Title of Invention	A MICROLISA DENGUE DEVICE FOR DETECTION OF DENGUE IGG ANTIBODIES
Abstract	The present invention relates to a microlisa dengue device for the analyzing the presence of dengue IgG antibodies present in human serum or plasma. It is a process for the preparation of the microlisa dengue device for the detection of IgG antibodies present in human serum or plasma. More specifically it is a kit for analyzing the presence of dengue IgG antibodies in human serum or plasma.

Full Text

Field of the invention The present invention relates to the Dengue IgG ELISA device for analyzing the presence of Dengue IgG antibodies in human serum and plasma. More specifically the present invention relates to the process for making the Dengue IgG microlisa device for the detection of IgG antibodies present in human serum or plasma. The present invention also relates to a kit for analyzing the presence of Dengue IgG antibodies in human serum or plasma. The Enzyme Linked Immuno Assay (ELISA) is recommended as the screening method for the detection of Dengue IgG antibodies. Dengue IgG Microlisa is developed to detect IgG antibodies to dengue. The detection of dengue IgG antibodies are indication of secondary dengue viral infection. It is an in vitro- qualitative enzyme immunoassay for the detection of IgG antibodies to dengue in human serum or plasma and is used as a screening test for the testing of collected blood samples suspected for Dengue. 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 diseases while Dengue Hemorrhage Fever can cause life threatening complications. In early acute phase of infection, the specific IgM class antibodies are the first to appear in serum or plasma 1- 4 weeks after the onset of the symptoms and last for up to 3 months. IgG class antibodies appear later and remain elevated throughout the patient's life. Detection of IgM is indicative of the recent current infection and IgG is a marker for past exposure to the causative agent. Specific IgM or IgG responses to particular infectious agent can be measured by antibody based diagnostic tests such as ELISA, immunochromatography, particle agglutination early acute phase of the infection, the specific IgM class antibodies are ELISA, biosensor or other similar assays. The first epidemic occurred simultaneously in Asia, Africa and North America in 1780. The diseases was 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 lOOO.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. 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). 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 the 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;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. Journal of clinical microbiology. Apr. 1979 P. 498-502. Immunoglobulin G and M - specific enzyme linked Immunosorbent assay for detection of dengue anibodies.Bv David Dittmar. Timothy J. Clearv and Albert Castro. Department of Pathology. It says "an enzyme - linked immunsorbent assay for the detection of antibodies to dengue virus is described. This method co-relates with the Hemagglutination Inhibition technique. The enzyme- linked immunosorbent assay can also be specific for human immunoglobulin -M antibodies when a n chain specific antiglobulin enzyme conjugate and fractionated serum are employed. By using this technique, Dengue immunoglobulin M- antibodies were demonstrated in an infant suspected of having a recent dengue infection". Journal of Clinical Microbiology. Nov.2005. p.5784-5786. Use of four Dengue Virus Antigens for Determination of Dengue Immune Status by Enzyme- Linked Immunosorbent Assay of Immunoglobulin G Avidity. By Severine Matheus. Xavier Deparis. Bhetv Labeau. Josiane Lelarge. Jacques Morvan and Philippe Dussart: It says " We used an enzyme- linked immunosorbent assay (ELISA) of immunoglobulin G avidity to determine the dengue immune status of 105 pairs of serum samples from patients infected with dengue virus. This study shows that a simple avidity test, for which only one acute- phase serum sample is required, is potentially more useful than the hemagglutination inhibition test for the discrimination of primary form secondary dengue virus infection, whatever the type of dengue antigen used." Description of the invention The present invention is a ELISA test for the qualitative and differential detection of IgG antibodies to dengue in human serum and plasma. The object of the present invention is to provide a device and a test kit for the differential detection of IgG antibodies to dengue present in human serum or plasma. Yet another object of the present invention is to provide a microtiter device and a test kit to a man of ordinary prudence, which will take a short time to perform the test, Brief description with figures Figure 1 shows the microtiter wells. Detailed description: Principle The test is performed in a 8X12 wells which contains an 8 X 12 matrix of 96 wells, each of which are about 1 cm high and 0.7 cm in diameter. Dengue IgG Microlisa test is an enzyme immunoassay based on "Capture ELISA". Antihuman IgG antibodies are coated on to microtiter wells. Specimens and controls are added to the microtiter wells and incubated. IgG Antibodies of dengue if present in the sample, will bind to antihuman IgG antibodies adsorbed on to the surface of the wells. The plates are then washed to remove unbounded material. Horseradish Peroxidase ( HRPO) conjugated dengue antigen (serotype Den 1-4) is added to each well. This dengue antigen conjugate will bind to dengue specific IgG antibodies which complexes with antihunman IgG antibodies. Finally substrate solution containing chromogen and hydrogen peroxide is added to the wells and incubated. A blue colour will develop in proportion to the amount of dengue antibodies present in the specimen. The colour reaction is stopped by the stop solution. The enzyme substrate reaction is read by EIA reader for absorbance at a wavelength of 450 nm. If the sample does not contain dengue IgG antibodies, then the enzyme conjugate will not bind and the solution in the wells will be either colourless or only a faint background colour develops. Interpretation of result After adding the substrate, if the sample is blue in colour, then the patient's sample is reactive for dengue IgG antibodies. If there is no colour to the solution, then the sample is not reactive to the dengue IgG antibodies. Stop solution such as acids are added to stop the reaction. After adding the stop solution, if the solution in the micro titer well is yellow in colour, the sample is reactive for dengue IgG antibodies and if there is no yellow colour, then the sample is non reactive for Dengue IgG antibodies. Specimen collection and handling Human serum or plasma samples should be used for the test. While preparing the serum samples, remove the serum from the clot as soon as possible to avoid hemolysis. Fresh serum or plasma samples are preferred. Specimen should be free of microbial contamination and may be stored at 2 to 8°C for one week or frozen at -20°C or lower. Repeated freezing and thawing should be avoided. Frozen sample Dengue IgG Microlisa test is best used with fresh samples that have not been frozen and thawed. Allow the frozen samples to thaw in a vertical position in a rack. Don't shake the sample. This allows the particles to settle in the bottom. The sample should be centrifuged. Preparation of Reagents Prepare the reagents during the assay procedure. Reagents and the samples should be at room temperature before beginning the assay and can remain at room temperature during testing. Return the reagents to room temperature after testing. Antihuman IgG antibodies. Bring the pack to room temperature before opening to prevent condensation on the microwells. Break off the required number of strips needed for the assay and place in the well holder. Take the strip holder with the required number of strips, taking in to account that two each of the negative, positive controls and calibrator should be included in the run while opening the fresh kit. However for one or two strips each of negative and positive controls and calibrator and for more strips each of negative and positive controls and calibrator should be included in each subsequent runs. Sample Preparation: a. Tube dilution Mark the tubes carefully for the proper identification of the samples. Dilute the serum samples to be tested, with sample diluent 1:100 in separate tubes.( 1ml diluent + lOul serum samples) b. Microwell dilution i. Pipette lOOjul of sample diluent in to the microwell. ii. Add lu.1 of serum sample to be tested. iii Ensure thorough mixing of the sample to be tested. Preparation of working wash buffer Prepare at least 25 ml of buffer for each strip by adding 1 ml concentrated buffer with 24 ml of water. Mix well before use. Preparation of working conjugate Dilute conjugate concentrate 1:10 in conjugate diluent. Preparation of wash Buffer Take 800- 900ml of distilled water and add Phosphate buffer of lOmM and 0.15M normal saline (NaCl) to the distilled water. Stir the solution in a magnetic stirrer to obtain a homogeneous solution. Add 0.03 to 0.06 % of TritonX-100 to the above solution to make the volume up to 1000ml.Adjust the pH of the solution to 7.0 to 7.4. Preparation of the working chromogenic substrate solution./ TMB) Dilute TMB concentrate is in the ratio 1:10 in substrate solution, i.e. TMB Diluent. The 10X solution may crystallize during storage. TMB Concentrate (IPX) TMB means 3,3,5,5, Tetra Methyl Benzedene, which is dissolved in Dimethyl sulphoxide which is used as an indicator. TMB Diluent Buffer solution containing the substrate is the TMB Diluent. Stopsolution Acids are the stop solution. According to the present invention, the process of detection of IgG antibodies in human serum or plasma are as follows: 1. Leave A-l well as blank which is to test the quality of the substrate. 2. Add 100^1 negative control(ready to use) in each well No. B-l & C-l respectively. 3. Add lOOul Positive Control in D-l and E-l wells. 4. Add lOOuJ calibrator in F-l andG-1 wells. 5. Add 100(0,1 of sample diluent in each sample well starting from HI followed by the addition of 1 pi sample. 6. Apply cover seal and incubate in an incubator at 37°C+_ 2°C for 60 minutes +__ 1 minute. 7. Take out the plate from the incubator and wash the wells 5 minutes with the working wash solution. 8. Add 1 OOul of working conjugate solution in each well excluding A- 1. 9. Apply cover seal and incubate at 37°C +_ 2°C for 60 minutes+_ 2 minutes. 10. Aspirate and wash the well. 11. Add lOOpl of the working substrate solution in each well including the A-l well. 12.1ncubate at room temperature for 30 minutes in dark. 13. Add 50 JJL! of stop solution and read the result within 30 minutes. According to the present invention, Dengue IgG microlisa device for the detection of dengue IgG antibodies in human serum or plasma comprises of 8 XI2 matrix of 96 wells about one centimeter high and 0.7 centimeter in diameter characterized in that the antihuman IgG antibodies are added to the microtiter wells where in the specimens and the control are added to the microtiter wells followed by the addition of enzyme conjugate. In an embodiment of the present invention, a process for making the Dengue IgG microlisa device for the analysis of the presence of the dengue IgG antibodies in human serum or plasma comprises the following steps. 1. Dissolving dilute antihuman IgG antibodies in the ratio of 200 to 800 nanograms in the buffer solution and followed by mixing it thoroughly. 2. Stirring the solution at 50 to 100 rpm in a magnetic stirrer to obtain a homogeneous solution. 3. Dispensing the solution in the wells in the range of 50- 200 ul. 4. Incubating the wells overnight at 2 to 10°C. 5. Washing the wells with the buffer solution of pH 7.2 to 7.6 at least 4 to 5 times. 6. Blocking the washed wells with protein solution made in 10% BSA solution. 7. Removing the unutilized solution by inverting the wells and drying the wells at room temperature thus obtaining the device. In another embodiment of the present invention, the enzyme conjugate is dengue antigen linked with an enzyme such as HRPO prepared by the following steps. a. A 20 mg of the enzyme such as HRPO dissolved in 1ml of distilled water. b. Taking 0.08 molar per iodate and mixing in the ratio 1: 10 with HRPO. c. Shaking and leaving the mixture at room temperature for 60 minutes. d. Mixing with the activated HRPO solution obtained with 10 mg of dengue antigen. e. Allowing the solution to incubate for 2 hours at ambient temperature. f. Adding 1:20 ratio of sodium borohydride solution with the above solution and leaving it to incubate for 30 minutes at ambient temperature; allowing the reaction between antigens and antibodies with HRPO. g. Adding 1:20 ratio Ethanolamine. h. Allowing the solution to incubate for 30 minutes followed by dialyzing. i. Adding buffer solution i.e. BSA in PBS at the percentage 5% to 10% thus obtaining the enzyme conjugate. In still another embodiment of the present invention, the said negative control (-) is normal human sera diluted in saline solution which is negative for dengue, HIV, HCV and HBV and the positive control (+) is human sera diluted in saline solution which is positive to IgG antibodies of Dengue and negative for HCV, HBV and HIV. Example 1: 20 mg of enzyme such as HRPO dissolved in 1 ml of distilled water followed by taking 0.08 molar sodium per iodate and mixing in the ratio 1:10 with HRPO; shaking and leaving the mixture at room temperature for 60 minutes; mixing with activated HRPO solution obtained with 10 mg of recombinant dengue antigen ; allowing the solution to maturize for 2 hours at ambient temperature; adding 1:20 ratio of sodium borohydride solution with the above solution and leaving the same to maturize for 30 minutes at ambient temperature; allowing the reaction between the antigens and antibodies with HRPO; adding 1:20 ratio Ethanolamine; allowing the solution to maturize for 30 minutes followed by dialyzing; i.e. BSA in PBS at the percentage 9% to obtain the enzyme conjugate. I Claim, 1. A Microlisa Dengue device for the detection of dengue IgG antibodies in human serum and plasma comprising 8X12 matrix of 96 wells about one centimeter high and 0.7 centimeter in diameter characterized in that the microtiter wells comprises antihuman IgG antibodies. 2. The process for making Microlisa Dengue device for analyzing the presence of dengue IgG antibodies in human serum and plasma comprising the following steps: a. Dissolving dilute antihuman IgG in the ratio of 200 to 800 nanograms in a buffer solution and followed by mixing it thoroughly; b. Stirring the solution at 50- 100 rpm in a magnetic stirrer to obtain a homogeneous solution. c. Dispensing the solution in the wells in the range of 50 to 200µL. d. Incubating the wells overnight at 2- 10°C. e. Washing the wells with the buffer solution of pH 7.2 to 7.6 at least 4 to 5 times. f. Blocking the washed wells with protein solution made in 10% BSA solution; g. Removing the unutilized solution by inverting the wells and drying the wells at room temperature thus obtaining the device. 3. A process as claimed in claim 2 where in the enzyme conjugate is dengue antigen linked with an enzyme such as HRPO prepared by the following steps: a. A 20mg of enzyme such as HRPO dissolved in 1ml of distilled water. b. Taking 0.08molar sodium per iodate and mixing in the ratio 1:10 with HRPO. c. Shaking and leaving the mixture at room temperature for 60 minutes. d. Mixing with activated HRPO solution obtained with 10 mg dengue antigen. e. Allowing the solution to maturize for 2 hrs at ambient temperature. f. Adding 1:20 ratio of borohydride solution with the above solution and leaving it to maturize for 30 minutes at ambient temperature; allowing the reaction between antigens and antibodies with HRPO. g. Adding 1:20 ratio ethanolamine; h. Allowing the solution to maturize for 30 minutes followed by dialyzing; i. Adding buffer solution i.e., BSA in PBS at the percentage 5% to 10% thus obtaining the enzyme conjugate. 4. A device as claimed in claim 1 which is a Microlisa Dengue device for the analysis of dengue IgG antibodies in human serum or plasma as here in before described. 5. A process for making the Microlisa Dengue device for analyzing the presence of IgG antibodies in human serum or plasma as here in before described.

Documents:

1688-del-2007-abstract.pdf

1688-del-2007-claims-(26-09-2008).pdf

1688-del-2007-claims.pdf

1688-DEL-2007-Correspondence-Others-(26-09-2008).pdf

1688-del-2007-correspondence-others-1.pdf

1688-del-2007-correspondence-others.pdf

1688-del-2007-description (complete).pdf

1688-del-2007-drawings.pdf

1688-del-2007-form-1.pdf

1688-del-2007-form-18.pdf

1688-DEL-2007-Form-2-(26-09-2008).pdf

1688-del-2007-form-2.pdf

1688-del-2007-form-26.pdf

1688-del-2007-form-3.pdf

1688-del-2007-form-5.pdf

1688-del-2007-form-9.pdf