Title of Invention	A DEVICE AND KIT FOR ANALYZING THE PRESENCE OF ANTI DENGUE IGM AND ANTI DENGUE IGG IN A SAMPLE AND A PROCESS THERE OF.
Abstract	The present invention relates to the novel diagnostic test kit for the diagnosis of antibodies to dengue in human serum or plasma. Particularly, it is a composition for the preparation of the Dengue device. More particularly, the present invention relates to the process of developing a novel device for the detection of anti dengue IgM and IgG antibodies present in human serum and plasma.

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Field of the Invention The present invention relates to the novel diagnostic kit for analyzing the presence of antibodies to dengue in human serum or plasma. Specifically it is a composition for the preparation of dengue device. More specifically, the present invention relates to the process of developing a novel device for the detection of IgM and IgG antibodies present in human serum and plasma. Background Dengue fever and dengue hemorrhage fever are acute febrile diseases found in the tropics. It is a serious health problem. It is caused by one of four closely related viruses serotypes of genus flavivirus, family flaviviridae. Each serotype is sufficiently different that there is no cross protection and epidemics caused by multiple serotypes can occur. It is transmitted to humans by the mosquito Aedes aegypti. The Dengue virus contains 70 viruses including yellow fever viruses and encephalitides. Illness may be complicated hemorrhagic fever. This infectious disease is manifested by a sudden fever with severe headache, muscle and joint pains and rashes. The dengue rash is characteristically bright red and usually appears first on the lower limbs and chest, in some patients it spreads to cover most of the body. The classic dengue fever lasts about 6 to 7 days. Clinically the platelet count will drop until the patient's temperature is normal. Dengue virus can cause two types of diseases: dengue fever and dengue Hemorrhage fever. Dengue Fever is a self-limiting febrile disease while Dengue Hemorrhage Fever can cause life-threatening complications. The first epidemic occurred simultaneously in Asia, Africa and North America in 1780. The diseases were identified and named in 1779. A global pandemic began in Southeast Asia in the 1950s and by 1975 DHF had become a leading cause of death among children in many countries in that region. Dengue was the most important mosquito born diseases affecting human after Malaria. Significant out brakes of dengue fever tend to occur every five to six years. In the year 2003, there were 6 deaths from dengue shock syndrome. With the proper medical treatment the mortality rate for dengue can therefore be brought down to less than 1 in 1000.However more than 400 cases and 22 deaths were reported due to dengue fever in the Indian capital. By October 7, 2006, reports were 337 cases of mosquito borne virus and a death toll of 49. According to the World Health Organizations estimates, there may be currently as many as 100 million cases of dengue fever every year. About 2.5 billion people in over a hundred tropical and sub-tropical countries, representing 40% of the world's population, are now at risk from dengue. The history of dengue virus vaccine development goes back more than 60 years beginning with the attempts in the 1920 to inactive dengue virus from infectious human plasma with ox, bile or formalin ( Simons, J.S.etal In 3 Brown W.H. Monographs 29; The Philippines; Manila Bureau of printing 1: 489,1931). There is no effective antiviral therapy for the treatment of dengue infections (Leyssen,P,De Clercq, E.,ccnd Neyts,J.2000). Some of the recent attempts to find out the vaccine against the detection of dengue virus met some success. But it has been contended that such approaches are neither feasible nor practical due to the relatively poor growth of these virus in culture and their antigenic stability after purification. Thus the vaccine described was not likely to be practical or suitable for use in humans. Therefore in view of the above there is a need for the dengue vaccine for the detection which have cured all the defects of the previous vaccines hence the present invention with high sensitivity and specificity came. Prior Arts US Patent No: 6190859 An inactivated dengue virus vaccine to immunize and protect humans against dengue fever is described. The vaccine is based on dengue viruses which have been propagated to high titers in suitable cells, purified and inactivated under conditions which destroy infectivity but preserve immunogenicity, a high level of which is demonstrated in animal models. Uses of the inactivated dengue virus for detecting antibodies to dengue and kits there for are also described. US Patent No: 7,041,255 relates to a pair of dengue virus-specific primers for use in a reverse transcriptase-polymerase chain reaction to detect dengue virus. US Patent No: 5939254 relates to specific primers that amplify a portion of the 3'-noncoding regions of dengue virus types 1, 2, 3 and 4, and a method of using these primers in a rapid reverse transcriptase-polymerase chain reaction (RT-PCR) for specific detection of dengue viruses, but not other flaviviruses, is disclosed. US Patent No:5968732 Isothermal transcription based assay for the detection and genotyping of dengue virus. An isothermal transcription based amplification assay for dengue virus RNA uses primer combinations for sequences within the envelope gene or the 3' non-coding region of the virus and a probe. Probes may be specific for a serotype of dengue virus. US Patent No: 5824506 relates to the derivation of peptide antigen from the dengue virus type-2 glycoprotein NS1. 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. This patent discloses the usage of the peptide antigens for the detection of the dengue. These patents don't disclose a fast, cost effective, simple and rapid diagnostics that combine sensitivity and specificity. It doesn't provide a method of detection of dengue for a man of ordinary skill to perform. The present invention is a rapid solid phase immuno- chromatographic assay for the qualitative and differential detection of IgG and IgM antibodies to dengue virus in human serum or plasma or whole blood. The object of the present invention is to provide a test device and the kit, which detects the IgM and IgG antibodies present in human serum or plasma or whole blood. In the present device, the control line helps the people to know that the reagents are working properly. The other object of the present invention is to provide a test device and a kit, which enables everyone to detect the infection of dengue virus in human serum or plasma or whole blood. Another object of the present invention is to provide a novel, inexpensive, simple, rapid diagnostic device and the kit, which detects the dengue IgM and 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 the dengue virus infection or anti dengue virus antibodies in the serum or plasma or whole blood of an individual suspected of having disease. Yet another object of the present invention is to provide a test device and kit to the man of ordinary prudence, which will take a short time to perform the test. The test device is for in vitro diagnostic use only and is intended as an aid in the presumptive differential diagnosis between primary and secondary dengue infection. The test kit includes a dengue card, assay buffer, sample loop, transfer dropper and sample cuvette. Summary of the invention: Objects of the present invention are achieved by providing dengue proteins, one is designed to detect the anti dengue IgM and the other is designed to detect anti dengue IgG present in the human serum or plasma or whole blood. The present invention also discloses a simple and rapid dengue strip test device. There are two formats in which test can be performed. In format 1, the line/dot for the detection of anti dengue IgM is coated with monoclonal or polyclonal antihuman IgM and the line or dot for the detection of anti dengue IgG is coated with monoclonal or polyclonal antihuman IgG. The conjugate/ signal reagent used for the corresponding anti dengue IgM line is dengue recombinant antigen conjugate or dengue viral lysate or a mixture of both conjugated to metal colloid eg. Colloidal gold or modified microspheres and the corresponding conjugate used in the conjugate pad for the anti dengue IgG line is dengue recombinant antigen conjugate or dengue viral lysate or a mixture of both conjugated to metal colloid or modified microspheres. The control line is coated with anti species IgG for eg.Goat anti Rabbit IgG, Goat anti Mouse IgG etc and the conjugate used for the control line is species IgG for eg. Rabbit IgG or mouse IgG. In format 2, the line / dot for the detection of anti dengue IgG and anti dengue IgM are coated with dengue recombinant antigen or dengue viral lysate or a mixture of both. The corresponding conjugate used for the detection of the anti dengue IgG is the monoclonal /polyclonal antihuman IgG and the conjugate used for the detection of the anti dengue IgM is monoclonal /polyclonal anti human IgM. The control line is coated with anti species IgG for eg.Goat anti Rabbit IgG, Goat anti Mouse IgG etc and the conjugate used for the control line is species IgG for eg, Rabbit IgG or mouse IgG. Brief description of the invention with figures Figure 1 shows the bi-directional test device. Figure 2 shows the presence of both IgM and IgG antibodies Figure 3 shows the presence of IgM antibody. Figure 4 shows the presence of IgG antibody. Figure 5 shows the device is non reactive for both IgM and IgG and the test performed is valid. Figure 6 shows the dengue device with two cassettes. Detailed description The present invention describes a device and the kit for the detection of the presence of anti dengue IgM and anti dengue IgG antibodies in human serum, plasma and whole blood. The present invention relates to the test kit for the earlier detection of the dengue antibodies present in the human serum or plasma or whole blood. It comprises of a bi-directional device which has a base strip and membrane made of cellulose material, preferably cellulose nitrate is mounted on the base. The casing comprises of top cover and a bottom cover adapted to be pressed with each other. A sample well is provided in the middle of the top cover through which the sample is dropped in to the membrane (Figure 1). Figure 1 shows front view plan of the bi-directional diagnostic device for analysis of analyte in a sample fluid comprising of analyte and non analyte materials whereby the test device is impregnated with four lines/dots. The lines for the detection of anti dengue IgM and anti dengue IgG are impregnated with monoclonal or polyclonal anti-human IgM and anti human IgG respectively and are capable of immobilization against solvent transport by the sample. The control line/dot positioned upstream is coated with anti species IgG for eg. goat anti-mouse IgG and conjugate used for the control line is species IgG for eg, Rabbit IgG or mouse IgG. The concentration of coating for the test lines/dots are 0.1 mg/ml to 5mg/ml preferably between 0.5mg/ml to 2mg/ml and for control line the concentration of protein is 0.1 mg/ml to 2 mg/ml preferably 0.5mg/ml to 1 mg/ml. In the device as shown in the figure 1, the sample is added to the sample well at the center of the top of the device. The IgM line and the control line is on one side and the IgG line and the control line is on the other side which is interchangeable. This device is also used for the detection of dengue as explained in the formats 1& 2. In the device as shown in the figure 6, two cassettes are positioned as shown in the figure. In this case, the sample is added to the sample well on both the cassettes. Control line and the line for the detection of anti dengue IgM are positioned on the upper cassette and the control line and the line for the detection of anti dengue IgG are positioned at the lower cassette which are inter changeable. In an embodiment of the present invention, the test device is prepared as follows: In format 1 in this device, for anti dengue IgG detection, the test line is coated with the Monoclonal or polyclonal anti human IgG and the corresponding conjugate / signal reagent used in the conjugate pad is the Dengue recombinant antigen conjugate or dengue viral lysate conjugate or a mixture of both conjugated to metal colloid or modified microspheres. For the detection of anti dengue IgM, the line is coated with monoclonal or polyclonal antihuman IgM and the corresponding conjugate / signal reagent used in the conjugate pad is that of dengue recombinant antigen or dengue viral lysate or a mixture of both conjugated to metal colloid or modified microspheres. Phosphate buffer solution is used for dispensing the dengue antibodies on the nitro cellulose membrane. The concentration of coating for the test lines/dots are 0.1 mg/ml to 5mg/ml preferably between 0.5mg/ml to 2mg/ml and for control line, the concentration of coating is 0.1 mg/ml to 2mg/ml preferably 0.5mg/ml to 1 mg/ml. Format 1: Principle When human serum or plasma or whole blood is added to the test device, dengue IgG and IgM antibodies in the specimen sample react with coloured conjugate particles coated with the dengue proteins. If the sample contains anti dengue Immunoglobulin G or anti dengue Irnrnunoglobulin M, these antibodies react with the Dengue antigen conjugate forming antibody antigen conjugate complex. As this complex migrates along the length of the test, the anti- dengue IgG or IgM antibody present in the Antigen Conjugate Antibody complex is captured by relevant anti human IgG or anti human IgM test lines or dots on the nitrocellulose membrane forming a coloured line/ dot at the IgG or IgM region of the test device. The intensity of the line/dot will vary depending upon the amount of the antibody present in the sample. The appearance of the colour in the specific test region should be considered as positive for that particular antibody type. A colored procedural control will always develop in the test device to indicate that the reagents are functioning properly. In format 2 in the above bi-directional device (fig-1) the line for the detection of anti dengue IgG is coated with dengue recombinant antigen or dengue viral lysate or a mixture of both and the corresponding conjugate/ signal reagent used in the conjugate pad is the monoclonal/ polyclonal anti human IgG conjugated to metal colloid or modified microspheres. For the detection of anti dengue IgM, the line is coated with the dengue recombinant antigen or dengue viral lysate or a mixture of both and the corresponding conjugate / signal reagent used in the conjugate pad is the monoclonal or polyclonal antihuman IgM conjugated to metal colloid or modified microspheres. The concentration of coating for the lines/dots are 0.1 mg/ml to Smg/ml preferably between 0.5mg/ml to 2mg/ml and for control line the concentration of the coating is O.lmg/ml to 2mg/ml preferably 0.5mg/ml to 1 mg/ml. . Format 2: Principle When a sample is added to sample well in the device, anti dengue IgG and IgM antibodies present in the sample react with coloured conjugate coated with anti human IgG/ anti human IgM antibody and this reacts with the antigen in the line or membrane to form antigen antibody complex which gives a coloured line / dot which shows the presence of anti dengue IgM and IgG antibodies in the sample. If the control line / dot and the line / dot for the detection of anti dengue IgM only appears in colour, the sample is reactive for dengue IgM antibodies which is primary infection. If the control line as well as the IgG line for the detection of IgG antibody appears in colour, the sample is reactive for dengue IgG antibodies which is the secondary infection. If both the IgG and IgM line /dot and the control line /dot appears in colour, the sample is reactive for both the dengue IgG and dengue IgM antibodies which is secondary stage of infection. If the control line / dot alone appears in colour, the sample is non reactive for the dengue and workability of the device is perfectly right. Cassette shown in the figure 6 is also used for the detection of the antibodies to dengue as explained in the above two formats. Interpretation of the result The device is as shown in the figure 1. When neither the control line nor the IgM or IgG line appears, the test should be treated as invalid. (Figure 1) The appearance of coloured line/dot in both the control region 'C' and the test region IgM region *M* and IgG region 'G' indicates that the sample is reactive for both IgM and IgG antibodies. This is indicative of a secondary dengue infection.(Figure 2) The appearance of the coloured line in both the control region 'C' and the IgM region 'M' indicates that the sample is reactive for IgM antibodies which is indicative of primary dengue infection. (Figure 3) The appearance of the coloured line in both the control region 'C' and the IgG region 'G' indicates that the sample is reactive for IgG antibodies which is indicative of the secondary dengue infection. (Figure 4) The appearance of the coloured line in both the control regions 'C' only indicate that the sample is non reactive for the dengue antibodies and the test procedure is perfect. (Figure 5) Sample collection Human serum or plasma or whole blood should be used as samples. If the specimen or plasma or whole blood specimens cannot be tested immediately, they should be refrigerated at 2 - 8°C. For storage for more than 3 days, freeze the specimen at -20°C or below. Specimens containing precipitate or particulate matter may yield inconsistent test results. Such specimens should be centrifuged and the clear supernatant should only used for testing. In an embodiment of the present invention, the assay buffer is prepared as follows: Buffer used for the analysis of dengue IgM and Dengue IgG antibodies in the sample comprising HEPES buffer at an effective concentration of 5mM toSOOmM; protein stabilizers like gelatin, albumin, or non immune animal serum are added at an effective concentration such that the final concentration of the same should be 0.01% to 5 % preferably at 2%; Non ionic detergent selected from the group consisting of Polyethylene Sorbitan monolaurate and Polyethylene ethers are added at an effective concentration of 0.05% to 2%, preferably at 1% ; Anionic surfectant selected from the group consisting of Caprylic acid or Laurlyl sulphate is added at an effective concentration of 0.01% to 0.5% preferably at 0.25%; a high molecular weight polymer selected form the group consisting of Polyethylene glycols, polyvinyl alcohol, Polyvinyl pyrrolidone are added at an effective concentration of 0.1% to 2% preferably at 1%; preservatives like Sodium Azide or Thimerosal are added such that the concentration of the same should be 0.01% to 0.2 %; such that the pH of the composition is in the range of about 6 to 9 preferably 7 to 8. Test Procedure Take the required number of cassettes from the foil pouch and add a mixture of the assay buffer and the sample to the sample well of the device. Allow reaction to take place during the next 20 minutes and read the result. I claim; 1. A test device and a test kit for the simultaneous detection of the presence of dengue IgM antibodies and dengue IgG antibodies in human serum or plasma or whole blood comprises nitrocellulose membrane coated with plurality of lines such that line for detection of anti dengue IgM antibodies is coated with monoclonal or polyclonal anti human IgM, line for detection of anti dengue IgG antibodies is coated with monoclonal or polyclonal anti human IgG and the membrane is mounted on a strip having sample pad, absorbent pad and conjugate pad impregnated with conjugate and the kit comprises of assay buffer. 2. A device as claimed in claim 1 wherein line for detection of dengue IgG is coated with monoclonal or polyclonal antihuman IgG. 3. A device as claimed in claims 1 and 2 wherein the conjugate used is dengue recombinant antigen conjugated to metal colloid or modified microsphere or dengue viral lysate conjugated to metal colloid or modified microsphere or a mixture of both. 4. A device as claimed in claim 1 wherein line for detection of dengue IgM antibody is coated with monoclonal antihuman IgM or polyclonal antihuman IgM. 5. A device as claimed in claims 1 and 4 wherein the conjugate used is dengue recombinant antigen conjugated to metal colloid or modified microsphere or dengue viral lysate conjugated to gold colloid or modified microsphere or a mixture of both. 6. A device as claimed in claim 1 wherein line for detection of dengue IgG is coated with dengue recombinant antigen or dengue viral lysate or a mixture of both. 7. A device as claimed in claims 1 and 5 wherein conjugate used is monoclonal anti human IgG conjugated to metal colloid or modified microsphere or polyclonal antihuman IgG conjugated to metal colloid or modified microsphere. 8. A device as claimed in claim 1 wherein the line for detection of dengue IgM antibody is coated with dengue recombinant antigen or dengue viral lysate or a mixture of both. 9. A device as claimed in claims 1 and 7 wherein the conjugate used is monoclonal antihuman IgM conjugated to metal colloid or modified microsphere or polyclonal antihuman IgM conjugated to metal colloid or modified microsphere. 10. A device as claimed in claim 1 wherein the concentration of antibodies and antigens used for coating test line is 0.1 mg/ml to 5 mg/ml preferably between 0.5 mg/ml to 2 mg/ml and the concentration for coating control line is 0.1 mg/ml to 2 mg/ml preferably 0.5 mg/ml to 1 mg/ml. 11. A kit as claimed in claim 1, wherein the assay buffer consist of: 5 mM to 300 mM HEPES buffer, pH 6 to 9 (preferably 7 to8); 0.01% to 5% (preferably 2%) protein stabilizers such as gelatin, albumin or non immune animal serum; 0.05% tp 2% (preferably 1%) Non ionic detergent from group consisting of Polyethylene Sorbiton monolaurate and Polyethylene ethers; 0.01% to 0.5% (preferably 0.25%) anionic surfactant from group consisting of Caprylic acid or Lauryl sulphate; 0.1% to 2% (preferably 1%) high molecular weight polymer selected from group consisting of Polyethylene glycols, polyvinyl alcohol, polyvinyl pyrollidone; 0.01% to 0.2 % preservatives like Sodium Azide or Thiomersal. 12. A device and kit for detection of IgG and IgM antibodies to dengue substantially described herein before with reference to and as illustrated in accompanying drawing."

Documents:

2480-del-2007-Correspondence-Others.pdf

2480-del-2007-Form-9.pdf

2484-DEL-2007--Post Grant Opposition-(20-12-2010).pdf

2484-del-2007-abstract.pdf

2484-del-2007-Assignment-(27-04-2011).pdf

2484-DEL-2007-Claims-(06-03-2009).pdf

2484-del-2007-claims.pdf

2484-del-2007-Correspondence Others-(27-04-2011).pdf

2484-DEL-2007-Correspondence-Others-(06-03-2009).pdf

2484-del-2007-correspondence-others-1.pdf

2484-del-2007-correspondence-others.pdf

2484-del-2007-description (complete).pdf

2484-del-2007-drawings.pdf

2484-del-2007-form-1.pdf

2484-del-2007-Form-16-(27-04-2011).pdf

2484-del-2007-form-18.pdf

2484-del-2007-form-2.pdf

2484-del-2007-form-3.pdf

2484-del-2007-form-5.pdf

2484-DEL-2007-Post-Grant-Opposition-(18-11-2010).pdf

2484-DEL-2007-Post-Grant-Opposition-(20-09-2010).pdf