FORM 2
THE PATENT ACT 1970 (39 of 1970)
The Patents Rules, 2003 COMPLETE SPECIFICATION See Section 10, and rule 13)

1. TITLE OF INVENTION
A SEROLOGICAL TEST KIT FOR QUALITATIVE DETECTION of Igm ANTIBODIES TO DENGUE VIRUS;
2. APPLICANT (S)

a) Name
b) Nationality
c) Address

TRANSASIA BIO-MEDICALS LTD.
INDIAN Company
TRANSASIA HOUSE,
8, CHANDIVALI STUDIO ROAD,
MUMBAI - 400 072
MAHARASHTRA

3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which it is to be performed : -

A serological test kit for qualitative detection of IgM antibodies to dengue virus
Field of the invention
The present invention relates to a serological test kit for in-vitro detection of dengue virus antibodies in human plasma and/ or serum. More specifically, it relates to a novel, diagnostic kit for the detection of anti-Dengue-IgM antibodies to four different types of dengue virus in the human serum and/or plasma. The diagnostic kit of the present invention comprises mixture of three recombinant antigens of dengue virus as coating material on solid phase. The present invention also relates to a method for preparing a serological test kit for detecting the anti-Dengue-IgM antibodies in a human plasma and/ or serum. Further, it also relates to in vitro method for detecting anti-Dengue-IgM antibodies to four different types of dengue virus in the human serum and/or plasma.
Background of the invention
Dengue is an acute febrile tropical disease and the virus mat causes it, is an arbovirus, which is transmitted by mosquitoes. The vector of said disease is Aedes aegypti, which most commonly leaves its larvae in domestic and peri-domestic areas. The dengue virus has been classified into four different antigenic types (Den-1, Den-2, Den-3 and Den-4).
The dengue virus infections are serious health hazards in many areas of the world. The dengue virus can cause two forms of disease, Dengue Fever (DF) and Dengue Hemorrhagic Fever (DHF). While DF is a self-Hmiting febrile disease, DHF may lead to life-threatening complications. Laboratory diagnosis of dengue virus infection has mainly depended upon the isolation of dengue virus using mosquito cell cultures or inoculation of mosquitos and detection of anti-dengue antibody by ELISA. But the isolation of the dengue virus is tedious, time consuming and generally requires several days or weeks, which is not always possible in emergency like conditions.
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Polymerase Chain Reaction (PCR) has potential for sensitive, specific and rapid detection of minute quantities of certain genetic material in clinical specimens that provides an attractive approach for the rapid diagnosis of dengue virus infection. Several methods of Reverse Transcriptase (RT)-PCR using different pairs of primers for the dengue viruses and different approaches for the detection of amplification products have been previously reported. However, most of these methods require more than 24 hours for analysis. Rapid RT-PCR methods for detection of dengue viremia have been reported, but they have not been shown to detect all four dengue serotypes in clinical specimens. RT-PCR followed by hybridisation to serotype-specific probes, or RT-PCR followed by nested PCR, have been used for this purpose. Although these methods are sensitive, they still require considerable time and labour. Therefore, above all methods are not always reliable and cannot be used routinely in the countries to which the dengue virus prevails predominantly, for the reasons of high cost and sophisticated equipments. In order to overcome these drawbacks of the prior art methods, a serological test comprising detecting anti-Dengue antibodies, such as IgG, IgM or both, in the human plasma and/or serum, which are specific for all four dengue virus antigens using ELISA technique may be used.
Generally, the dengue virus infection is caused by four closely related serotype of dengue virus -1, -2, -3 and -4. The immunological status of the infected individual determines whether an anti-Dengue response is primary or secondary. In primary infection, the anti-Dengue-IgM appears as early as 3 to 5 days after the onset of illness and peak around 16 days after infection. Anti-Dengue-IgG antibodies appear around 21 days after infection and persist for several years. Therefore, in the serological test kit of the present invention, three recombinant antigens of Dengue virus have been used for the detection of anti-Dengue-IgM antibodies to four different types of Dengue virus in the human serum and/or plasma.
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Object of the invention
Therefore, it is an object of the present invention to provide a serological test kit for detecting anti-Dengue-IgM antibodies to four closely related serotypes of Dengue virus -1 (Den-1), -2 (Den-2), -3 (Den-3) and -4 (Den-4) in the human plasma and/or serum of. A method for preparing a serological test kit for detection of anti-Dengue-IgM antibodies of four different types of Dengue virus antigens is also one of the object of the present invention. As per the further object of the present invention, there is also provided, an in vitro method for detecting anti-Dengue-IgM antibodies to said serotypes of Dengue virus.
Statement of the invention
According to one objects of present invention, there is provided a serological test kit comprises a mixture of three recombinant Dengue virus antigens coated on to a solid support for selectively detecting anti-Dengue-IgM antibodies of Dengue virus in human serum and/or plasma with a high degree of specificity and sensitivity. The kit of the present invention also comprises immunochemically acceptable reagents for detecting anti-Dengue-IgM antibodies of Dengue virus in human serum and/or plasma. The immunochemically acceptable reagents for detecting anti-Dengue-IgM antibodies of Dengue virus comprises an enzyme linked conjugate-M, a Dengue positive control, a Dengue negative control, a colour reagent, a sample diluent, a stopping solution and a washing solution.
The present invention also provides, a method for preparing a serological test kit. The method comprises coating over a solid support a mixture of recombinant Dengue virus antigens and providing immunochemically acceptable reagents required for detecting anti-Dengue-IgM antibodies of Dengue virus in human serum and/or plasma. The method of coating comprises immobilizing on to the solid support a plurality of layers of the mixture of recombinant Dengue antigens in bicarbonate buffer; blocking said antigens using blocking solution, comprising

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phosphate buffer; and stabilising using stabilizing solution, comprising phosphate buffer saline of pH 7.4.
There is also provided, an in vitro method for detecting anti-Dengue-IgM antibodies of Dengue virus in human serum and/or plasma using the serological test kit of the present invention. The method comprises contacting a sample with three recombinant Dengue virus antigens coated on to the solid support to bind to target IgM antibodies in the sample; simultaneously or subsequently to step of contacting, binding at least one said IgM specific recombinant antigen with a conjugated label, thereby reducing amount of substrate of an enzyme linked to the conjugate-M; and detecting the enzyme activity, whereby the presence of the target IgM antibody is determined in the sample.
Detailed description of the invention
The diagnostic kit of the present invention is a solid phase immunoassay kit that utilizes the mixture of recombinant Dengue antigens for detecting anti-Dengue-IgM antibodies against at least any one of four closely related serotypes of Dengue virus -1, -2, -3 and -4 present in human serum and/or plasma of biological sample.
In more particularly embodiment of the invention, the serological test kit of the present invention comprises immobilized on to the solid support the mixture recombinant Dengue antigens for detecting anti-Dengue-IgM antibodies against at least any one of four closely related serotypes of Dengue virus of types Den-1, Den-2, Den-3 and Den-4 present in human serum and/or plasma of biological sample obtained form the patient.
In most particular embodiment of the invention, the serological test kit of the present invention comprises a microtitre plate coated with mixture of three recombinant Dengue virus antigens selected form recombinant Dengue, Ver. 1, recombinant Dengue, Ver. 2 and recombinant Dengue, Chimeric for detecting anti-Dengue-IgM antibodies of at least any one of four closely related serotypes of Dengue virus of types Den-1, Den-2, Den-3 and Den-4. Three recombinant Dengue virus antigens

selected are recombinant Dengue, Ver. 1, recombinant Dengue, Ver. 2 and recombinant Dengue, Chimeric are coated in bicarbonate buffer with a goat anti-Human IgM [u specific], blocked in blocking solution, comprising phosphate buffer and bovine serum albumin (BSA) and stabilized with stabilizing solution, comprising phosphate buffer saline (PBS) of pH 7.4.
In another most preferred embodiment of the invention, the serological test kit for detecting anti-Dengue-IgM antibodies in the in the human serum and/ or plasma, which comprises at least three coating on to the solid support of mixture of goat anti-human-IgM [u specific] with three recombinant Dengue virus antigens selected from recombinant Dengue, Ver. 1, recombinant Dengue, Ver. 2 and recombinant Dengue, Chimeric for detecting anti-Dengue-IgM antibodies of any one of four closely related serotypes Dengue virus of types Den-1, Den-2, Den-3 and Den-4.
In one preferred embodiment, the buffer solutions used in the present invention are mixed with surfactant, stabilizer, sodium chloride and preservative.
In more preferred embodiment, the stabilizer used in the said buffer solutions is a protein stabilizer preferably bovine serum albumin (BSA).
In another more preferred embodiment, the surfactant used in the said buffer solutions may be selected from the non-ionic, anionic and Zwitterionic surfactant; preferably the surfactant used is Non-ionic surfactant.
In still another more preferred embodiment, the preservative used in the said buffer solutions is selected from Thimerosal and gentamycin.
In different embodiment, the enzyme linked conjugate-M comprises goat anti-human-IgM-HRPO in conjugate diluent and along with thimerosal and gentamycin.
In another different embodiment, the Dengue positive control is an inactivated anti-Dengue containing human serum with thimerosal and gentamycin.

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In still another different embodiment, the Dengue negative control is an inactivated normal human serum with thimerosal and gentamycin as preservatives.
In a preferred embodiment, the colour reagent is 3,3', 5,5'-tetra methyl benzidine dimethyl sulfoxide and H2O2 along with thimerosal and gentamycin.
In another preferred embodiment, the sample diluent is a TRIS buffer, animal serum, BSA, Tween-20, TritonXlOO, with thimerosal and gentamycin as preservatives.
In still another preferred embodiment, the stopping solution is a concentrated phosphoric acid and deionized water.
In another embodiment, the washing solution concentrate is a TRIS buffer, NaCl, Tween-20 in deionized water.
In accordance with another object of the present invention, there is provided a method of preparing the serological test kit for detecting anti-Dengue-IgM antibodies of Dengue virus in human serum and/or plasma. The method comprises coating on to a solid support the mixture of three recombinant Dengue virus antigens for detecting anti-Dengue-IgM antibodies against any one of four closely related serotypes of Dengue virus type -1, -2, -3 and -4 in bicarbonate buffer and thereafter blocking and stabilizing said antigens using blocking and stabilizing solutions; providing a coupling product as an enzyme linked conjugate-M obtained by covalently binding an goat anti-human-IgM-HRPO; and providing solutions of a Dengue positive control, a Dengue negative control, a colouring reagent, a sample diluent, a stopping solution and a washing solution.
In a preferred embodiment of the said process, the method of coating comprises immobilizing on to the solid support a plurality of layers of the mixture of three recombinant Dengue antigens in bicarbonate buffer blocking said antigens using blocking solution, comprising phosphate buffer; and stabilising using stabilizing solution, comprising phosphate buffer saline of pH 7.4.
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In another preferred embodiment of the said process, the immobilized antigens are three recombinant Dengue virus antigens for detecting anti-Dengue-IgM antibodies against any one of four closely related serotypes of Dengue virus type -1 (Den-1), -2 (Den-2), -3 (Den-3) and -4 (Den-4).
In still another preferred embodiment of the said process, the immobilized antigens are recombinant Dengue virus antigens selected from recombinant Dengue, Ver. 1, recombinant Dengue, Ver. 2 and recombinant Dengue, Chimeric .
In a different preferred embodiment of the said process, the enzyme-linked conjugate-M selected for detecting anti-Dengue-IgM antibodies is goat anti-human-IgM-HRPO.
In still different embodiment of the invention, the method comprises preparing a solution of an inactivated anti-Dengue containing human serum in thimerosal and gentamycin as a Dengue positive control.
In still another different embodiment of the invention, the method comprises preparing a solution of an inactivated normal human serum in thimerosal and gentamycin as a Dengue negative control.
In a distinct embodiment of the method, preparing the colouring reagent comprises preparing a solution of 3,3', 5,5'-tetra methyl benzidine dimethyl sulfoxide inH2O2 thimerosal and gentamycin.
In one more preferred embodiment of the invention, the method comprises preparing a solution of a sample diluent containing a TRIS buffer, animal serum, BSA, Tween-20, Triton X100, thimerosal, gentamycin and bovine immunoglobulin.
In a separate embodiment of the invention, the method comprises preparing a solution of a stopping solution containing a concentrated phosphoric acid and deionized water.
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In another separate embodiment of the invention, the method comprises preparing a washing solution concentrate containing a TRIS buffer, NaCl, Tween-20 in deionized water.
Pursuant to another aspect of the invention, an in vitro method for detecting anti-Dengue-IgM antibodies of Dengue virus in human serum and/or plasma using the serological test kit of the present invention is provided. The method comprises contacting a serum and/or plasma sample containing anti-Dengue-IgM-antibodies with three recombinant Dengue virus antigens that are bound to the surface of a solid support, thereby forming a stable complex, if anti-Dengue-IgM antibodies are present; subsequently binding at least one said IgM specific recombinant antigen with a conjugated label, thereby reducing amount of substrate of an enzyme linked to the conjugate-M; and detecting the enzyme activity for the presence of target IgM antibodies in the sample.
In more preferred embodiment of the invention, the in vitro method for the detecting anti-Dengue-IgM antibodies of Dengue virus in human serum and/or plasma comprises: presenting a coated mixture of three recombinant Dengue virus antigens, which are selected from recombinant Dengue, Ver. 1, recombinant Dengue, Ver. 2 and recombinant Dengue, Chimeric as a solid phase and an enzyme linked conjugate-M; contacting a given quantity of liquid sample containing anti-Dengue-IgM antibodies with said solid phase antigens, thereby forming a stable complex and determining the enzyme activity of the conjugate-M added for the presence of anti-Dengue-IgM antibodies.
In most preferred embodiment of the invention, the in vitro method for the detecting anti-Dengue-IgM antibodies in human serum and/or plasma comprises: presenting set of coated mixtures of three recombinant Dengue virus antigens selected from recombinant Dengue, Ver. 1, recombinant Dengue, Ver. 2 and recombinant Dengue, Chimeric immobilized on to a solid support and an enzyme linked conjugate-M obtained by covalently binding an goat anti-human-IgM-HRPO to an enzyme; contacting a liquid sample containing anti-Dengue-IgM antibodies with said

recombinant Dengue virus antigens, thereby forming a stable complex with anti-Dengue-IgM antibodies present in the sample or Dengue positive control and determining the enzyme activity of the conjugate-M for the presence of anti-Dengue-IgM antibodies against either at least any one or four closely related serotypes of Dengue virus, Den-1, Den-2, Den-3 and Den-4.
In preferred embodiment of the present invention, for the detecting anti-Dengue-IgM antibodies of Dengue virus in human serum and/or plasma, the foregoing reagents are employed in predetermined amounts.
In another embodiment of the present invention, the solid carrier to which antigen is bound may be any water-insoluble, water-insuspensible, solid carrier. An example of suitable solid carrier includes microtitre plate. The immunological component may be bound to the solid carrier by covalent bonds or by adsorption. The advantage of the use of a solid carrier is that no centrifugation step is needed for the separation of solid and liquid phase.
In still another embodiment of the present invention, the antibody enzyme conjugate-M consists of the immunological component covalently linked to one or more enzyme molecules. Such linking can be achieved either by direct condensation or by using external bridging molecules, in accordance with methods known to those skilled in the art. Thus, the reagents such as carbodiimides, diisocyanates, glutaric aldehyde and bis-diazobenzidine can effect the production of enzyme coupling products employing a covalent bond.
The choice of the enzyme that is to form a part of the coupling product is determined by properties such as the specific binding activity i.e. a high conversion rate increases the sensitivity of the test system and the simplicity of determination of the enzyme. The determination of an enzyme catalysing a conversion, in which coloured reaction components are involved, is simple. Such colorimetric determinations can be automatic in a simple manner. It is also possible to employ enzymes catalysing those conversions in which reaction components are involved that can be determined

spectrophotometrically. These determinations are also suitable for automation, which is an additional advantage. As an enzyme suitably employed in the present invention is peroxidase, preferably Horse radish peroxidase.
Any further modifications in and/or improvements in any aspect of the embodiments of this invention will also fall under the scope of this invention. In view of the foregoing description and example, it will become apparent to those of ordinary skill in the art that equivalent modification thereof may be made without departing from the spirit and scope of this invention. Various features of the invention hereinbefore described are set forth in the foregoing claims.
Examples
The following examples serve to illustrate the use of the invention, but are not to be regarded as limitations for the scope of the invention:
Example: 1 - Detection of anti-Dengue-IgM antibodies
A mixture of three recombinant Dengue virus antigens is coated onto the wells of a microplate. When human plasma and/or serum added to sample diluent containing well, the bound Dengue virus antigens will form a stable complex, if anti-Dengue-IgM antibodies are present in the sample. Followed by a wash step, goat anti-human-IgM-HRPO is added to the wells. A second wash step will remove free goat anti-human-IgM-HRPO. Colour reagent containing the substrate of HRPO is then added to the wells. Wells containing negative control samples will remain colourless and blue colour will develop in wells containing positive controls and test specimens containing IgM antibodies to Dengue virus. Upon addition of stopping solution, blue colour changes to yellow. The intensity of yellow colour is directly proportional to the amount of bound anti-Dengue-IgM antibodies in the respective wells.
Example: 2 - Test procedure for detecting anti-Dengue-IgM antibodies
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(a) Bring all the reagents and test specimens at room temperature before use; (b) except blank, add l00ml of sample diluent to each well, in each run, there will be one blank, one negative control and three positive controls, add l0ml of control and test specimens to the respective wells, mix properly with pipettor and cover the plate with black cover and incubate 1.0 hr at 20-30°C; (c) wash the plate as per microplate washing procedure known to person skilled in the art; (d) add 50ul conjugate-M to each well (except blank well) and cover the plate with black cover and incubate for 15 minutes at 20-30°C; (e) repeat the step (c) for washing; (f) add 50ml of colour reagent to each well and cover the plate with black cover and incubate for 15 minutes in dark at 20-30°C; (g) add 100ml of stopping buffer to each well; and (h) read absorbance at 450 nm and deduct the blank absorbance from the control and test wells.
Example: 3 - Calculation for cut-off value determination
Blank value: Absorbance of blank values should be less than 0.1. Positive Control: Absorbance of individual positive control should be grater than 0.5. Negative Control: Absorbance of individual negative control should be less than 0.2.
PCx: Average value of positive controls.
Calculation of PCx:
For example:
PC Absorbance
1 0.970
2 0.975
3 0.980
4 PCx: (0.97+0.975+0.98)/3=0.975
Cut-off value formula: l.0xPCx Cut-off vale: 1.0x0.975=0.975
Interpretation of results:

Non-reactive: If the absorbance of the test serum and/or plasma is less than the 0.8 times of cut-off value, then the sample is considered as non-reactive.
Doubtful: If the absorbance of the test serum and/or plasma is in between 0.8 times to 1.0 times of cut-off value, then the sample is considered as doubtful.
Reactive: If the absorbance of the test serum and/or plasma is greater than the cutoff value, then it is considered as initial reactive.

WE CLAIM:
1. A serological test kit for selectively detecting anti-Dengue-IgM antibodies in human serum and/or plasma comprises coated onto a solid support a mixture of three recombinant Dengue virus antigens for four different types of Dengue virus; and an immunochemically acceptable reagents selected form an enzyme linked conjugate-M, a Dengue positive control, a Dengue negative control, a colour reagent, a sample diluent, a stopping solution and a washing solution required for the detection of said anti-Dengue-IgM antibodies, wherein said solid support contains plurality of immobilized set of goat anti-human-IgM [u specific] with three recombinant antigens of Dengue virus selected from recombinant Dengue, Ver. 1, recombinant Dengue, Ver. 2 and recombinant Dengue, Chimeric.
2. The serological test kit as claimed in claim 1, in which the mixture of three immobilized recombinant antigens of Dengue virus are selected for detecting anti-Dengue-IgM antibodies of at least any one of four closely related serotypes of four different types of Dengue virus selected from Den-1, Den-2, Den-3 and Den-4.
3. The serological test kit as claimed in claim 1, in which the coated solid support is a microtitre plate with plurality of micro-wells and having at least three immobilized coatings of a homogenous mixture of three recombinant antigens for detecting four closely related serotypes of said Dengue virus.
4. The serological test kit as claimed in claim 1, in which the mixture of three recombinant Dengue virus antigens are immobilised in bicarbonate buffer and blocked with blocking solution of phosphate buffer and bovine serum albumin, and stabilized with stabilizing solution of phosphate buffer saline of pH 7.4.
5. The serological test kit as claimed in claim 1, in which the enzyme linked conjugate-M for detecting anti-Dengue-IgM antibodies of at least any one of

four closely related serotypes of four different Dengue virus comprises goat anti-human-IgM-HRPO in conjugate diluent with thimerosal and gentamycin.
6. The serological test kit as claimed in claim 1, in which the Dengue positive control is an inactivated anti-Dengue containing human serum with thimerosal and gentamycin as preservatives.
7. The serological test kit as claimed in claim 1, in which the Dengue negative control is an inactivated normal human serum with thimerosal and gentamycin.
8. The serological test kit as claimed in claim 1, in which the colour reagent is 3,3'/ 5,5'-tetra methyl benzidine dimethyl sulfoxide with H2O2, thimerosal and gentamycin.
9. The serological test kit as claimed in claim 1, in which the sample diluent is a TRIS buffer, animal serum, BSA, Tween-20, TritonXl00, with thimerosal and gentamycin.
10. The serological test kit as claimed in claim 1, in which the stopping solution is a concentrated phosphoric acid with deionized water.
11. The serological test kit as claimed in claim 1, in which the washing solution concentrate is a TRIS buffer, NaCl, Tween-20 with deionized water.
12. A method for preparing a serological test kit for detecting anti-Dengue-IgM antibodies to four different types of Dengue virus in the human serum and/or plasma as claimed in claims 1 to 11 comprises: coating onto a solid support a mixture of three recombinant Dengue virus antigens selected from recombinant Dengue, Ver. 1, recombinant Dengue, Ver. 2 and recombinant Dengue, Chimeric in bicarbonate buffer; blocking said three recombinant antigens using a blocking solution of phosphate buffer and bovine serum albumin; stabilizing said antigens using a stabilizing solution of phosphate

buffer saline of pH 7.4; providing an enzyme liked conjugate-M obtained by covalently binding of a goat anti-human-IgM-HRPO; and preparing homogenous solutions of a Dengue positive control, a Dengue control, a colouring agent, a sample diluent, a stopping solution and a washing solution.
13. An in vitro method for detecting anti-Dengue-IgM antibodies to four different types of Dengue virus in the human serum and/ or plasma using a serological test kit of claims 1 to 11 comprises: presenting set of coated mixture with three recombinant Dengue virus antigens selected from recombinant Dengue, Ver. 1, recombinant Dengue, Ver. 2 and recombinant Dengue, Chimeric onto a solid support and conjugate-M with a goat anti-human-IgM-HRPO; contacting a liquid sample containing anti-Dengue-IgM antibodies with said recombinant antigens of solid support, thereby forming a stable complex with anti-Dengue-IgM antibodies present in said sample or a Dengue positive control; and determining an enzyme activity of the conjugate-M for the presence of anti-Dengue-IgM antibodies of four different types of Dengue virus in the sample.
Dated this 13lh day of October, 2006.
^-SLTRESH VAZIRANI CHAIRMAN AND MANAGING DIRECTOR TRANSASIA BIO-MEDICALS LTD.
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