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 KIT FOR DIAGNOSIS OF DENGUE VIRUS INFECTION;

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 kit for diagnosis of dengue virus infection
Field of the invention :
The present invention relates to a diagnostic kit for detection of Dengue virus antibodies in human plasma and/or serum. Particularly, it relates to a novel kit for the detection of anti-Dengue IgM and IgG antibodies to four different types of Dengue virus, in the human serum and/or plasma, in a single assay system and in one attempt. 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 diagnostic kit for detecting the anti-Dengue IgM and IgG antibodies, in a human plasma and/or serum, in a single assay system and in one attempt. Further, it also relates to in vitro assay for detecting anti-Dengue IgM and IgG antibodies to four different types of dengue virus in a single assay system and in one attempt.
Background of the invention :
Dengue is an acute febrile tropical disease and the Flavi virus that causes it, is an arbovirus (arthropod-borne virus), 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-limiting febrile disease, DHF may lead to life-threatening complications. Laboratory diagnosis of Dengue virus infection has mainly depended upon the isolation oi Dengue virus using mosquito cell cultures or inoculation of mosquitoes and detection of anti-Dengue antibody by microscopy or serotyping assay. But the isolation of the dengue virus is tedious, time consuniing


and generally requires several days or weeks, which is not always possible in emergency like conditions.
Polymerase Chain Reaction (PCR) has potential for sensitive, specific and rapid detection of minute quantities of 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 hybridization 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 labor. 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 any of the four closely related serotype of Dengue virus -1, -2, -3 and -4. The immunological status of the infected individual determines whetlier 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 peaks around 16 days after infection. Anti-Dengue-IgG antibodies appear around 21 days after infection and persist for several years. Therefore, in the solid phase diagnostic kit of the present invention, three recombinant antigens of Dengue virus have been used for the detection of anti-Dengue IgM and IgG antibodies to four different types of Dengue virus in the human serum and/or plasma.


Object of the invention :

Therefore, it is an object of the present invention to provide a solid phase diagnostic kit for detecting anti-Dengue IgM and IgG 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, in a single assay system and in one attempt. A method for preparing a solid phase diagnostic kit for detection of anti-Dengue IgM and IgG antibodies of four different types of Dengue virus antigens in a single assay system and in one attempt is also one of the objects of the present invention. As per the further object of the present invention, there is also provided, an in vitro assay for detecting anti-Dengue IgM and IgG antibodies to said serotypes of Dengue virus in a single assay system and in one attempt.
Statement of the invention :

According to one object of present invention, there is provided a diagnostic kit for detecting anti-Dengue IgM and IgG antibodies to four different types of dengue virus, in the human serum and/or plasma, in a single assay system and in one attempt The kit comprises an impregnated combination of three recombinant Dengue virus antigens with goat anti-human-IgM [u specific] onto a solid support for selectively detecting anti-Dengue IgM and IgG antibodies of Dengue virus in human serum and/ or plasma, in a single assay system and in one attempt. The kit of the present invention also comprises immunochemically acceptable reagents for detecting anti-Dengue IgM and IgG antibodies of Dengue virus in human serum and/or plasma. The immunochemically acceptable reagents for detecting anti-Dengue IgM and IgG antibodies of Dengue virus comprise an enzyme linked conjugate-[M+G], a Dengue positive control, a Dengue negative control, a colour reagent, a sample diluent, a stopping solution and a washing solution.
4

A method for preparing a diagnostic kit is provided for detection of anti-Dengue IgM and IgG antibodies to four different types of Dengue virus in a single assay system and in one attempt. The preparation method comprises impregnating over a solid support a combination of three recombinant Dengue virus antigens and goat anti-human-IgM [u specific]; and providing immunochemically acceptable reagents required for detecting anti-Dengue IgM and IgG antibodies of Dengue virus in human serum and/or plasma. The method of impregnating comprises coating on to the solid support a plurality of layers of homogenous solution of goat anti-human-IgM [u specific] and three recombinant Dengue antigens in bicarbonate buffer; blocking said combination antigens using blocking solution, comprising phosphate buffer; and stabilizing it using stabilizing solution, comprising phosphate buffer saline of pH 7.4.
In another aspect of the present invention, an in vitro assay for detecting anti-Dengue IgM and IgG antibodies of Dengue virus, in human serum and/or plasma, in a single assay system and in one attempt using a diagnostic kit of the present invention is also provided. The assay method comprises contacting a sample with three recombinant Dengue virus antigens in conjunction with goat anti-human-IgM [u specific] impregnated onto the solid support to spot anti-Dengue IgM and IgG antibodies in the sample; simultaneously binding said IgM and IgG specific recombinant antigen with a conjugated label, thereby reducing amount of substrate of an enzyme linked to the conjugate-[M+G]; and detecting the enzyme activity, whereby the presence of anti-Dengue IgM and IgG antibodies is determined in the sample.
Detailed description of the invention :
The diagnostic kit of the present invention is a solid phase serological assay kit, in which a specific combination of three recombinant Dengue virus antigens along with goat anti-human-IgM [u specific] is used for selective detection of anti-Dengue IgM


and IgG antibodies to four closely related serotypes of Dengue virus -1, -2, -3 and -4, in biological sample under testing, in a single assay and in one attempt.
In more particular embodiment of the invention, the diagnostic kit comprises coated onto the solid support the combination of goat anti-human-IgM [u specific] and three recombinant Dengue virus antigens for detecting anti-Dengue IgM and IgG antibodies to at least any one of the four closely related serotypes of Dengue virus of four different types, for example, Den-1, Den-2, Den-3 and Den-4, in human serum and/or plasma, in a single testing assay of one attempt.
In most particular embodiment of the invention, the diagnostic kit comprises a microtitre plate with plurality of micro-wells that is impregnated by plurality of coating of homogenous mixture of three recombinant Dengue virus antigens selected form recombinant Dengue, Ver. 1, recombinant Dengue, Ver. 2 and recombinant Dengue, Chimeric and goat anti-human-IgM [u specific] for simultaneous detection of anti-Dengue IgM and IgG antibodies of four closely related serotypes of Dengue virus of four different types, such as, for example Den-1, Den-2, Den-3 and Den-4 in a single testing. The combination of a goat anti-Human IgM [u specific] and three recombinant Dengue virus antigens selected from recombinant Dengue, Ver. 1, recombinant Dengue, Ver. 2 and recombinant Dengue, Chimeric in bicarbonate buffer are adhered onto the micro-wells of microtitre plate and blocked using a blocking solution, containing phosphate buffer and bovine serum albumin (BSA). These antigens are further stabilized using stabilizing solution, comprising phosphate buffer saline (PBS) of pH 7.4.

In another most preferred embodiment of the invention, the diagnostic kit for simultaneous detection of anti-Dengue IgM and IgG antibodies in the human serum and/or plasma comprises at least three coating of homogenous mixture of goat anti-human-IgM [u specific] in conjunction with said recombinant antigens of the types, such as, recombinant Dengue, Ver. 1, recombinant Dengue, Ver. 2 and recombinant Dengue, Chimeric onto the micro-wells of microtitre plate.
6

In one preferred embodiment of the present invention, the buffer solutions described hereinbefore are being stabilized prior to use by mixing with surfactant, stabilizer, sodium chloride and preservative. The preferred stabilizer used in the said buffers is bovine serum albumin (BSA). The surfactant is selected from the group of non-ionic, anionic and Zwitterionic surfactant. The preferred surfactant used is Non-ionic surfactant. A combination of thimerosal with gentamycin is being preferred as preservative for buffer solutions used.
In separate embodiment, the enzyme linked conjugate-[M+G] comprises goat anti-human-IgM+IgG[Fc]-HRPO in conjugate diluent and along with thimerosal and gentamycin.
In another separate embodiment, the Dengue positive control is an inactivated anti-Dengue antibodies containing human serum with thimerosal and gentamycin.
In still another separate embodiment, the Dengue negative control is an inactivated normal human serum with thimerosal and gentamycin as preservatives.
In a distinct embodiment, the colour reagent is 3,3', 5,5'-tetra methyl benzidine dimethyl sulfoxide and H2O2 along with thimerosal and gentamycin.
In another distinct embodiment, the sample diluent is a TRIS buffer, animal serum, BSA, Tween-20, Triton X 100, MgCl2 with thimerosal and gentamycin as preservatives.
In still another distinct 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 diagnostic kit for simultaneously detecting anti-Dengue IgM and IgG antibodies of Dengue virus in human serum and/or plasma. The method comprises impregnating over a solid support a combination of three recombinant Dengue virus antigens and goat anti-human-IgM [u specific] for detecting anti-Dengue IgM and IgG antibodies against any one of four closely related serotypes of Dengue virus type -1, -2, -3 and -4 in bicarbonate buffer; blocking and stabilizing said antigens using blocking and stabilizing solutions; providing a coupling product as an enzyme linked conjugate-[M+G] obtained by covalently binding an goat anti-human-IgM + IgG[Fc]-HRPO; and providing solutions of immunochemically acceptable reagents selected from 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 impregnating comprises coating on to the solid support a plurality of layers of homogenous solution of goat anti-human-IgM [u specific] and three recombinant Dengue antigens in bicarbonate buffer; blocking said combination antigens using blocking solution, comprising phosphate buffer; and stabilizing it using stabilizing solution, comprising phosphate buffer saline of pH 7.4.
In another preferred embodiment of the said process, the coated antigens are three recombinant Dengue virus antigens for detecting anti-Dengue IgM and IgG 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). Preferably, said coated recombinant antigens are recombinant Dengue virus antigens selected from recombinant Dengue, Ver. 1, recombinant Dengue, Ver. 2 and recombinant Dengue, Chimeric.

In another preferred embodiment of the said process, the enzyme-linked conjugare-[M+G] selected for detecting anti-Dengue IgM and IgG antibodies is goat anti-human-IgM+IgG[Fc]-HRPO.
8

In still another preferred embodiment of the invention, the method comprises preparing a solution of an inactivated anti-Dengue antibodies containing human serum with thimerosal and gentamycin as a Dengue positive control.
In one preferred embodiment of the invention, the method comprises preparing a solution of an inactivated normal human serum with thimerosal and gentamycin as a Dengue negative control.
In one more preferred embodiment of the method, preparing the colouring reagent comprises preparing a solution of 3,3'/ 5,5'-tetra methyl benzidine dimethyl sulfoxide with H2O2 along with thimerosal and gentamycin.
In a separate 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 X 100, MgCl2, thimerosal, gentamycin and bovine immunoglobulin.
In another preferred embodiment of the invention, the method comprises providing a solution of a stopping solution containing a concentrated phosphoric acid and deionized water.
In still another embodiment of the invention, the method comprises preparing a washing solution concentrate containing a TRIS buffer, NaCl, 7 ween-20 in deionized water.
According to another aspect of the invention, there is also provided an in vitro assay for simultaneous detection of anti-Dengue IgM and IgG antibodies of Dengue virus, in a single assay system and in one attempt, in human serum and/or plasma using the diagnostic kit of the present invention. The assay method comprises contacting a serum and/or plasma sample containing anti-Dengue IgM and IgG antibodies with microplate containing goat anti-human-IgM [u specific] with three recombinant


Dengue virus antigens, thereby forming a stable complex, if anti-Dengue IgM and IgG antibodies are present; simultaneously binding at least one said IgM and IgG specific recombinant antigens with a conjugated label, thereby reducing amount of substrate of an enzyme linked to the conjugate-[M+G]; and detecting the enzyme activity for the presence of target IgM and IgG antibodies in the sample.
In more preferred embodiment of the invention, the in vitro assay for simultaneous detecting of anti-Dengue IgM and IgG antibodies of Dengue virus in human serum and/or plasma comprises: providing a microplate containing a mixture of three recombinant Dengue virus antigens selected from recombinant Dengue, Ver. 1, recombinant Dengue, Ver. 2 and recombinant Dengue, Chimeric with goat anti-Human-IgM [u specific] as a solid phase; further providing an enzyme linked conjugate-[M+G] obtained by covalently binding an goat anti-human-IgM and IgG[Fc]-HRPO to an enzyme; contacting a given quantity of liquid sample containing anti-Dengue IgM and IgG antibodies with said recombinant Dengue virus antigens, thereby forming a stable complex with anti-Dengue IgM and IgG antibodies present in the sample or Dengue positive control and determining the enzyme activity of the conjugate-[M+G] for the presence of anti-Dengue IgM and IgG of 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 simultaneously detecting anti-Dengue IgM and IgG antibodies of Dengue virus, in human serum and/or plasma, in a single assay and one attempt, the foregoing reagents are employed in predetermined amounts.
In different embodiment of the present invention, the solid support to which said recombinant Dengue virus antigens are attached may be any water-insoluble, water-insuspensible, solid support. An example of suitable solid support is microtitre plate. The immunological component may be bound to the solid carrier by covalent bonds


or by adsorption. The advantage of such microtitre plate is that no centrifugation is required for separation of solid and liquid phase.
In another different embodiment of the present invention, the antibody enzyme conjugate-[M+G] consists of the immunological component covalently linked to one or more enzyme molecules. The linking can be achieved either bv direct condensation or using external bridging molecules as per the methods known in the art. Thus, the reagents like carbodiimides, diisocyanates, glutaric aldehyde and bis-diazobenzidine can be used for production of enzyme coupling products employing a covalent bond.
The choice of an enzyme that is used with coupling product is determined by the properties such as specific binding activity i.e. a high conversion rate, as it increases the sensitivity of the test kit. 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 spectrophotometrickily. 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.

11

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 - Simultaneous detection of anti-Dengue IgM and IgG antibodies
A mixture of three recombinant Dengue virus antigens and goat anti-human-IgM [u specific] 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 and IgG antibodies are present in the sample. Followed by a wash step, goat anti-human-IgM+IgG[Fc]-HRPO is added to the wells. A second wash step will remove free goat anti-human-IgM+IgG[Fc]-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 and IgG antibodies to Dengue virus antigens. 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 and IgG.antibodies in the respective wells.
Example : 2 - Test procedure for detecting anti-Dengue IgM and IgG antibodies
(a) Bring all the reagents and test specimens at room temperature before use; (b) except blank, add lOOul of sample diluent to each well, in each run, there will be one blank, one negative control and three positive controls, add lOul 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+G] 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 50ul of colour reagent to each well and cover the plate with black cover and incubate for


minutes in dark at 20-30°C; (g) add l00ul 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 greater 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 PCx: (0.97+0.975+0.98)/3=0.975 Cut-off value formula: l.OxPCx Cut-off vale: 1.0 x 0.975=0.975
Interpretation of results:
If the absorbance of the test serum and/or plasma is less than 0.8 times of cut-off value, then the sample is considered as non-reactive. If the absorbance of the test serum and/or plasma is in between 0.8 to 1.0 times of cut-off value, then the sample is considered as doubtful. If the absorbance of the test serum and/or plasma is greater than the cut-off value, then it is considered as initial reactive.

13

WE CLAIM :
1. A diagnostic kit for simultaneously detecting anti-Dengue IgM and IgG antibodies in human serum and/or plasma, in a single assay and in one attempt, comprises an impregnated combination of three recombinant Dengue virus antigens for four different types of Dengue virus with goat anti-human-IgM [u specific] onto a solid support; and an immunochemically acceptable reagents selected form an enzyme linked conjugate-[M+G], 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 and IgG antibodies, wherein plurality of impregnated set of three recombinant antigens of Dengue virus selected from recombinant Dengue, Ver. 1, recombinant Dengue, Ver. 2 and recombinant Dengue, Chimeric with goat anti-human-IgM [u specific] are present said solid support.
2. The diagnostic kit as claimed in claim 1, wherein the impregnated recombinant antigens of Dengue virus are selected for simultaneous detection of anti-Dengue IgM and IgG antibodies 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 diagnostic kit as claimed in claim 1, wherein the solid support is a microtitre plate with at least three coatings of a homogenous mixture of said recombinant Dengue virus antigens and goat anti-human-IgM [u specific] for detecting four closely related serotypes of said Dengue virus.
4. The diagnostic kit as claimed in claim 1, wherein the recombinant Dengue virus antigens are coated with goat anti-human-IgM [u specific] 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 diagnostic kit as claimed in claim 1, wherein the enzyme linked conjugate-[M+G] comprises goat anti-human-IgM+IgG[Fc]-HRPO in conjugate diluent with thimerosal and gentamycin.
6. The diagnostic kit as claimed in claim 1, wherein the Dengue positive control comprises an inactivated anti-Dengue antibodies containing human serum with tiiimerosal and gentamycin.
7. The diagnostic kit as claimed in claim 1, wherein Dengue negative control comprises an inactivated normal human serum with thimerosal and gentamycin.
8. The diagnostic kit as claimed in claim 1, wherein the colour reagent is 3,3', 5,5'-tetra methyl benzidine dimethyl sulfoxide with H2O2, and thimerosal and gentamycin.
9. The diagnostic kit as claimed in claim 1, wherein the sample diluent comprises a TRIS buffer, animal serum, BSA, Tween-20, Triton X 100, MgCb with thimerosal and gentamycin.
10. The diagnostic kit as claimed in claim 1, wherein the stopping solution comprises a concentrated phosphoric acid and deionized water.
11. The diagnostic kit as claimed in claim 1, wherein the washing solution concentrate comprises a TRIS buffer, NaCl, Tween-20 with deionized water.



12. A method for preparing a diagnostic kit for simultaneous detection of anti-Dengue IgM and IgG antibodies to four different types of Dengue virus in the human serum and/or plasma as claimed in claims 1 to 11 comprises: impregnating over a solid support a combination of three recombinant Dengue virus antigens selected from recombinant Dengue, Ver. 1, recombinant Dengue, Ver. 2 and recombinant Dengue, Chimeric, goat anti-human-IgM [u specific] 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 a coupling product as an enzyme linked conjugate-[M+G] obtained by covalently binding an goat anti-human-IgM+IgG[Fc]-HRPO; and preparing solutions of immunochemically acceptable reagents selected from a Dengue positive control, a Dengue negative control, a colouring reagent, a sample diluent, a stopping solution and a washing solution.
16

13. An in vitro assay for detecting anti-Dengue IgM and IgG antibodies to four different types of Dengue virus in the human serum and/ or plasma using a diagnostic kit of claims 1 to 11 comprises: providing a microplate containing a mixture of three recombinant Dengue virus antigens selected from recombinant Dengue, Ver. 1, recombinant Dengue, Ver. 2 and recombinant Dengue, Chimeric with goat anti-Human-IgM [u specific] as a solid phase; further providing an enzyme linked conjugate-[M+G] obtained by covalently binding an goat anti-human-IgM and IgG[Fc]-HRPO to an enzyme; contacting a given quantity of liquid sample containing anti-Dengue IgM and IgG antibodies with said recombinant Dengue virus antigens, thereby forming a stable complex with anti-Dengue IgM and IgG antibodies present in the sample or Dengue positive control and determining the enzyme activity of the conjugate-[M+G] for the presence of anti-Dengue IgM and IgG of antibodies of four closely related serotypes of Dengue virus, Den-1, Den-2, Den-3 and Den-4 in the sample.

FOR TRANSASIA BIO-MEDICALS LTD.

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RAVINDRAYEVLE ICE PRESIDENT-LEGAL, IP AND COMPANY SECRETARY

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