilized with anti dengue NS1 antibodies. The other strip is coated with control line and two test lines, one for detection of anti dengue IgM antibodies and the other for the detection of anti dengue IgG antibodies. The test line on second strip meant for detection of anti dengue IgM antibodies is immobilized with anti human IgM antibodies and test line meant for detection of dengue IgG antibodies is immobilized with anti human IgG antibodies.
When samples are added through sample well on sample pad, it will migrate laterally through strips. The analytes if present in sample will bind to the corresponding signal reagent (conjugate) forming a complex. The complex will further move on strip and will bind with corresponding capture reagent immobilized on the strip, leading to formation of coloured line.
The first strip having control line and two test lines meant for detection of anti dengue IgG and anti dengue IgM antibodies is coated as follows.
1 mg/ml to 2.5 mg/ml of monoclonal anti human IgG and/or polyclonal anti human IgG antibodies are added to 10 mM to 50 mM sodium citrate buffer; pH 5.0 to 7.0 containing 150 mM Sodium chloride and mixed. The mixture is then dispensed at position of test line meant for detection of anti dengue IgG antibodies. The antibodies can be biotinylated using method well known in art and these biotinylated antibodies can then be immobilized at respective position on membrane.
0.5 mg/ml to 1.5 mg/ml of monoclonal anti human IgM and/or polyclonal anti human IgM antibodies are added to 10 mM to 50 mM Sodium citrate buffer; pH 5.0 to 7.0 containing 150 mM sodium chloride and mixed. The mixture is then dispensed at position of test line meant for detection of anti dengue IgM antibodies. The antibodies can be biotinylated using method well known in art and these biotinylated antibodies can then immobilized at respective position on membrane.

The control line is immobilized with anti species IgG in phosphate buffer saline having
pH 7.5.
The strip is then dried at 37°C for 1 to 4 hours.
The second strip having line for dengue NSl antigen detection and control line is coated
as follows.
1 mg/ml to 3 mg/ml of monoclonal anti dengue NSl antibody and/or polyclonal anti
dengue NSl antibody are added to 10 mM to 50 mM MOPS buffer; pH 7.0 to 8.0
containing 150 mM sodium chloride and mixed. The mixture is then dispensed at position
of test line meant for detection of dengue NSl antigen. The antibodies can be biotinylated
using method well known in art and these biotinylated antibodies can then immobilized at
respective position on membrane.
The control line is immobilized with anti species IgG in phosphate buffer saline having
pH 7.5.
The strip is then dried at 37°C for 1 to 4 hours.
The conjugate used for detection of anti dengue IgG antibody and anti dengue IgM antibodies in human serum or plasma or whole blood is Dengue viral lysate conjugated to gold cold colloid or modified microsphere and/or dengue recombinant antigen conjugated to gold colloid or modified microsphere.
The conjugate used for detection of dengue NSl antigen consist of monoclonal anti dengue NSl antibodies conjugated to gold colloid or modified microsphere and/or polyclonal anti dengue NSl antibody conjugated to gold colloid or modified microsphere.
The assay buffer used for performing the test consist of lOmM to 50 mM Disodium hydrogen phosphate, lOmM to 50 mM Sodium dihydrogen phosphate, 20mM TRIS, 0.5 M to 2 M Potassium chloride, 0.5%-3% Goat albumin or sheep albumin, 0.2% preservative like sodium Azide and l%-3% zwitterionic detergent such as CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1 -propanesulfonate); the assay buffer has pH 7 to 8.

Format 4
In this format, the device is as shown in figure 4. The casing comprises of rectangular top cover and bottom cover, tightly pressed with each other. A circular hole is provided in the top cover at the centre of the device. The bottom receptacle is provided with absorbant pad. The membrane is cut from the cellulose material and is mounted on an absorbant pad housed in casing, exposed through the hole in centre of top cover
The device is a flow through test device whereby membrane is coated with four dots. The first dot is immobilized with anti dengue NSl antibodies and is meant for detection of dengue NSl antigen in the sample. The second dot is immobilized with anti human IgM antibodies and is meant for detection of anti dengue IgM antibodies in the sample. The third dot is immobilized with anti human IgG antibodies and is meant for detection of dengue IgG antibodies in sample. The fourth dot is control dot and is meant for testing the workability of reagent.
The sample is added to the membrane through circular hole provided on top cover. The analytes if present in the sample will bind to the corresponding capture reagent immobilized as dots on membrane forming a complex. When conjugate solution is added to the device, the conjugate will bind to the corresponding complex leading to formation of coloured dot.
The membrane with test dots for detection of anti dengue IgG antibodies, anti dengue IgM antibodies and dengue NSl antigen is coated as follows.
Each spot/dot for detection of anti dengue IgG antibody is coated with 250 nanogram to 5 ug, preferably 1 jig to 2 (j,g of monoclonal antihuman IgG antibody and/or polyclonal anti human IgG antibody.
For coating the dot for detection of anti dengue IgG antibodies, 250 nanogram to 5ug, preferably 1 jxg to 2 u£ monoclonal anti human IgG and/or polyclonal antihuman IgG antibodies are added to 10 mM to 50 mM Sodium citrate buffer; pH 5.0 to 7.0 containing 150 mM Sodium chloride and 100 nanogram to 500 nanogram Bovine Serum albumin and mixed. The mixture is then dispensed at position of test dot meant for detection of anti dengue IgG antibodies.

Each spot/dot for detection of anti dengue IgM antibody is coated with 250 nanogram to 10 ug, preferably 3 ug to 5 ug monoclonal anti human IgM antibody and/or polyclonal anti human IgM antibody.
For coating the dot for detection of anti dengue IgM antibodies, 250 nanogram to 10 ug, preferably 3 ug to 5 ug monoclonal anti human IgM antibody and/or polyclonal anti human IgM antibody are added to l0mM to 50 mM sodium citrate buffer; pH 5.0 to 7.0 containing 150 mM Sodium chloride and 100 nanogram to 500 nanogram Bovine serum albumin and mixed. The mixture is then dispensed at position of dot for detection of anti dengue IgM antibodies.
Each spot/dot for detection of dengue NSl antigen is coated with 500 nanogram to 5 ug,
preferably 2 ug to 3 ug monoclonal anti dengue NSl antibody and/or polyclonal anti
dengue NS1 antibody.
For coating the dot for detection of dengue NSl antigen, 500 nanogram to 5 ug,
preferably 2 ug to 3 ug monoclonal anti dengue NSl antibody and/or polyclonal anti
dengue NSl antibody are added to 10 mM to 50 mM MOPS buffer; pH 7.0 to 8.0
containing 150 mM Sodium chloride and 100 nanogram to 500 nanogram Bovine Serum
albumin and mixed. The mixture is then dispensed at position of dot meant for detection
of dengue NS 1 antigen.
The control dot is immobilized with anti species IgG in phosphate buffer saline having
pH 7.5.
The membrane is then dried at 37°C for at least 1 hour.
The conjugate used for detection of anti dengue IgG antibodies, anti dengue IgM antibodies and dengue NSl antigen comprises dengue viral lysate conjugated to gold colloid or modified microsphere and/or dengue recombinant antigen conjugated to gold colloid or modified microsphere; And monoclonal anti dengue NSl antibody conjugated to gold colloid or modified microsphere and/or polyclonal anti dengue NSl antibody conjugated to gold colloid or modified microsphere.

Biotinylated dengue recombinant antigen and/or biotinylated dengue viral lysate and biotinylated monoclonal anti dengue NS1 antibodies and/or biotinylated polyclonal anti dengue NS1 antibodies can also be used for detection of anti dengue IgG and anti dengue IgM antibodies dengue NS1 antigen followed by addition of conjugate. The conjugate then comprises streptavidin conjugated to gold colloid or modified microsphere.
The sample diluent used for the test consist of 10 mM to 50 mM Tris buffer, pH 7.0 to 8.5 containing 1 M to 3 M Sodium Chloride, 0.02% to 0.2% Casein, 0.2% to 0.5% polyoxyethylene sorbitan monolaurate, 0.05% to 0.2% polyoxyethylene-23-lauryl ether, 0.2 % Sodium Azide and 0.01% to 0.05 %Merthiolate.
Format 5
In this format, the device used is as shown in figure 5. The casing comprises of rectangular top cover and bottom cover, tightly pressed with each other. Two circular holes are provided in the top cover of the casing. The bottom receptacle is provided with absorbant pad. Two circular membranes are cut from the cellulose material and are mounted on the absorbant pad housed in bottom part of casing, which are exposed through the holes of top cover.
The device is a flow through test device whereby two membranes are used. The first membrane is immobilized with dot for detection of dengue NS1 antigen and a control dot. The second membrane is immobilized with dots for detection of anti dengue IgM antibodies and anti dengue IgG antibodies; and a control dot. The dot on first membrane meant for detection of dengue NS1 antigen is immobilized with anti dengue NS1 antibodies. The dot on second membrane meant for detection of dengue IgG antibodies is immobilized with anti human IgG antibodies and the dot meant for detection of dengue IgM antibodies is immobilized with antihuman IgM antibodies.
The samples are added to the membrane through holes in the top cover. The analytes if present in the sample will bind to the corresponding dot of immobilized capture reagent forming a complex. When conjugate solution is added to the device, the conjugate will bind to the corresponding complex leading to formation of coloured dot.

The membrane with test dot for detection of dengue NSl antigen and control dot is
coated as follows.
Each spot/dot for detection of dengue NSl antigen is coated with 500 nanogram to 5 µg,
preferably 2 µg to 3 µg monoclonal anti dengue NSl antibody and/or polyclonal anti
dengue NS1 antibody.
For coating the dot for detection of dengue NSl antigen, 500 nanogram to 5 µg,
preferably 2 µg to 3µg monoclonal anti dengue NSl antibody and/or polyclonal anti
dengue NSl antibody are added to 10 mM to 50 mM MOPS buffer; pH 7.0 to 8.0
containing 150 mM Sodium chloride and 100 nanogram to 500 nanogram Bovine Serum
albumin and mixed. The mixture is then dispensed at position of dot meant for detection
of dengue NSl antigen.
The control dot is immobilized with anti species IgG in phosphate buffer saline having
pH 7.5.
The membrane is then dried at 37°C for at least 1 hour.
The membrane with test dot for detection of anti dengue IgG antibodies, anti dengue IgM
antibodies and a control dot is coated as follows.
Each spot/dot for detection of anti dengue IgG antibody is coated with 250 nanogram to 5
µg, preferably lµg to 2 µg of monoclonal antihuman IgG antibody and/or polyclonal anti
human IgG antibody.
For coating the dot for detection of anti dengue IgG antibodies, 250 nanogram to 5µg,
preferably 1µg to 2µg monoclonal anti human IgG and/or polyclonal antihuman IgG
antibodies are added to 10 mM to 50 mM Sodium citrate buffer; pH 5.0 to 7.0 containing
150 mM Sodium chloride and 100 nanogram to 500 nanogram Bovine Serum albumin
and mixed. The mixture is then dispensed at position of test dot meant for detection of
anti dengue IgG antibodies.
Each spot/dot for detection of anti dengue IgM antibody is coated with 250 nanogram to 10 µg, preferably 3 µg to 5 µg monoclonal anti human IgM antibody and/or polyclonal anti human IgM antibody.

For coating the dot for detection of anti dengue IgM antibodies, 250 nanogram to 10 ug,
preferably 3 µg to 5 µg monoclonal anti human IgM antibody and/or polyclonal anti
human IgM antibody are added to l0mM to 50 mM sodium citrate buffer; pH 5.0 to 7.0
containing 150 mM Sodium chloride and 100 nanogram to 500 nanogram Bovine serum
albumin and mixed. The mixture is then dispensed at position of dot for detection of anti
dengue IgM antibodies.
The control dot is immobilized with anti species IgG in phosphate buffer saline having
pH 7.5.
The membrane is then dried at 37°C for at least 1 hour.
The conjugate used for detection of dengue NSl antigen consist of monoclonal anti dengue NSl antibody conjugated to gold colloid or modified microsphere and/or polyclonal anti dengue NSl antibody conjugated to gold colloid or modified microsphere. Biotinylated monoclonal anti dengue NSl antibodies and/or biotinylated polyclonal anti dengue NSl antibodies can also be used, followed by addition of conjugate. The conjugate then comprises streptavidin conjugated to gold colloid or modified microsphere.
The conjugate used for detection of anti dengue IgG antibodies and anti dengue IgM antibodies is dengue viral lysate conjugated to gold colloid or modified microsphere and/or dengue recombinant antigen conjugated to gold colloid or modified microsphere. Biotinylated dengue viral lysate and/or biotinylated dengue recombinant antigens can also be used, followed by addition of conjugate. The conjugate then comprises streptavidin conjugated to gold colloid or modified microsphere.
The sample diluent used for the test consist of 10 mM to 50 mM Tris buffer, pH 7.0 to 8.5 containing 1 M to 3 M Sodium Chloride, 0.02% to 0.2% Casein, 0.2% to 0.5% polyoxyethylene sorbitan monolaurate, 0.05% to 0.2% polyoxyethylene-23-lauryl ether, 0.2 % Sodium Azide and 0.01% to 0.05 %Merthiolate.

The invention can be described more clearly with reference to following examples but
these are for illustrative purpose only and same should not be construed to restrict the
scope of invention.
Example 1
For lateral flow immunochromatographic test device, 1 mg/ml of monoclonal antihuman
IgG is added to 20 mM Sodium citrate buffer, pH 5.5, containing 150 mM Sodium
Chloride, and mixed. The mixture is then dispensed at position of test line meant for
detection of anti dengue IgG antibodies.
1.2 mg/ml of biotinylated monoclonal antihuman IgM antibody is added to 50 mM
Sodium citrate buffer, pH 7.0, containing 150 mM Sodium Chloride, and mixed. The
mixture is then dispensed at position of test line meant for detection of anti dengue IgM
antibodies.
2.5 mg/ml of polyclonal anti dengue NS1 antibody is added to 10 mM MOPS buffer, pH
8.0, containing 150 mM Sodium Chloride and mixed. The mixture is then dispensed at
position of test line meant for detection of dengue NS1 antigen.
The strip is then dried at 37°C for 1 to 4 hours.
The conjugate used for detection of anti dengue IgG antibody, anti dengue IgM antibody
and dengue NS1 antigen is dengue viral lysate and dengue recombinant antigen
conjugated to modified microsphere and monoclonal anti dengue NS1 antibody
conjugated to modified microsphere (conjugation of antibody to modified microsphere is
well known in art).
The conjugate is dried on conjugate release pads and impregnated on strip adjacent to
sample pad.
Example 2
For bidirectional and two strip lateral flow immunochromatographic device as described in format 2 and 3, the strip for detection of anti dengue IgG and anti dengue IgM is coated as follows.
For coating line for detection of anti dengue IgG antibodies, 1.5 mg/ml of biotinylated polyclonal anti human IgG antibody is added to 25 mM sodium citrate buffer, pH 7.0,

containing 150 mM Sodium chloride and mixed. The mixture is then dispensed at
position of test line meant for detection of dengue IgG line.
For coating test line meant for detection of anti dengue IgM antibodies, 1 mg/ml of
monoclonal anti human IgM is added to 10 mM Sodium citrate buffer, pH 6.0 containing
150 mM Sodium chloride and mixed. And 1.5 mg/ml of polyclonal anti human IgM is
added to 20 mM Sodium citrate buffer, pH 5.0, containing 150 mM Sodium chloride.
Mix both the solution together. Dispense the mixture at position of test line meant for
detection of anti dengue IgM antibodies.
The strip is then dried at 37°C for 1 to 4 hours.
The conjugate used for detection of anti dengue IgG antibodies and anti dengue IgM
antibodies is dengue viral lysate and dengue recombinant antigen conjugated to gold
colloid. The conjugate is dried on conjugate release pad and is impregnated on strip
adjacent to sample pad.
The strip for detection of dengue NS1 antigen is coated as follows.
1.5 mg/ml of monoclonal anti dengue NS1 antibody is added to 25 mM MOPS buffer, pH
7.5, containing 150 mM Sodium Chloride. And 1 mg/ml of biotinylated polyclonal anti
dengue NS1 antibody is added to 10 mM MOPS buffer, pH 7.0, containing 150 mM
Sodium Chloride. Mix both the solution together and dispensed the mixture at position of
test line meant for detection of dengue NS1 antigen. The strip is then dried at 37°C for 1
to 4 hours.
The conjugate used is monoclonal anti dengue NS1 antibody conjugated to gold colloid.
The conjugate is dried on conjugate release pad and impregnated on the strip.
Example 3
For two holes flow through device as described in format 5, the membrane for detection of anti dengue IgG antibody and anti dengue IgG antibody is coated as follows. For each spot to be immobilized for detection of anti dengue IgG antibody, prepare 10 mM Sodium Citrate buffer, pH 7.0, containing 150 mM Sodium chloride and 200 nanogram Bovine Serum Albumin. To this add 1.3µgof monoclonal anti human IgG

antibody. The mixture is then dispensed at position of test dot meant for detection of anti dengue IgG antibodies.
For each spot to be immobilized for detection of anti dengue IgM antibody, prepare 25
mM Sodium citrate buffer of pH 6.5 containing 150 mM Sodium chloride and add 100
nanogram of BSA. To this buffer add 3µg of polyclonal anti human IgM antibodies. The
mixture is then dispensed at position of test dot meant for detection of dengue IgM
antibodies.
The conjugate used for detection of anti dengue IgG and anti dengue IgM antibody is
dengue recombinant antigen conjugated to gold colloid.
The membrane for detection of dengue NSl antigen is coated as follows.
For each spot to be immobilized for detection of dengue NS1 antigen, prepare 25 mM
MOPS buffer, pH 7.0, containing 150 mM Sodium chloride and add 300 nanogram BSA.
To this add 2.4 µg of monoclonal anti dengue NSl antibody. The mixture is then
dispensed at position of test dot meant for detection of dengue NSl antigen.
The conjugate used for detection of dengue NSl antigen is polyclonal anti dengue NSl
antigen conjugated to gold colloid.

I claim:
1. A kit for the simultaneous and differential detection of the presence of dengue NS1 antigen, dengue IgM antibodies and dengue IgG antibodies in human serum or plasma or whole blood comprising of assay buffer, sample diluent and a device either in immunochromatography or in flow through assay; having nitrocellulose membrane coated with plurality of lines/dots; such that line/dot for detection of anti dengue IgM antibodies is coated with anti human IgM, line/dot for detection of anti dengue IgG antibodies is coated with anti human IgG and line/dot for detection of dengue NS1 antigen is coated with anti dengue NS1 antibody as herein described; such that in lateral flow immunochromatographic format, the membrane is mounted on a strip having sample pad, absorbant pad and conjugate pad; and in flow through assay device, conjugate is in liquid form or in lyophilized form and membrane is mounted onto absorbant pad and is exposed through hole/s in the top cover; and the kit further comprising of assay buffer or sample diluent.
2. A kit as claimed in claim 1, wherein the lateral immunochromatography, the line for detection of dengue NS1 antigen is coated by adding 1 mg/ml to 3 mg/ml monoclonal anti dengue NS1 antibody or polyclonal anti dengue NS1 antibody or a mixture thereof to 10 mM to 50 mM MOPS buffer; pH 7.0 to 8.0 containing 150 mM Sodium chloride; mixing the solution; dispensing the mixture at position of test line meant for detection of dengue NS1 antigen.
3. A kit as claimed in claim 1, wherein the lateral flow immunochromatography, the line meant for detection of anti dengue IgM antibody is coated by adding 0.5 mg/ml to 1.5 mg/ml of monoclonal anti human IgM or polyclonal anti human IgM antibodies or a mixture thereof to 10 mM to 50 mM Sodium citrate buffer, pH 5.0 to 7.0 containing 150 mM sodium chloride; mixing the solution; dispensing the mixture at position of test line meant for detection of anti dengue IgM antibodies.

4. A kit as claimed in claim 1, wherein the lateral flow immunochromatography, the test line meant for detection of anti dengue IgG antibody is coated by adding 1 mg/ml to 2.5 mg/ml of monoclonal anti human IgG or polyclonal anti human IgG antibodies or a mixture thereof to 10 mM to 50 mM sodium citrate buffer; pH 5.0 to 7.0 containing 150 mM Sodium chloride; mixing the solution; dispensing the mixture at position of test line meant for detection of anti dengue IgG antibodies.
5. A kit as claimed in claims 1 and 2, wherein the lateral flow immunochromatography, the conjugate used for detection of dengue NS1 antigen consist of monoclonal anti dengue NS1 antibodies conjugated to gold colloid or modified microsphere or polyclonal anti dengue NS1 antibody conjugated to gold colloid or modified microsphere or a combination thereof.
6. A kit as claimed in claim 1, 3 and 4, wherein the lateral flow immunochromatography, the conjugate used for detection of anti dengue IgG antibodies and anti dengue IgM antibodies consist of Dengue viral lysate conjugated to gold cold colloid or modified microsphere and/or dengue recombinant antigen conjugated to gold colloid or modified microsphere.
7. A kit as claimed in claims 1, 2, 3 and 4, wherein the assay buffer consisting of lOmM to 50 mM Disodium hydrogen phosphate, lOmM to 50 mM Sodium dihydrogen phosphate, 20mM TRIS, 0.5 M to 2 M Potassium chloride, 0.5%-3% Goat albumin or sheep albumin, 0.2% preservative like sodium Azide and l%-3% zwitterionic detergent such as CHAPSO (3-[(3-cholamidopropyl)dimethyl ammonio] -2-hydroxy-l-propanesulfonate);assay buffer has pH 7 to 8.
8. A kit as claimed in claim 1, wherein flow through assay, the test dot for detection of dengue NS1 antigen is coated by adding 500 nanogram to 5 jag, preferably 2 ug to 3 ug monoclonal anti dengue NS1 antibody or polyclonal anti dengue NS1 antibody or a mixture thereof to buffer comprising 10 mM to 50 mM MOPS; pH 7.0 to 8.0, 150 mM Sodium chloride and 100 nanogram to 500 nanogram Bovine

Serum albumin; mixing the solution; dispensing the mixture at the position of dot meant for detection of dengue NS1 antigen.
9. A kit as claimed in claims 1, wherein flow through assay, the test dot for detection of anti dengue IgG is coated by adding 250 nanogram to 5ug, preferably 1 ug to 2 ug monoclonal anti human IgG or polyclonal antihuman IgG antibodies or a mixture thereof to buffer comprising 10 mM to 50 mM Sodium citrate; pH 5.0 to 7.0, containing 150 mM Sodium chloride and 100 nanogram to 500 nanogram Bovine Serum albumin; mixing the solution; dispensing the mixture at the position of test dot meant for detection of anti dengue IgG antibodies.
10. A kit as claimed in claims 1, wherein flow through assay device, the test dot for detection of anti dengue IgM is coated by adding 250 nanogram to 10 ug, preferably 3 ug to 5 ug monoclonal anti human IgM antibody or polyclonal anti human IgM antibody or a mixture thereof to buffer comprising lOmM to 50 mM sodium citrate; pH 5.0 to 7.0, containing 150 mM Sodium chloride and 100 nanogram to 500 nanogram Bovine serum albumin; mixing the solution; dispensing the mixture at position of dot meant for detection of anti dengue IgM antibodies.
11. A kit as claimed in claims 1 and 8, wherein the conjugate used for the detection of dengue NS1 antigen consisting of monoclonal anti dengue NS1 antibody conjugated to gold colloid or modified microsphere or polyclonal anti dengue NS1 antibody conjugated to gold colloid or modified microsphere or a mixture thereof; OR streptavidin conjugated to gold colloid or modified microsphere, such that biotinylated monoclonal anti dengue NS1 antibody or biotinylated polyclonal anti dengue NS1 antibody or a mixture of two are added prior to the addition of streptavidin conjugate.
12. A kit as claimed in claim 1, 9 and 10, wherein the conjugate used for detection of anti dengue IgG and anti dengue IgM consisting of dengue viral lysate conjugated

to gold colloid or modified microsphere or dengue recombinant antigen conjugated to gold colloid or modified microsphere or a combination thereof; Or streptavidin conjugated to gold colloid or modified microsphere, such that biotinylated dengue viral lysate or biotinylated dengue recombinant antigens or a mixture of two are added prior to addition of streptavidin conjugate.
13. A kit as claimed in claims 1, 8, 9 and 10, wherein the sample diluent consisting of 10 mM to 50 mM Tris buffer, pH 7.0 to 8.5 containing 1 M to 3 M Sodium Chloride, 0.02% to 0.2% Casein, 0.2% to 0.5% polyoxyethylene sorbitan monolaurate, 0.05% to 0.2% polyoxyethylene-23-lauryl ether, 0.2 % Sodium Azide and 0.01% to 0.05 % Merthiolate.
14. A kit for detection of dengue NSl antigen, anti dengue IgG antibodies, anti dengue IgM antibodies substantially described herein with reference to and as illustrated in accompanying drawing.

Documents:

488-del-2008-abstract-(27-02-2009).pdf

488-del-2008-Claims-(14-08-2014).pdf

488-DEL-2008-Claims-(26-06-2009).pdf

488-del-2008-claims-(27-02-2009).pdf

488-del-2008-Correspondence-Others-(24-09-2014).pdf

488-DEL-2008-Correspondence-Others-(26-06-2009).pdf

488-DEL-2008-Correspondence-Others-(27-02-2009).pdf

488-DEL-2008-Correspondence-Others-(5-1-2010).pdf

488-del-2008-correspondence-others.pdf

488-DEL-2008-Description (Complete)-(26-06-2009).pdf

488-del-2008-description (complete)-(27-02-2009).pdf

488-del-2008-description (provisional).pdf

488-DEL-2008-Drawings-(26-06-2009).pdf

488-DEL-2008-Drawings-(27-02-2009).pdf

488-del-2008-drawings.pdf

488-DEL-2008-Form-1-(27-02-2009).pdf

488-del-2008-form-1.pdf

488-DEL-2008-Form-2-(26-06-2009).pdf

488-DEL-2008-Form-2-(27-02-2009).pdf

488-del-2008-form-2.pdf

488-DEL-2008-Form-3-(27-02-2009).pdf

488-del-2008-form-3.pdf

488-DEL-2008-Form-5-(27-02-2009).pdf

488-del-2008-form-5.pdf

488-DEL-2008-Pre Grant Opposition- (18-11-2009).pdf

488-del-2008-Pre-Grant Oposition-(20-01-2014).pdf

488-del-2008-Pre-Grant Opposition-(27-05-2014).pdf

488-del-2008-Pre-Grant Opposition-(30-05-2014).pdf

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Patent Number

262648

Indian Patent Application Number

488/DEL/2008

PG Journal Number

36/2014

Publication Date

05-Sep-2014

Grant Date

02-Sep-2014

Date of Filing

29-Feb-2008

Name of Patentee

MAHAJAN ; LALIT

Applicant Address

N-118, GREATER KAILASH, PART-1, NEW DELHI, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	MAHAJAN ; LALIT	N-118, GREATER KAILASH, PART-1, NEW DELHI, INDIA.

PCT International Classification Number

G01N33/574

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA