FORM 2

THE PATENT ACT (39 of 1970)

1970

The Patents Rules, 2003 COMPLETE SPECIFICATION
(See Section 10, and rule 13)
1. TITLE OF INVENTION
A QUALITATIVE IN VITRO DIAGNOSTIC KIT FOR SIMULTANEOUS DETECTION OF ANTI-HIV AND HCV ANTIBODIES AND HBsAg ANTIGENS
2. APPLICANT(S)

a) Name
b) Nationality
c) Address

TRANSASIA BIO-MEDICALS LTD.
INDIAN Company
TRANSASIA HOUSE,
8, CHANDIVALI STUDIO ROAD,
MUMBAI - 40 0 0 72
MAHARASHTRA

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

A qualitative in vitro diagnostic kit for simultaneous detection of anti-HIV and HCV antibodies and HBsAg antigens
FIELD OF THE INVENTION
The present invention relates to a qualitative in vitro diagnostic kit for simultaneous detection of antibodies to human immunodeficiency virus (HIV) and hepatitis C virus (HCV), and hepatitis B virus (HBV) antigens in human serum and/or plasma. More particularly, it relates to an in vitro diagnostic kit for simultaneously detecting anti-glycoprotein antigen (gp) antibodies to HIV-1 and/or HIV-2 antigens, anti-HCV antibodies to HCV antigens and hepatitis B surface antigens (HBsAg) in human serum and/or plasma. The present invention also relates to a process for the preparation of said kit and an in vitro method for detecting anti-HIV-1 or HIV-2 antibodies, anti-HCV antibodies and HBsAg in the human and/or plasma samples.
BACKGROUND & PRIOR ART OF THE INVENTION
Significant population in the world is infected with HIV. The human serum and/or plasma of patients suffering from HIV infection show presence of different anti-HIV antibodies. Among these various antigens, antibodies to glycoprotein 41 (gp41) of HIV-1 and glycoprotein 36 (gp36) of HIV-2 antigens have been found the most specific and sensitive. Anti-gp41 and anti-gp36 antibodies are absent in non-infected healthy individuals. Patients suffering from HIV infection show presence of these antibodies against said antigens and these antibodies can be detected by number of serological assays.
With the advent of AIDS, viral testing has become a matter of great importance. The HIV-1 and HIV-2 viruses are highly transmissible that ultimately causes death of the infected person. Even today there is no treatment for the AIDS at present, therefore, the detection of anti-gp41 and/or gp36 antibodies corresponding to HIV-1 and/or HIV-2 in serum and/or plasma of infected individuals is important in epidemiological studies and to prevent further spreading of these fatal viruses.


Among other challenges to be faced, one is protection of blood products from contamination by the causative agent of AIDS, HIV-1 and/or HIV-2. An evidence of epidemic as a effect of HIV-1 is widespread and has been reported almost in all corners of the world as compared to the effects of HIV-2 that is particularly reported in few hundred to few thousand people, mainly restricted to West African and European countries. Blood transmission and its products are the major sources of these viral infections. It is, therefore, equally necessary to identify the potential blood donors who are being infected with HIV-1 and HIV-2 so that at least onwards transmission of these viruses could be prevented. Practically, screening the donors for the presence of anti-gp41 and gp36 antibodies to HIV-1 and HIV-2 antigens can make it possible.
Also, about 5% of the world population is also infected with another fatal virus, HBV, which is responsible for both hepatitis B and hepatocellular carcinoma. One out of every ten persons is infected with Hepatitis B infection that develops some form of chronic liver disease and becomes a long-term carrier of HBV. At present various stages after HBV infection; presence of Hepatitis B surface antigen (HBsAg) in serum and plasma is identified. HBsAg is a set of lipoprotein having molecular weight ranging from 22 kD to 96 kD that constitute the envelop of the virus. The HBsAg is the first detectable marker in HBV infected serum and/ or plasma samples and being datable during the whole jaundice phase and becomes undetectable after the appearance of anti-HBsAg antibodies in the serum and/or plasma.
Further, HCV is a main aetiological agent of non-A and non-B Hepatitis, which account for greater than 90% of post-transfusion hepatitis case. HCV is a spherical virus of about 30-60 mm in diameter with single positive standard RNA and relates to a 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. Chronic infection is a major cause of chronic liver disease and hepatocellular carcinoma worldwide. It is therefore, highly necessary to correctly diagnose HCV infection, and to follow and treat HCV infection. Such diagnosis necessitated the

requirement of sensitive tools including antibody assay and genomic assay. The present HCV test kits 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.
In the prior art, number of immunological assays have been reported for the detection of antigens as well as antibodies of HIV-1, HIV-2, HBV and HCV; components of HIV-1, HIV-2, HBV and HCV; and their associated antigens and antibodies in the human serum and/or and plasma. However, in the field of diagnostic what is important is assay, which is used for detection should be sensitive and reliable because the incorrect diagnosis can lead to serious consequences. This is especially true in the case of the determination of anti-HIV and HCV antibodies and HBsAg and their associated antigens and antibodies because the transmission of the HIV, HBV and HCV via blood donors constitutes a significant public health risk.
Enzyme linked immunosorbent assays based on the viruses are presently available, but those require special equipments and expertise and are very expensive to perform. In addition, they produce significant number of false positive results. Immunofluorescence tests are comparatively less expensive and easier to perform than the above said assays, but those require the use of a fluorescence microscope. Also, radioimmunoassay in their various forms are available, but these methods have several problems like requirement of special equipments, trained staff, need for extra safety measures to protect against harmful radiation and short half-life span of radioactive labelling element.
At present, infections of HIV, HBV and HCV are global burden in health care system. Currently the detection of these viral infections in blood banks, hospitals and pathological laboratories are based on serological screening of donor samples i.e., three independent immunoassays require to be performed to detect anti-HIV and HCV antibodies and HBsAg in the human and/ or plasma samples. It is therefore highly necessary to offer an in vitro diagnostic kit for the simultaneous detection of anti-HIV-1, anti-HIV-2 and anti-HCV antibodies and HBsAg in the

human serum and/or plasma that is easy to handle and perform the assay, ready to use within reasonable time and not requires special equipments and expertise. Therefore, inventors of the present invention have proposed a qualitative in intra diagnostic by utilizing a mixture of recombinant or synthetic glycoprotein antigens of HIV-1 and HIV-2, a mixture of affinity purified anti-HBsAg antibodies to HBsAg and a mixture of recombinant or synthetic antigens of HCV for the simultaneous detection of anti-HIV-1 and HIV-2 antibodies, HBsAg antigens and anti-HCV antibodies in the human serum and/or plasma.
OBJECT OF THE INVENTION
Therefore, an object of the present invention is to offer a qualitative in intra diagnostic kit for the simultaneous detection of antibodies to HIV and HCV and HBsAg antigens in the human serum and/or plasma.
Therefore, another object of the present invention is to provide a qualitative in vitro diagnostic kit for the simultaneous detection of anti-HIV-1 and HIV-2 antibodies, HBsAg and anti-HCV antibodies in the human serum and/or plasma.
Therefore, still another object of the present invention is to provide a qualitative in vitro diagnostic kit for the simultaneous detection of anti-gp41 and gp36 antibodies to HIV-1 and HIV-2 antigens, HBsAg antigens and anti-HCV antibodies to HCV antigens in the human serum and/or plasma.
Therefore, yet another object of the present invention is to provide a simple, cost effective and reliable in vitro diagnostic kit for screening and detecting anti-gp41 and gp36 antibodies, HBsAg and anti-HCV antibodies in the human serum and/ or plasma samples.
Therefore, further object of the present invention is to provide a qualitative in vitro diagnostic kit for the simultaneous detection of anti-gp41 and gp36 antibodies to HIV-1 and HIV-2 antigens, HBsAg of HVB and anti-HCV antibodies to HCV

antigens in the human serum and/or plasma that is free from the risks and problems associated with the prior art assays.
Therefore, yet further object of the present invention is to provide a qualitative in vitro diagnostic kit for the simultaneous detection of anti-gp41 and gp36 antibodies, HBsAg and anti-HCV antibodies in the human serum and/or plasma, which comprises a mixture of recombinant or synthetic glycoprotein antigens of HIV-1 and HIV-2, a mixture of affinity purified anti-HBsAg antibodies to HBsAg and a mixture of recombinant or synthetic antigens of HCV.
Therefore, another object of the present invention is to provide a process for the preparation a qualitative in vitro diagnostic kit for the simultaneous detection of anti-HIV-1 and HIV-2 antibodies, HBsAg and anti-HCV antibodies in the human serum and/or plasma.
Therefore, yet another object of the present invention is to provide an in vitro diagnostic method for the simultaneous detection of anti-HIV-1 or HIV-2 antibodies or HBsAg or anti-HCV antibodies in the human serum and/or plasma samples.
STATEMENT OF THE INVENTION
Therefore, in accordance with a foremost object of the present invention, a qualitative in vitro diagnostic kit for the simultaneous detection of anti-HIV-1 and HIV-2 antibodies HBsAg and anti-HCV antibodies in the human serum and/or plasma is provided, which comprises a solid support or matrix coated with a mixture of recombinant or synthetic HIV-1 and HIV-2 glycoprotein antigens, a mixture of specific anti-HBsAg antibodies and a mixture of recombinant or synthetic antigens of HCV; anti-human IgG and IgM and anti-HBsAg conjugated enzymes; and any other immunochemical reagents that are required for detecting the said antibodies and antigens and performing the said assay.
Therefore, in accordance with another object of the present invention, a process for preparing a qualitative in vitro diagnostic kit for the simultaneous detection of anti-

HIV-1, anti-HIV-2, anti-HCV antibodies and HBsAg in the human serum and/or plasma is also provided, which comprises preparing a solid support or matrix that is coated with a mixture of recombinant or synthetic HIV-1 and HIV-2 glycoprotein antigens, a mixture of specific anti-HBsAg antibodies to HBsAg and a mixture of recombinant or synthetic antigens of HCV; preparing anti-human IgG and IgM and anti-HBsAg conjugated enzymes; and preparing immunochemical reagents that are required for detecting the said antibodies and antigens and performing the said assay.
Therefore, in accordance with another object of the present invention, an in vitro diagnostic method for the simultaneous detection of anti-HIV-1 and anti-HIV-2 antibodies, HBsAg and anti-HCV antibodies in the human serum and/or plasma is provided, which comprises contacting a human serum and/or plasma sample with a solid support or matrix, which contains a coated mixture of recombinant or synthetic glycoprotein antigens of HIV-1 and HIV-2, mixture of anti-HBsAg antibodies to HBsAg and mixture of recombinant or synthetic antigens HCV and also contains anti-human IgG and IgM and anti-HBsAg conjugated enzymes; and detecting antigen-antibody complex formed therein using the immunochemical reagents required for detecting the said antibodies and antigens.
SUMMARY OF THE INVENTION
In accordance with one aspect, the qualitative in vitro diagnostic kit for the simultaneous detection of anti-HIV-1, anti-HIV-2 and anti-HCV antibodies and HBsAg in the human serum and/or plasma comprises:
(i) A solid support or matrix with plurality of microwells in which each well is being coated with a mixture of recombinant or synthetic glycoprotein antigens of HIV-1 and HIV-2, a mixture of affinity purified anti-HBsAg antibodies to HBsAg and a mixture of recombinant or synthetic antigens of HCV;

(ii) A pair of conjugated enzymes in which is one enzyme is conjugated with anti-human IgG and IgM and another is conjugated with anti-HBsAg antibodies; and
(iii) Solutions of immunochemical reagents that are required for detecting said antibodies and antigens and performing the assay, which include anti-HIV positive control, HBsAg positive control, anti-HCV positive control, negative control, colour reagent, sample diluent, enhancer, stopping solution and washing solution.
In accordance with another aspect, the process for preparing a qualitative in vitro diagnostic kit for the simultaneous detection of anti-HIV-1, anti-HIV-2 and anti-HCV antibodies and HBsAg in the human serum and/or plasma comprises:
(i) Preparing a solid support or matrix with plurality of microwells by coating a mixture of recombinant or synthetic gp41 and gp36 antigens of HIV-1 and HIV-2, a mixture of affinity purified anti-HBsAg antibodies and a mixture of recombinant or synthetic antigens of HCV using covalent bonding;
(ii) Preparing a pair of conjugated enzymes by conjugating one enzyme with anti-human IgG and IgM and another enzyme with anti-HBsAg antibodies using covalent linking;
(iii) Preparing immunochemical reagent by preparing solutions of anti-HIV positive control, HBsAg positive control, anti-HCV positive control, negative control, colour reagent, sample diluent, enhancer, stopping solution and washing solution.
In accordance with still another aspect, the in vitro diagnostic method for the simultaneous detection of anti-HIV-1 and HIV-2 antibodies, HBsAg and anti-HCV antibodies in the human serum and/or plasma samples comprises:
(i) Contacting human serum and/ or plasma samples with a solid support or matrix with plurality of microwells coated with a mixture of recombinant or

synthetic glycoprotein antigens of HIV-1 and HIV-2, a mixture of affinity purified anti-HBsAg antibodies to HBsAg and a mixture of recombinant or synthetic antigens of HCV;
(ii) Subsequently contacting said mixtures with conjugated enzymes in which one enzyme is linked with anti-human IgG and IgM and another is linked with anti-HBsAg antibodies; and
(iii) Detecting bound anti-HIV-1 or anti-HIV-2 or anti-HCV antibodies and HBsAg using immunochemical reagents required for detecting the said antibodies and antigens.
DESCRIPTION OF THE INVENTION
In one particular aspect of the invention, the qualitative in vitro diagnostic kit for the simultaneous detection of anti-HIV-1 and HIV-2 antibodies, HBsAg and anti-HCV antibodies in the human serum and/or plasma comprises (i) a solid support or matrix with plurality of microwells that are coated with a mixture of recombinant or synthetic glycoprotein antigens gp41 and gp36 of HIV-1 and HIV-2, respectively, a mixture of affinity purified goat anti-HBsAg antibodies to HBsAg and a mixture of recombinant or synthetic HCV core antigens, HCV NS3 antigens, HCV NS4 antigens and HCV NS5 antigens; (ii) two different conjugated enzymes in which one is separately linked with goat anti mouse IgG & IgM and Mouse anti-human IgG and IgM and another is linked with anti-HBsAg monoclonal antibodies; and (iii) solutions of immunochemical reagents that are required for detecting said antibodies and antigens and performing the assay, that include anti-HIV positive control, HBsAg positive control, anti-HCV positive control, negative control, colour reagent, sample diluent, enhancer, stopping solution and washing solution.
In another particular aspect of the invention, the process for preparing a qualitative in vitro diagnostic kit for the simultaneous detection of anti-gp41 and gp36 antibodies, HBsAg and anti-HCV antibodies in the human serum and/or plasma comprises (i) preparing a solid support or matrix with plurality of microwells coated


with a mixture of recombinant or synthetic glycoprotein antigens gp41 and gp36 of HIV-1 and HIV-2, a mixture of affinity purified goat anti-HBsAg antibodies to HBsAg and a mixture of recombinant or synthetic antigens of HCV core antigens, HCV NS3 antigens, HCV NS4 antigens and HCV NS5 antigens using covalent bonding; (ii) preparing two conjugated enzymes in which one is separately linked with mouse and goat anti-human IgG and IgM and another is separately linked with anti-HBsAg monoclonal antibodies using covalent linking; and (iii) preparing immunochemical reagents that are required for detecting said antibodies and antigens and performing the assay, which include anti-HIV positive control, HBsAg positive control, anti-HCV positive control, negative control, colour reagent, sample diluent, enhancer, stopping solution and washing solution by preparing their solutions in required solvents.
In yet another particular aspect of the invention, the in vitro diagnostic method for the simultaneous detection of anti-HIV-1 and HIV-2 antibodies, HBsAg and anti-HCV antibodies in the human serum and/or plasma samples comprises (i) contacting human serum and/or plasma samples under testing with a solid support or matrix with plurality of microwells that are coated with a mixture of recombinant or synthetic glycoprotein antigens gp41 and gp36 of HIV-1 and HIV-2, respectively, a mixture of affinity purified goat anti-HBsAg antibodies to HBsAg and a mixture of recombinant or synthetic antigens of HCV core antigens, HCV NS3 antigens, HCV NS4 antigens and HCV NS5 antigens; (ii) subsequently contacting said mixtures of antigens and antibodies with two different conjugated enzymes in which one enzyme is separately linked with mouse and goat anti-human IgG and IgM and another is separately linked with anti-HBsAg monoclonal antibodies; and (iii) detecting the bound anti-HIV-1 or anti-HIV-2 or anti-HCV antibodies and HBsAg using immunochemical reagents that are required for detecting the said antibodies and antigens.



DETAILED DESCRIPTION OF THE INVENTION
The present invention can be understood more readily by reference to following detailed description of specific embodiments of the diagnostic kit, process for preparing the said kit and in vitro method for detecting the said antibodies and antigens. Although, the invention has been described with reference to particular and specific embodiments, it is not intended that such details should be regarded as limitations to the scope of the invention. Unless otherwise indicated, all the technical and scientific terms used have the same meaning as commonly understood by the person skilled in the art to which this invention belongs.
In preferred aspect of the present invention, the in vitro diagnostic kit for simultaneous detection of anti-HIV-1 and HIV-2 antibodies, HBsAg and anti-HCV antibodies in the human serum and/or plasma comprises the solid support or matrix with plurality of microwells that is coated with a mixture of recombinant or synthetic glycoprotein antigens gp41 and gp36, preferably custom peptides 1 and 2 of gp41 antigen and custom peptide 3 of gp36 antigen for HIV-1 and HIV-2.
In next preferred aspect of the present invention, the in vitro diagnostic kit for simultaneous detection of anti-HIV-1 and HIV-2 antibodies, HBsAg and anti-HCV antibodies in the human serum and/or plasma comprises the solid support or matrix with plurality of microwells that is coated with a mixture of recombinant or synthetic HCV antigens, preferably recombinant HCV core antigens, recombinant HCV NS3 antigens, recombinant HCV NS4 antigens and recombinant HCV NS5 antigens.
In another preferred aspect of the present invention, the in vitro diagnostic kit for simultaneous detection of anti-HIV-1 and HIV-2 antibodies, HBsAg and anti-HCV antibodies in the human serum and/or plasma comprises the solid support or matrix with plurality of microwells that is coated with an affinity purified mixture of specific goat anti-HBsAg antibodies to HBsAg.

In still another preferred aspect of the present invention, the in vitro diagnostic kit for simultaneous detection of anti-HIV-1 and HIV-2 antibodies, HBsAg and anti-HCV antibodies in the human serum and/or plasma comprises the conjugated enzyme that is linked with mouse and goat anti-human IgG and IgM antibodies. The said anti-human IgG and IgM conjugate preferably provided with conjugate stabilizer and comprises of mouse anti-human-IgG-HRPO, mouse anti-human-IgM-HRPO, goat-anti-mouse-IgG-HRPO and goat-anti-mouse-IgG[H+L]-HRPO.
In yet another preferred aspect of the present invention, the in vitro diagnostic kit for simultaneous detection of anti-HIV-1 and HIV-2 antibodies and HBsAg antigens in human serum and/or plasma comprises the conjugated enzyme that is linked with anti-HBsAg monoclonal antibodies to HBsAg. The said anti-HBsAg conjugate is preferably anti-HBsAg monoclonal antibody linked with HRPO and provided with conjugate stabilizer.
In preferred aspect of the invention, the anti-HIV positive control of diagnostic kit comprises an inactivated anti-HIV human serum along with gentamycin and thimerosal as a preservative.
In another preferred aspect of the invention, the HBsAg positive control of diagnostic kit comprises an inactivated HBsAg containing human serum along with gentamycin and thimerosal.
In next preferred aspect of the invention, the anti-HCV positive control of diagnostic kit comprises an inactivated anti-HCV human serum along with gentamycin and thimerosal as a preservative.
In still another preferred aspect of the invention, the negative control of diagnostic kit comprises an inactivated normal human serum along with gentamycin and thimerosal.
In yet another preferred aspect of the invention, the colour reagent of diagnostic kit comprises 3,3',5,5'-tetramethyl benzidine, dimethyl sulfoxide and hydrogen peroxide.

In preferred aspect of the invention, the sample diluent of diagnostic kit comprises TRIS buffer, BSA, bovine IgG, Tween-20, urea and thimerosal.
In another preferred aspect of the invention, the enhancer of diagnostic kit comprises TRIS buffer, BSA, bovine IgG, NaCl, Tween-2 and thimerosal.
In still another preferred aspect of the invention, the stopping solution of diagnostic kit comprises concentrated phosphoric and deionized water.
In yet another preferred aspect of the invention, the washing solution of diagnostic kit comprises TRIS buffer, NaCl, Tween-20 and deionized water.
In an important aspect of present invention, the in vitro diagnostic kit for simultaneous detection of anti-HIV-1 and HIV-2 antibodies, HBsAg and anti-HCV antibodies in human serum and/or plasma is stored at temperature from 2°C to 8 °C.
In another important aspect of the present invention, the solid support or matrix of the diagnostic kit is a microtitre plates having plurality of microwells that is provided as an insoluble support or matrix. It is provided preferably in two different capacities as per as the number of microwells are concerned. The one said plate contains 96 microwells and another contains 192 microwells for testing maximum 96 and 192 test samples, respectively.
In separate aspect of the present invention, the process for preparing a qualitative in vitro diagnostic kit for simultaneous detection of antibodies to HIV-1, HIV-2 and HCV and HBsAg in the human serum and/or plasma comprises preparing a solid support or matrix with plurality of microwells coated with a mixture of recombinant or synthetic glycoprotein antigens of HIV-1 and HIV-2, a mixture of anti-HBsAg antibodies to HBsAg and a mixture of recombinant or synthetic antigens of HCV.
In preferred aspect of the invention, the process for preparing the solid support or matrix comprises coating onto the surfaces of the microwells of microtitre plate a mixture of recombinant or synthetic glycoprotein antigens gp41 and gp36 of HIV-1

and HIV-2, a mixture of affinity purified specific goat anti-HBsAg antibodies to HBsAg and a mixture of recombinant or synthetic antigens of HCV using non-covalent bonding.
In another preferred aspect of the invention, the process for preparing the diagnostic kit for simultaneous detection of antibodies to HIV-1, HIV-2 and HCV and HBsAg in the human serum and/or plasma comprises preparing conjugated enzymes in which one is separately linked with mouse and goat anti-human IgG and IgM and another is separately linked with anti-HBsAg monoclonal antibodies.
In more preferred aspect of the invention, the process for preparing the conjugated enzymes comprises separately linking the enzyme with mouse and goat anti-human IgG and IgM and with anti-HBsAg monoclonal antibodies to HBsAg using covalent linking so as to obtained anti-human IgG and IgM enzyme conjugate and anti-HBsAg enzyme conjugate. Most preferably, the said anti-human IgG and IgM enzyme conjugate comprises of mouse anti-human-IgG-HRPO, mouse anti-human-IgM-HRPO, goat-anti-mouse-IgG-HRPO and goat-anti-mouse-IgG[H+L]-HRPO. Similarly, the said anti-HBsAg conjugate most preferably comprises of anti-HBsAg monoclonal antibody linked with HRPO and provided with conjugate stabilizer.
In preferred aspect of the process for preparing microtitre plate, a mixture of recombinant or synthetic glycoprotein antigens gp41 and gp36 of HIV-1 and HIV-2, a mixture of affinity purified goat anti-HBsAg antibodies to HBsAg and a mixture of recombinant or synthetic antigens of HCV for coating onto the microwells are prepared by dissolving the said antigens and antibodies in to a bicarbonate buffer.
In another preferred aspect of the process for preparing microtitre plate, the said coated antigens and antibodies are blocked using a blocking solution comprising phosphate buffer, BSA and Trans-001.
In still another preferred aspect of the process for preparing microtitre plate, said the blocked antigens and antibodies are stabilised using a stabilising solution comprising PBS, Trans-002 and Tran-003 (Bovine IgG).

In yet another preferred aspect of the invention, the process for preparing the kit comprises separately preparing three different coating solutions, in which one includes a mixture of recombinant or synthetic glycoprotein gp41+gp36, the second includes a mixture of affinity purified goat anti-HBsAg antibodies to HBsAg and the third includes a mixture of recombinant or synthetic antigens of HCV; and incubating the said solutions at 37°C for 2 hours and subsequently at 25°C to 30°C for 4 hours in dehumidified room.
In separate preferred aspect of the preferred invention, the process for preparing the solutions of conjugated enzymes comprises mixing of said anti-human IgG and IgM conjugate and anti-HBsAg conjugate in conjugate diluent in appropriate proportions.
In different preferred aspect of the present invention, the process for preparing an anti-HIV positive control comprises mixing inactivated anti-HIV human serum in diluent with thimerosal and gentamycin in appropriate proportions.
In still different preferred aspect of the present invention, the process for preparing an HBsAg positive control comprises mixing inactivated HBsAg containing human serum in diluent with thimerosal and gentamycin in appropriate proportions.
In yet different preferred aspect of the present invention, the process for preparing an anti-HCV positive control comprises mixing inactivated anti-HCV human serum in diluent with thimerosal and gentamycin in appropriate proportions.
In another preferred aspect of the invention, the process for preparing a HIV-negative control comprises mixing of inactivated normal human serum in diluent with thimerosal and gentamycin in appropriate proportions.
In still another preferred aspect of the invention, the process for preparing a colour reagent comprises mixing of 3,3', 5,5' tetramethyl benzidine in dimethyl sulfoxide and H2O2 with thimerosal and gentamycin in appropriate proportions.


In' yet another preferred aspect of the invention, the process for preparing a sample diluent comprises mixing BSA, bovine IgG, Tween-20 and urea in TRIS buffer with thimerosal and gentamycin in appropriate proportions.
In preferred aspect of the invention, the process for preparing an enhancer comprises mixing BSA, bovine IgG, NaCl and Tween-20 in TRIS buffer with thimerosal and gentamycin in appropriate proportions.
In another preferred aspect of the invention, the process for preparing a stopping solution comprises mixing concentrated phosphoric acid with deionized water in appropriate proportions.
In yet another preferred aspect of the invention, the process for preparing a washing solution comprises mixing TRIS buffer, sodium chloride, Tween-20 in deionized water in appropriate proportions.
In different aspect of the present invention, an in vitro diagnostic method for the simultaneous detection of antibodies to HIV-1, HIV-2 and HCV and HBsAg in the human serum and/or plasma comprises contacting human serum and/or plasma samples with a solid support or matrix with plurality of microwells that are coated with a mixture of recombinant or synthetic gp41 and gp36 antigens, a mixture of affinity purified goat anti-HBsAg antibodies to HBsAg and a mixture of recombinant or synthetic antigens of HCV, thereby forming antigen-antibody complex, if anti-gp41 or anti-gp36 or anti-HCV antibodies or HBsAg present in human serum and/or plasma samples; subsequently adding both the conjugated enzymes that are separately linked with mouse and goat anti-human IgG and IgM and anti-HBsAg monoclonal antibodies, thereby developing a blue colour in the wells of positive controls and test samples by adding colour reagent; and detecting bound anti-gp41 or anti-gp36 antibodies or HBsAg or anti-HCV antibodies spectrophotometrically by adding stoping solution, thereby changing the blue colour into yellow, the intensity of which is directly proportional the amount of bound anti-gp41 or anti-gp36 or anti-HCV antibodies or HBsAg.

In more preferred aspect of the present process, when the human blood serum and/or plasma sample is added to the microwells containing coated mixture of recombinant or synthetic glycoprotein antigens gp41 and gp36, mixture of specific goat anti-HBsAg antibodies and mixture of recombinant or synthetic antigens of HCV, the coated antigens and antibodies form stable complexes with anti-gp41 or anti-gp36 antibodies, HBsAg or anti-HCV antibodies, if present in the samples, followed by the washing step, the said anti-HBsAg and anti-human IgG and IgM-HRPO conjugated enzymes are added to the wells, after second washing step, the free enzyme conjugates are being removed, the colour reagent containing the substrate of HRPO is then added to the wells, the wells containing negative control samples will remain colourless and blue colour is developed in wells containing positive controls and test specimen, upon addition of stopping solution, the blue colour changes to yellow, the intensity of which is directly proportional to the amount of bound anti-gp41, anti-gp36 antibodies, anti-HCV or HBsAg.
In another preferred aspect of the invention, the process comprises contacting positive control and test samples with a mixture of recombinant or synthetic gp41 and gp36 antigens, a mixture of anti-HbsAg antibodies to HbsAg and a mixture of recombinant or synthetic antigens of HCV, which are coupled with the microwells of plate, thereby generating stable antigen-antibody complexes that confine and separate anti-gp41, anti-gp36, anti-HCV antibodies or HbsAg in the positive control and test samples and measuring the amount of said antibodies and antigens using colouring agent by reading the intensity of yellow colour spectrophotometrically.
In most preferred aspect, the method comprises confining the antigen-antibody complexes so formed due to interaction between said antigens and antibodies and conjugated enzymes by adding colour agent, thereby producing the blue colour due to catalytic reaction by conjugated enzymes and measuring the intensity of yellow colour produced by adding stopping solution, which is directly proportional to the amount of said antibodies and antigens into the test sample.

EXAMPLES
The following examples serve to illustrate the invention by way of best method of performing the invention and not to be regarded as limitations to the scope and boundaries of the invention.
Example: 1 - Detection of anti-gp41, anti-gp36 & anti-HCV antibodies and HBsAg
Adding positive controls, anti-gp41, anti-gp36, HBsAg or anti-HCV containing samples to the wells containing sample diluent and said coated antigens and antibodies, thereby forming stable antigen-antibody complex, if said anti-gp41, anti-gp36 or anti-HCV antibodies and HBsAg are present in the samples; followed by a wash step, adding conjugated enzymes; followed by a second wash step that removes unbound enzyme conjugates, adding colour reagent containing substrate for HRPO of the conjugates, thereby developing a blue colour in the wells containing positive control and test samples containing anti-gp41, anti-gp36, anti-HCV antibodies or HBsAg; adding stopping solution, thereby changing the blue colour to yellow; and finally measuring an intensity of the yellow colour spectrophotometrically for detecting the amount of anti-gp41, anti-gp36, anti-HCV antibodies or HBsAg antigens.
Example: 2 - Process steps for detecting antibodies to HIV & HCV & antigens of HBV
(i) Bring all the reagents and test specimens at room temperature before use, (ii) keep one blank [150ml sample diluent + 50ml conjugate], three negative controls, one positive control for HIV, one positive control for HBsAg and one positive control for HCV in each run, (iii) add 100ml of controls to each well, (iv) except blank, add 125ml of sample diluent to each well, (v) add 25(11 of test samples to respective well, (vi) mix properly and cover the plate with black cover and incubate for 1 hour at room 30 - 37°C, (vii) wash the plate as per known microplate washing procedure, (viii) add 25ml of enhancer, 60ml of anti-HBsAg conjugate and 65ml anti-human IgG and IgM conjugate to each well including blank, (ix) cover the plate with black cover and

incubate for 30 minutes at 30 - 37°C, (x) repeat the washing step, (xi) add 60ml of colour reagent to each well and cover the plate with black cover and incubate for 15 minutes at 20 - 30°C, (xii) add 100ml of stopping buffer to each well and (f) read absorbance at 450 nm [using 620/630/650 nm as reference wavelengths] and deduct the blank absorbance from the control and test wells.
Example: 3 - Calculation for cut-off value determination
In case blank, the absorbance of blank should be less than 0.2. In case of positive control, the absorbance of individual positive control should be grater than 1.0. And in case of negative control, the absorbance of individual negative control should be less than 0.2.
NCx : Average value of negative controls.
Calculation of NCx :
For example:
NC Absorbance
1 0.03
2 0.033
3 0.036
NCx : (0.03+0.033+ 0.036)/3 = 0.033
Cut-off value formula: 0.2 + NCx
Cut-off vale: 0.2 + 0.033= 0.233
INTERPRETATION OF RESULTS:
In case of non-reactive samples, if the absorbance of the test serum and/or plasma is less than the cut-off value, then the sample is considered as non-reactive. In case of

reactive samples, 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 initial reactive sample should be retested as duplicate and if the absorbance of duplicate retest results are 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. The repeatedly reactive specimens found by using the diagnosis kit of the present invention, must be further confirmed with a confirmatory test or assay. Also, limitation of the test performed using the kit of the present invention is that the non-reactive result does not preclude the possibility of HIV or HBV or HCV infection.

WE CLAIM:
1. A qualitative in vitro diagnostic kit for simultaneous detection of anti-HIV-1
and HIV-2 antibodies, HBsAg and anti-HCV antibodies in human serum and/or plasma comprises:
(i) A solid support or matrix with plurality of microwells in which each well is being coated with a mixture of recombinant or synthetic glycoprotein antigens of HIV-1 and HIV-2, a mixture of affinity purified anti-HBsAg antibodies to HBsAg and a mixture of recombinant or synthetic antigens of HCV;
(ii) A pair of conjugated enzymes in which is one enzyme is conjugated with anti-human IgG and IgM and another is conjugated with anti-HBsAg antibodies; and
(iii) Solutions of immunochemical reagents that are required for detecting said antibodies and antigens and performing the assay, which include anti-HIV positive control, HBsAg positive control, anti-HCV positive control, negative control, colour reagent, sample diluent, enhancer, stopping solution and washing solution.
2. The in vitro diagnostic kit according to claim 1, in which said solid support or matrix is preferably coated with a mixture of synthetic gp41 and gp36 antigens of HIV-1 and HIV-2, a mixture of affinity purified goat anti-HBsAg antibodies to HBsAg and a mixture of recombinant antigens HCV.
3. The in vitro diagnostic kit according to claims 1 and 2, in which said solid support or matrix is more preferably coated with a mixture of synthetic gp41 [custom peptides 1 and 2] and gp36 [custom peptide 3] of HIV-1 and HIV-2.
4. The in vitro diagnostic kit according to claims 1 and 2, in which said solid support or matrix is more preferably coated with specific goat anti-HBsAg antibodies to HBsAg.

5. The in vitro diagnostic kit according to claims 1 and 2, in which said solid support or matrix is more preferably coated with a mixture of recombinant HCV core antigens, recombinant HCV NS3 antigens, recombinant HCV NS4 antigens and recombinant HCV NS5 antigens of HCV.
6. The in vitro diagnostic kit according to claim 1, in which said one enzyme, preferably HRPO is conjugated with mouse and goat anti-human IgG and IgM and another HRPO is conjugated with anti-HBsAg monoclonal antibodies.
7. The in vitro diagnostic kit according to claims 1 and 6, in which said anti-human IgG and IgM HRPO preferably comprises mouse anti-human-IgG-HRPO, mouse anti-human-IgM-HRPO, goat-anti-mouse-IgG-HRPO and goat-anti-mouse-IgG[H+L]-HRPO and are provided with conjugate stabilizer.
8. The in vitro diagnostic kit according to claims 1 and 6, in which said anti-HBsAg HRPO preferably comprises anti-HBsAg monoclonal antibody linked with HRPO and is provided with conjugate stabilizer.
9. The in vitro diagnostic kit according to claim 1, in which said anti-HIV positive control comprises an inactivated anti-HIV human serum with gentamycin and thimerosal.
10. The in vitro diagnostic kit according to claim 1, in which said HBsAg positive control comprises an inactivated HBsAg containing human serum with gentamycin and thimerosal.
11. The in vitro diagnostic kit according to claim 1, in which said anti-HCV positive control comprises an inactivated anti-HCV human serum with gentamycin and thimerosal.
12. The in vitro diagnostic kit according to claim 1, in which said negative control comprises an inactivated normal human serum with gentamycin and thimerosal.

13. The in vitro diagnostic kit according to claim 1, in which said colour reagent comprises 3,3',5,5'-tetramethyl benzidine, dimethyl sulfoxide and hydrogen peroxide.
14. The in vitro diagnostic kit according to claim 1, in which said sample diluent comprises TRIS buffer, BSA, bovine IgG, Tween-20, urea and thimerosal.
15. The in vitro diagnostic kit according to claim 1, in which said enhancer comprises TRIS buffer, BSA, bovine IgG, NaCl, Tween-2 and thimerosal.
16. The in vitro diagnostic kit according to claim 1, in which said stopping solution comprises concentrated phosphoric and deionized water.
17. The in vitro diagnostic kit according to claim 1, in which said washing solution comprises TRIS buffer, NaCl, Tween-20 and deionized water.
18. A process for preparing a qualitative in vitro diagnostic kit for simultaneous detection of anti-HIV-1 and HIV-2 antibodies, HBsAg and anti-HCV antibodies in human serum and/or plasma comprises:
(i) Preparing a solid support or matrix with plurality of microwells by coating a mixture of synthetic gp41 and gp36 antigens of HIV-1 and HIV-2, a mixture of affinity purified anti-HBsAg antibodies to HBsAg and a mixture of recombinant antigens of HCV using non-covalent bonding;
(ii) Preparing a pair conjugated enzymes by conjugating one enzyme with anti-human IgG and IgM and another enzyme with anti-HBsAg antibodies using covalent linking; and
(iii) Preparing immunochemical reagent by preparing solutions of anti-HIV positive control, HBsAg positive control, anti-HCV positive control, negative control, colour reagent, sample diluent, enhancer, stopping solution and washing solution.

19. An in vitro diagnostic method for simultaneous detection of anti-HIV-1 and HIV-2 antibodies, HBsAg and anti-HCV antibodies in human serum and/or plasma comprises:
(i) Contacting human serum and/or plasma sample with a solid support or matrix having plurality of microwells coated with a mixture of synthetic gp41 and gp36 antigens, a mixture of specific goat anti-HBsAg antibodies and a mixture of recombinant HCV, thereby forming antigen-antibody complex, if anti-gp41, anti-gp36 antibodies, HBsAg or anti-HCV antibodies are present;
(ii) Subsequently adding a pair of conjugated enzymes, in which one conjugated with mouse and goat anti-human IgG and IgM and another conjugated with anti-HBsAg monoclonal antibodies and adding a colour reagent, thereby developing a blue colour in the wells of positive controls and test sample; and
(iii) Detecting bound anti-gp41, gp36 antibodies, HBsAg or anti-HCV antibodies spectrophotometrically by adding stoping solution, thereby changing the blue colour into yellow and measuring the intensity of yellow colour, which is directly proportional to amount of anti-gp41, anti-gp36 antibodies, HBsAg or anti-HCV antibodies.
Dated this 18th day of September, 2007.