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 DIAGNOSTIC KIT FOR DETECTION
HUMAN SERUM AND/OR PLASMA;

OF ANTI-HCV ANTIBODIES IN

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 diagnostic kit for detection of anti-HCV antibodies in human serum and/or plasma
FIELD OF INVENTION
The present invention relates to a diagnostic kit for in-vitro detection of Hepatitis C Virus antibodies in the human plasma and serum.
More specifically, the subject invention relates to a novel/ diagnostic kit for the detection of antibodies for Hepatitis C Virus in the human serum and plasma.
The diagnostic kit of the subject invention comprises synthetic peptides and recombinant proteins of Hepatitis C virus as coating material on solid phase.
PRIOR ART
The present HCV test kits as available for the detection of HCV antibodies in human serum or plasma, lacks in providing desired specificity and sensitivity, thus occasionally not able to detect the HCV at the early stages.
Hepatitis C Virus was identified in 1989 as the main aetiological agent of non-A, non-B Hepatitis accounting for greater than 90% of post-transfusion hepatitis case. Hepatitis C Virus is a spherical virus of about 30-60 mm in diameter with single positive standard RNA and is related to the family flaviviridae. It is considered to be the major cause of acute chronic hepatitis/ liver cirrhosis and hepatocellular carcinoma throughout the world.
HCV infection is a major cause of chronic liver disease and hepatocellular carcinoma. It is therefore, important to make a precise and accurate diagnosis, and to follow and treat hepatitis C infection,

Chronic infection is a major cause of chronic liver disease and hepatocellular carcinoma worldwide. It is therefore necessary to correctly diagnose hepatitis C infection. Such diagnosis necessitated the requirement of sensitive tools including antibody assay and genomic assay.
A series of sensitive and specific tests are available for the diagnosis and evaluation of patients with HCV infection. C100-3 peptide have been used in the first generation enzyme linked immunosorbent assay to detect anti-HCV. This prototype test for antibodies to HCV was found to be very valuable in the diagnosis and study of the epidemiology of HCV. It/ however/ lacked the sensitivity/ especially in the early diagnosis of acute hepatitis. The non-specificity observed in the available tests led to the development of second generation enzyme linked immunosorbent assay. The second regeneration assay incorporated several recombinant viral proteins or peptides and found to had better sensitivity and specificity then the first generation. The third generation assay for testing of anti-HCV was established to overcome the drawbacks found in second generation/ where peptides of NS5 were added to the peptide used in second generation.
The results found in third generation assay were not 100%, hence to achieve 100% results of specificity and sensitivity/ the fourth generation assay for testing anti-HCV has been developed to correctly diagnose Hepatitis C infection.
The test for antibodies to Hepatitis C Virus was proved to be highly valuable in the diagnosis of Hepatitis C Virus and study the infection/especially in the early diagnosis of Hepatitis C Virus before transfusion. Finding elevated serum ALT levels and presence of anti-HCV in serum and/or plasma can easily make the diagnosis of Hepatitis C.

The recombinant DNA technique has been used to encode the genome of the Hepatitis C Virus. The genome encodes for three structural proteins and several nonstructural proteins.
The first and second generation anti Hepatitis C Virus uses synthetic peptide and recombinant viral proteins from non-structural proteins. However/ they are associated with both false positive and negative results.
To overcome this drawback, the third generation antibody test using a greater range of antigens from core, N53, NS4 and NS5 regions of the HCV genome allowing the detection of specific antibodies to multiple viral epitopes and thus providing greater sensitivity and better specificity was developed. The use of these additional antigens results in early detection of antibodies during seroconversion following HCV infection.
The third generation Hepatitis C antibody test using recombinant antigens and HCV synthetic peptides is associated some times with false positive results due to cross reactivity with certain undesirable substances which are not participating in the detection of antibodies but are resulting in giving false positive results.
To overcome this drawback of false results and to achieve 100% reliable results, the fourth generation antibody test using a greater range of recombinant antigens from the subtype level of HCV core antigens/ HCV NS3 antigens, HCV NS4 antigens, and HCV NS5 antigens regions of the HCV genome allowing the detection of specifics antibodies to multiple viral epitopes is devised, thus providing greater sensitivity and better specificity are used. The use of these selected subtypes results in early detection of antibodies during seroconversion following HCV infection.
A new combination or formulation of antigens have been evolved to overcome the drawbacks existed in the conventionally available diagnostic methods.

This new combination of recombinant antigens comprises Non Structural Protein Subtype Three (NS3), Non Structural Protein Subtype Four (NS4)/ Non Structural Protein Subtype Five (NS5) and Core. Where the core is a structural Protein.
OBJECT OF THE INVENTION
Therefore/ it is an object of the present invention to provide an improved diagnostic kit for the detection antibodies of Hepatitis C antigen.
It is also object of the present invention to provide a method for preparing a diagnostic kit for detection of hepatitis C antibodies.
It is another object of the present invention to provide a method for detection of antibodies of Hepatitis C antigen.
STATEMENT OF THE INVENTION
According to objects of present invention/ there is provided a diagnostic kit comprising coated on to a solid support recombinant HCV antigens from the subtype level of 2-10 subtypes of putative core, and other non-structural proteins regions of the virus to selectively detect all subtypes of Hepatitis C virus in human serum and/or plasma with a high degree of specificity and sensitivity. The diagnostic kit further comprises immunochemically acceptable reagents required for detection of antibodies of Hepatitis C, such as/ an enzyme linked conjugate/ an anti-HCV positive control, a HCV negative control/ a colour reagent/ a sample diluent/ a stopping solution and a washing solution. Also/ provided is a process for preparing said diagnostic kit, comprising coating over a solid s'lpport recombinant HCV antigens and providing the reagents required for detection of anti-HCV antibodies. There is also provided/ a process for detection of anti-HCV antibodies in human plasma and/ or serum.

DETAILED DESCRIPTION OF THE INVENTION
In preferably aspect of the present invention, it relates to the fourth generation antibody test for the HCV/ utilizing a unique combination of recombinant HCV antigens from subtype level of 2-10 subtypes of putative core/ and other nonstructural proteins regions of the virus to selectively detect all subtypes of Hepatitis C virus in human serum and/or plasma.
In more preferred aspect of the invention, the diagnostic kit of the present invention comprises recombinant antigens of core HCV/ such as/ core/NS3/ NS4 and NS5.
In the most preferred aspect of the invention/ the recombinant antigens are HCV core antigens/ HCV NS3 antigens/ HCV NS4 antigens, and HCV NS5 antigens.
More specifically, present invention relates to a diagnostic kit for the detection of anti-HCV antibodies in human serum and/or plasma comprising recombinant antigens of HCV/ such as/ HCV core antigens, HCV NS3 antigens/ HCV NS4 antigens and HCV NS5 antigens coated on to the solid support/ such as microtitre plate; an enzyme linked conjugate/ such as/ an anti-human IgG[Fc]-HRPO; an anti-HCV positive control; a HCV negative control; a colour reagent; a sample diluent; a stopping solution and a washing solution/ wherein said solid support having at least three coatings of a homogenous mixture of said HCV recombinant antigens.
Most specifically/ the diagnostic kit of the present invention comprises a microtitre plate coated with plurality of coating of recombinant HCV antigens/ such as/ HCV core antigens, HCV NS3 antigens, HCV NS4 antigens and HCV NS5 antigens; an enzyme linked conjugate/ such as/ anti-human IgG[Fc]-HRPO. The said recombinant HCV antigens are coated in bicarbonate buffer/ blocked in blocking solution/ comprising phosphate buffer and Bovine Serum Albumin and stabilized with stabilizing solution, comprising PBS. At least one buffer solution used in the

present invention is selected form the phosphate group mixed with surfactant/ stabilizer, sodium chloride and preservative.
At least one buffer is selected from the group consisting of Disodium Hydrogen Phosphate and Sodium Dihydrogen Phosphate having molar ratio of 8-12 millimolar each.
The stabilizer used in the said buffer solution is a Protein Stabilizer preferably Bovine Serum Albumin.
The surfactant may be selected from the non-ionic/ anionic and witterionic surfactant; preferably the surfactant used is Non-ionic surfactant.
The preservative used in the said buffer solution is selected from Thimerosal and gentamycin.
The enzyme linked conjugate comprises anti-human IgG[Fc-HRPO] in conjugate diluent with Thimerosal and gentamycin.
The anti-HCV positive control is an inactivated HCV containing human serum along with thimerosal and gentamycin.
The HCV negative control is an inactivated normal human serum along with thimerosal and gentamycin.
The colour reagent is 3/3'/ SA'-tetra methyl benzidine dimethyl sulfoxide along with H202/ thimerosal and gentamycin.
The sample diluent is a TRIS buffer along with NaCI/ Tween-20/ thimerosal and gentamycin.

The stopping solution is a concentrated phosphoric acid and deionized water.
The washing solution concentrate is a TRIS buffer along with NaCI, Tween-20 and deionized water.
According to another aspect of the invention/ a method for preparing a diagnostic kit for detection of antibodies of HCV comprises: coating on to a solid support recombinant antigens of HCV/ such as, core NS3, NS4 and NS5 in bicarbonate buffer; providing a coupling product as an enzyme liked conjugate obtained by covalently binding an anti-human IgG[Fc]-HRPO; blocking said antigens using blocking solution containing phosphate buffer; further stabilizing said antigens using stabilizing solution and providing solutions of an anti-HCV positive control, a negative control, a colouring agent, a sample diluent, a stopping solution and a washing solution.
In said process, the antigens are recombinant antigens of HCV, such as, HCV core antigens, HCV NS3 antigens, HCV NS4 antigens and HCV NS5 antigens, and the enzyme linked conjugate is anti-human IgG[Fc]-HRPO.
The method comprises preparing a solution of an inactivated HCV containing human serum in thimerosal and gentamycin as an anti-HCV positive control.
The method comprises preparing a solution of an inactivated normal human serum in thimerosal and gentamycin as a HCV negative control.
The method comprises preparing a solution of 3,3', 5,5'-tetra methyl benzidine dimethyl sulfoxide in H2O2, thimerosal and gentamycin as a colouring reagent.
The method comprises preparing a solution of a sample diluent containing a phosphate buffer, BSA, Tween-20, thimerosal/ gentamycin and Tran-003.

The method comprises preparing a solution of a stopping solution containing a concentrated phosphoric acid and deionized water.
The method comprises preparing a solution of a washing solution concentrate containing a TRIS buffer/ NaCI/ Tween-20 and deionized water.
According to another aspect of the invention, a method for detection of anti-HCV antibodies comprises contacting a liquid sample/ for example serum and/ or plasma/ containing antibodies against HCV antigen to recombinant HCV antigen that are bound to the surface of a solid carrier. The liquid sample is then incubated with this coated solid carrier for a period of 0.5 to 35 hours, usually at a temperature between 4°C and 5°C. After washing/ the foregoing solid phase is contacted with covalently linked enzyme of the conjugate in the liquid phase. After removing unbound fraction/ determining the enzyme activity for the presence of anti-HCV antibodies in the sample.
More preferably/ the method for the detection of anti-HCV antibodies comprises: providing a recombinant antigens of HCV/ such as/ HCV core antigens/ HCV NS3 antigens/ HCV NS4 antigens and HCV NS5 antigens coated on to the solid support as a solid phase and a coupling product such as an enzyme linked conjugate obtained by covalently binding an anti-human IgG[Fc]-HRPO to an enzyme in a liquid phase; contacting a given quantity of liquid sample with anti-HCV antibody with said solid phase antigens/ thereby forming a stable complex and determining the enzyme activity for the presence of anti-HCV antibodies in the sample.
Most preferably, the method for the detection of anti-HCV, antibodies using the diagnostic kit of the present invention comprises: providing set of recombinant antigens of HCV/ such as/ core/ NS3/ NS4 and NS5 coated on to a solid support and a coupling product as an enzyme linked conjugate; contacting a liquid sample containing HCV with said recombinant antigens/thereby forming a stable complex with anti-HCV antibodies present in the sample or anti-HCV positive control and

determining the enzyme activity of the conjugate for the presence of anti-HCV antibodies in the sample.
The binding partner is employed in an insoluble form. The determination can take place by adding the binding partner in an insoluble form/ or it can be added in a dissolved form/ and in solubilized afterwards. The binding partner is preferably a protein capable of binding the antibody specifically.
After an incubation period/ usually from 0.5 to 25 hours, and at a temperature between 4°C and 5°C/ and washing/ the enzymatic activity bound to the solid phase is determined/ which activity is a measure of the presence of the antibody in the liquid sample tested.
For the detection of the antibody/ the foregoing reagents are employed in predetermined amounts.
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.
The antibody enzyme conjugate 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 production of enzyme coupling products employing a covalent bond can be effected by reagents such as carbodiimides/diisocyanates/ glutaric aldehyde and bis-diazobenzidine.
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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 catalyzing 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 catalyzing those conversions in which reaction components are involved that can be determined spectrophotometrically or fluorimetrically. These determinations are also suitable for automation/ which is an additional advantage.
As an enzyme suitable for the method of the invention can be employed peroxidase. Horse radish peroxidase is preferred.
Addition of positive control or HCV containing human serum or plasma and/or will form s stable complex with the bound antigen present in the well and with anti-human IgG[Fc]-HRPO.
EXAMPLE
The following examples serve to illustrate the practice of the invention/but are not to be regarded as limiting:
Example: 1 - Detection of anti-HCV antibody
Addition of positive control or HCV containing human serum or plasma will form a stable complex with the anti-HCV present in test or positive control specimen. After washing/ anti-human IgG[Fc]-HRPO is added to the wells. Only bound antigen-antibody complex present in the wells will react with the conjugate molecule. A second washing step will remove the unbound conjugate molecule. Addition of colour reagent/ will develop colour only in positive control wells and wells

containing HCV in test specimens. The intensity of development of colour is directly proportional to the presence of bound anti-HCV in the respective wells.
Example: 2 - Test procedure for detection of anti-HCV antibody
(a) Bring all the reagents and test specimens at room temperature before use; (b) except blank, add l00jil of sample diluent to each well, in each run/ there will be one blank/ three negative controls and one positive control/ add 10nl of control and test specimens to the respective wells, mix properly with pipettor and cover the plate with black cover and incubate 45 minutes at 20-30°C; (c) wash the plate as per microplate washing procedure known to person skilled in the art; (d) add 50ul conjugate to each well (except blank well)/ cover the plate with black cover and incubate for 15 minutes at 20-30°C; (e) add 50al of colour reagent to each well and cover the plate with black cover and incubate for 15 minutes in dark at 20-30°C; (e) add l00pl of stopping buffer to each well; and (f) 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 1.0. Negative Control: Absorbance of individual negative control should be less than 0.25 - 0.2. NCx: Average value of negative controls.
Calculation of NCx:
For example:
NC Absorbance
1 0.095
2 0.096
3 0.094
NCx: (0.095+0.096+0.094)/3=0.095 Cut-off value formula: 0.30+NCx
1 2

Cut-off vale: 0.30+0.015=0.395 Interpretation of results:
Non-reactive: If the absorbance of the test serum and/or plasma is less than the cutoff value/ then it is considered as non-reactive.
Reactive: If the absorbance of the test serum and/or plasma is equal or greater than the cut-off value/ then it is considered as initial reactive. This sample should be retested as duplicate. If the absorbance of duplicate retests result is less than cut-off value/ then the specimen is considered as non-reactive. If both of duplicate retest results are found reactive/ then the specimen is considered as repeatedly reactive.
Repeatedly reactive specimens found using a diagnostic kit of the present invention must be further confirmed with other tests e.g. western blot or indirect immunoflu orescent assay.

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WE CLAIMS:
1. A diagnostic kit for detection of anti-HCV antibodies in human serum and/or plasma comprising recombinant antigens of HCV/ such as/ core/ NS3/NS4/ NS5 coated on to a solid support/ wherein said solid support comprises plurality of coating of said HCV recombinant antigens and immunochemically acceptable reagents for the detection of anti-HCV antibodies/ such as/ an enzyme linked conjugate/ an anti-HCV positive control/ a HCV negative control/ a colour reagent/ a sample diluent/ a stopping solution and a washing solution.
2. The diagnostic kit as claimed in claim 1/ wherein the coated recombinant antigens are HCV core antigens/ HCV NS3 antigens/ HCV N54 antigens/ and HCV NS5 antigens.
3. The diagnostic kit as claimed in claim 1, wherein the enzyme linked conjugate is an anti-human IgG[Fc]-HRPO,
4. The diagnostic kit as claimed in claim 1, wherein the solid support is microtitre plate having at least three coatings of a homogenous mixture of said HCV recombinant antigens.
5. The diagnostic kit as claimed in claim \, wherein the said recombinant HCV antigens are coated in bicarbonate buffer/ blocked in phosphate buffer and Bovine Serum Albumin and stabilized with stabilizing solution.
6. A diagnostic kit as claimed in claim 1, wherein the anti-HCV positive control is an inactivated HCV containing human serum.

A diagnostic kit as claimed in claim 1/ wherein the HCV negative control is an inactivated normal human serum.
A diagnostic kit as claimed in claim 1/ wherein the colour reagent is 3,3',5/5/-tetra methyl benzidine dimethyl sulfoxide.
A diagnostic kit as claimed in claim \, wherein the sample diluent is a phosphate buffer along with BSA/ Tween-20/ thimerosal and gentamycin.
A diagnostic kit as claimed in claim 1, wherein the stopping solution is a concentrated phosphoric acid and deionized water.
11. A diagnostic kit as claimed in claim 1, wherein the washing solution is a TRIS buffer along with NaCI/ Tween-20 and deionized water.
12. A method for preparing the diagnostic kit for the detection anti-HCV antibodies as claimed in claims 1 to 11 comprises; coating on to a solid support recombinant antigens of HCV, such as/ core NS3/ NS4 and N55 in bicarbonate buffer; providing a coupling product as an enzyme liked conjugate obtained by covalently binding an anti-human IgG[Fc]-HRPO; blocking said antigens using blocking solution containing phosphate buffer; further stabilizing said antigens using stabilizing solution and providing solutions of an anti-HCV positive control, a negative control/ a colouring agent/ a sample diluent/ a stopping solution and a washing solution.
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13. An in vitro method for the detection of anti-HCV antibodies using the diagnostic kit as claimed in claims 1 to 11 comprises: providing set of recombinant antigens of HCV/ such as/ core/ NS3/ NS4 and NS5 coated on to a solid support and a coupling product as an enzyme linked conjugate; contacting a liquid sample containing HCV with said recombinant antigens/ thereby forming a stable complex with anti-HCV antibodies present in the sample or anti-HCV positive control and determining the enzyme activity of the conjugate for the presence of anti-HCV antibodies in the sample.
Dated this 3rd day of August, 2006.

SUKESH VAZIRANI
CHAIRMAN AND MANAGING DIRECTOR
TRANSASIA BIO-MEDICALS LTD.
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