FORM 2
THE PATENT ACT 1970 (39 of 1970)
The Patents Rules, 2003 COMPLETE SPECIFICATION See Section 10, and rule 13
TITLE OF INVENTION'
A DIAGNOSTIC KIT FOR SIMULTANEOUS SCREENING OF HIV-1 AND HIV-
ANTIBODIES AND ANTIGENS IN HUMAN SERtJM AND PLASMA


APPLICANT(S)
a) Name
b) Nationality
c) Address

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

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 simultaneous screening of HIV-1 and HIV-2 antibodies and antigens in human serum and plasma.
Field of the invention :
The present relates to a serological analysis of human serum and plasma using a kit of the present invention that is fourth generation ELISA kit for screening of antigen of human immunodeficiency virus (HIV-1 and HIV-2) and antibodies to HIV-1 and/or HIV-2 in human serum or plasma. More preferably, it relates to an in vitro kit for the screening of antigens of HIV-1 and/or HIV-2 and antibodies to HIV-1 and/or HIV-2 proteins in the human serum and plasma. Most preferably, it relates to a solid phase immunoassay kit for the screening and detecting of p24 antigens and antibodies to HIV-1 and/or HIV-2 in the human serum and plasma. The invention further relates to a method for the preparation of diagnostic kit of the prerent invention for the screening and detection of p24 antigens and antibodies against HIV-1 and/or HIV-2 proteins in the human serum and plasma. The invention also relates to an in vitro diagnostic method for the screening and detection of p24 antigens and antibodies to HIV-1 and HIV-2 proteins in the human serum and plasma.
Background and prior art of the invention :
Significant number of population in the world is being infected by HIV-1 and/or HIV-2. The HIV virus is divided into HIV-1 (group M and O) and HIV-2. All of HIV has been linked to AIDS. Patients suffering from HIV-1 and/or HIV-2 infection show presence of different anti-HIV-1 and/or HIV-2 antibodies in serum and plasma. A set of HIV-1 and/or HIV-2 specific synthetic peptides and antibodies to said HIV-1 and/or HIV-2 have been synthesized and used in the kits for screening and detecting anti-HIV-1 and/or HIV-2 antibodies and HIV-1 and/or HIV-2 antigens in the serum and plasma samples. Anti-HIV-1 and/or HIV-2 antibodies and HIV-1 and/or HIV-2 antigens are absent in healthy individuals. Patients suffering from HIV infection show the presence of anti-HIV-1 and/or HIV-2 antibodies and

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antigens of HIV-1 and/or HIV-2 and these antibodies or antigens can be detected. Subsequent to HIV-1 and/or HIV infection, serological analysis of human serum and plasma ensures the presence of said antibodies to HIV'-l and/or HIV-2 proteins and also HIV-1 and/or HIV-2 antigens. Therefore, it is highly desirable to detect simultaneously said antibodies to HIV-1 and/or HlV-2 proteins and antigens of HIV-1 and/or HIV-2 in the human serum and plasma. An appropriate technique for screening and detecting said antibodies and antigens in the human serum and plasma is solid phase immunoassay, which utilizes recombinant peptides antigens of HIV-1 and/or HIV-2 and anti- HIV-1 and/or HIV-2 monoclonal antibodies covalently bound or physically adsorbed to an insoluble matrix. The antibody-antigen complex so formed is then held by solid phase technique and bound fraction can be easily detected and estimated using colour reagent.
A living system responds to me presence of foreign antigen like protein, -virus, bacteria, etc by producing specific antibody against that particular antigen. Then, there is a specific reaction between antibody and antigen to form a complex. An antibody once produced is also capable of binding a hapten that is relatively a small and simple molecule, which may be determinant gfoup of given antigen and is capable of binding with specific antibody, but incapable of inducing an antibody production, unless it is bound to an antigen carrier. The binding interaction between antigen or hapten and its antibody is specific and sensitive. Other materials that show similar specific and sensitive binding interactions are enzymes and their substrates, hormones, vitamins, metabolites and phafrnacological agents and their receptors or binding substances and such other substances known in the art. These specific and sensitive binding reactions have given rise to a rapidly emerging analytical technique known as specific binding assay technique. In one such assay, the substances or group of substances to be determined, which may be referred as ligand, in a liquid sample is placed in competition with labelled form of the ligand or of binding analogue thereof for binding to binding reagent. Where an enzyme label is used and the binding reagent is antibody, the technique is known as an enzyme-immunoassay technique. Several alternative labelling materials are available for

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substituting the enzymes, such as radioisotopes, co-enzymes, enzyme substrates, enzyme-modulators like inhibitors and allosteric effectors, fluorescent molecules, and luminescent molecules, but these have inherent disadvantages of handling and test requires sophisticated instruments and trained manpower for accurate results.
The above system consists of antigen or hapten labelled with an enzyme marker, unlabelled native antigen in test sample and specific antibody, thereby there is competition between unlabelled antigen and labelled antigens for binding to limited amount of antibodies. Hence, greater the concentration of unlabelled antigen from the test sample less die labelled antigen will be bound to the antibodies. If the concentration of labelled antigen and antibody is fixed and the only variable is the level of unlabelled antigen, it becomes possible to establish an assay system for detecting unknown level of unlabelled antigens by physically separating the antigen-antibody complex from the remaining free antigens. The enzyme activity of the unknown is compared with standard values given by the range of known amount of antigens treated in die same manner.
In the prior art, many techniques are known for separating free unbound antigens or haptens from the complexes of antigen-antibody. One such technique is chromato-e lectio phoresis, which combines paper chromatography and paper electrophoresis. The paper with a high affinity for the free antigens, like Whatman paper, is used as carriers. This technique is discriminative and has been used in the assay of insulin, growth hormone, glucagons, parathyroid hormone, thyroid stimulating hormone and other peptide hormones, but it has number of disadvantages, which limit its use. One such disadvantage is limited amount of material can be applied to the absorbent and the separation and detection is laborious and time-consuming.
Other known technique is precipitation of antigen-antibody complexes, which involves use of salts, organic material or solvents under the conditions that do not affect free antigens. Among these, salts, materials and solvents used are ethanol, acetone, sodium sulphate, ammonium sulphate, dioxane, trichloroacetic acid, polyethylene glycol, etc. The use of salts, solvents or organic materials has advantage

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that the separation is immediate, and second incubation is not necessary, but the chemical precipitation technique causes co-precipitation of other proteins, which causes incomplete separation of two fractions.
The double antibody technique is also known and widely used for the separation of bound and free antigens. Using this technique, a second antibody that was raised against the first antibody is used to precipitate the primary antigen-antibody complex. Particularly, if the first antibody was raised in rabbit then the second antibody may be an antiserum to rabbit gamma globulin raised in goats, but the disadvantage of this technique is that use of second antibody requires an additional incubation.
Ion exchange and other resins are also known to use for binding free antigens by electrostatic forces and mainly used for the detection of small molecules, such as thyroid hormones. One technique of this type used for the separation of antigen-antibody complex from either free antigen employs a column packed with material that preferably adsorbs either free antigen or antigen-antibody complex. The incubated aqueous mixture is applied on to the head of such column and the column is then eluted. The radioactivity of either the column or the eluate is then determined and the content of the antigen in the starting solution is calculated from the count.
By yet another technique, free unbound antigens adsorbed onto adsorbent and then precipitated by centrifugation. Powdered talc (magnesium silicate), kaolin (aluminium silicate), QUSO (silica micro-granules), cellulose powder, etc are some of the simple adsorbents used for precipitation. Many separations are performed using adsorbent charcoal coated with dextran, which behaves rather like a sieve that allows the smaller molecules of free antigen to pass and these are bound by the charcoal, leaving the bound antigen in the solution, after the charcoal has been removed by centrifugation or filtration.
Also, the solid phase techniques are known for the separation of free and bound antigens that use antibodies covalently bound or physically adsorbed onto insoluble

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matrix, The formed antibody-antigen complex is held by the solid phase and the bound fraction can be easily separated from the free fraction by filtration.
In order to overcome aforesaid drawbacks, an in vitro serological method comprising screening and detecting anti- HIV-1 and/or HIV-2 antibodies and HIV-1 and/or HIV-2 antigens in the human plasma and/or serum using ELISA technique may be used. Therefore, the inventors of the present invention have invented a solid phase immunoassay kit and/or device that utilizes recombinant peptides antigens of HIV-1 and/or HIV-2 and anti- HIV-1 and/or HIV-2 monoclonal antibodies for screening and detecting anti-HIV-1 and/or HIV-2 antibodies and HIV-1 and/or HIV-2 antigens present in human serum and plasma, wherein said recombinant antigens and monoclonal antibodies are covalently bonded or physically adsorbed with a solid support for simultaneously screening and detecting the antibodies to HIV-1 and/oT HIV-2 proteins and antigens of HTV-1 and/or HIV-2.
An advantage of using the solid support like microwells containing microplate is that no centrifugation or filtration required for the separation of solid and liquid phases. The ELISA test able to detect HIV p24 antigen and HIV antibodies simultaneously allows obtaining an earlier diagnosis and reducing the risk of HIV transmission by blood donation. The kit and/or device of the present invention is a fourth generation solid-phase immunoassay, this designation referring to the ability of such assays to detect both the antibodies directed against HIV-1 and HIV-2, as also to detect the HIV-1 p24 antigen.
Object of the invention :
Accordingly an object of the present invention is to offer a diagnostic kit for the screening of antibodies to HIV-1 and/or HIV-2 proteins in the human serum and plasma.
Another object of the invention is to offer a diagnostic kit for the screening of HIV-1 and/or HIV-2 antigens in the human serum and/or plasma.
6

Still another object of the invention is to offer a diagnostic kit for the simultaneous screening and detection of antibodies against HIV-1 and/or HIV-2 proteins and HIV-1 and/or HIV-2 antigens in the human serum and/or plasma.
Yet another object of the present invention is to offer a diagnostic kit for the qualitative detection of antibodies to HIV-1 and/or HIV-2 and HIV-1 and/or HIV-2 antigens in the human serum and/or plasma.
Further object of present invention is to offer a diagnostic kit for the qualitative detection of antibodies against HIV-1 and/or HIV-2 proteins and HIV-1 and/or HIV-2 antigens in the human serum and/or plasma.
Still further object of the invention is to offer a diagnostic kit for simultaneous the screening of p24 antigens and anti-HIV-1 and HIV-2 antibodies in human serum and plasma samples.
Yet further object of the invention is to develop a simple, cost effective and reliable a diagnostic kit for the screening and detection both the antibodies directed against HIV-1 and/or HIV-2 proteins, and also p24 antigens of HIV-1.
Still different object of the invention is to offer a method for the preparation of a diagnostic kit for the screening and detection antibodies directed against HIV-1 and/or HIV-2 proteins and HIV-1 and/or HIV-2 antigens in the human serum and plasma, which is based on the solid phase immunoassay technique.
Yet separate object of the invention is to offer an in vitro diagnostic method for the simultaneous screening and detection of antibodies to HIV-1 and/or HIV-2 proteins and HIV-1. and/or HIV-2 antigens in the human serum and plasma samples.
Statement of the invention :
Therefore, in accordance with main object of the present invention there is provided, a diagnostic kit for the screening and detection of antibodies against HIV-1 and/or HIV-2 proteins and HIV-1 p24 antigens in the human serum and plasma, which

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utilizes recombinant antigens of HIV-1 and/or HIV-2 and monoclonal antibodies against HIV-1 and/or HIV-2 proteins for identifying of HIV-1 and/or HIV-2 antigens and monoclonal [human or mouse origin] antibodies to HIV-1 and/or HIV-2 proteins.
Further, in accordance with another object of the invention there is provided, a method for the preparation of a diagnostic kit for the screening and detection of antibodies against HIV-1 and/ or HIV-2 proteins and HIV-1 p24 antigens in the human serum and plasma, which is based on the solid phase immunoassay technique that utilizes recombinant antigens of HIV-1 and/or HIV-2 and monoclonal antibodies against HIV-1 and/or HIV-2 proteins for coating plurality of wells of solid support.
Also, in accordance with still another object of the invention there is provided, an in vitro diagnostic method for the screening and detection of antibodies against HIV-1 and/or HIV-2 proteins and HIV-1 p24 antigens in the human serum and plasma, which is based on the solid phase immunoassay technique that utilizes recombinant antigens of HIV-1 and/or HIV-2 and monoclonal [human or mouse origin] antibodies against HIV-1 and/or HIV-2 proteins.
Description of the invention :
The diagnostic kit for the screening and detection of antibodies against HIV-1 and/or HIV-2 proteins and HIV-1 p24 antigens in the human serum and plasma comprises a solid support in which recombinant antigens of HIV-1 and/ or HIV-2 and anti-HIV-1 and/or HIV-2 antibodies are coated or adsorbed onto plurality of microwells; conjugates selected from the group of biotinylated anti-p24 antibodies, streptavidin-enzyme conjugate and envelope antigen-enzyme conjugate; and immunological reagents selected form the group of HIV-1 antibody control, HIV-2 antibody control, HIV-1 p24 antigen control, HIV negative control, sample diluent, colour reagent, stopping solution and washing solution.


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feii^v

The method for the preparation of a diagnostic kit the screening and detection of antibodies against HIV-1 and/or HIV-2 proteins and HIV-1 p24 antigens in the human serum and plasma comprises preparing a solid support in which in which recombinant antigens of HIV-1 and/or HIV-2 and anti-HIV-1 and/or HIV-2 antibodies are coated or adsorbed onto plurality of microwells; providing biotinylated anti-p24 antibodies, streptavidin-enzyme conjugate, envelope antigen-enzyme conjugate; further providing HIV-1 antibody control, HIV-2 antibody control, HIV-1 p24 antigen control and HIV negative control; and preparmg solutions of diagnostic reagents like sample diluent, colour reagent, stopping solution and washing solution.
The in vitro diagnostic method for the screening and detection of antibodies against HIV-1 and/or HIV-2 proteins and HIV-1 p24 antigens in the human serum and plasma comprises contacting human serum and/or plasma samples with a solid support in which recombinant HIV-1 and/or HIV-2 antigens and anti-HIV-1 and/or HIV-2 antibodies are coated or adsorbed onto plurality of microwells, therpby forming stable complexes with envelope antigens and/or p24 antibodies attached to the microwells; identifying said antigen-antibody complexes formed by addition of biotinylated anti-p24 antibodies and streptavidrn-enzyme conjugate, and/or envelope antigen-enzyme conjugate; and detecting antibodies against HIV-1 and/or HIV-2 proteins and p24 antigens by addition of colour reagent that contains peroxidase substrate.
Detailed description of the invention :
What is claimed in the present invention can be understood with reference to the foregoing preferred and particular embodiments. Although, the subject matter has been disclosed with reference to particular and specific embodiments, it is not intended that such embodiments should be regarded as limitations to the scope of the invention. Also, unless otherwise specified, all the technical and scientific terms used herein before and after have the same meanings as commonly understood by the person skilled in the art to which this invention belongs or assigned to them.

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In preferred embodiment of the present invention, the diagnostic kit for the screening and detection of antibodies against HIV-1 and/or HIV-2 proteins and HIV-1 p24 antigens in the human serum and plasma comprises a microtitre plate as a solid support in which recombinant gp41 and recombinant gp36 antigens of HIV-1 and/or HIV-2 and monoclonal [human or mouse origin] anti-p24 antibodies for HIV-1 are coated or adsorbed onto plurality of microwells.
In more preferred embodiment of the invention, said microwells of microtitre plate are coated or adsorbed with a mixture of recombinant gp41 and recombinant gp36 antigens of HIV-1 and/or HIV-2 and monoclonal [human or mouse origin] anti-p24 antibodies for HIV-1 without affecting structural and functional integrity of said antigens and antibodies. Similarly, said antigens and antibodies of biotinylated anti-p24 antibodies, streptavidin-enzyme conjugate, envelope antigen-enzyme conjugate are coupled with enzymes and/or labels without affecting their structural and functional integrity.
In still preferred embodiment of the invention, said microtitre plate used as a solid support for the solid phase immunoassay comprises minimum 96 and maximum 126 microwells, which are iinmunochemically coated or adsorbed with a mixture of a mixture of recombinant gp41 and recombinant gp36 antigens of HIV-1 and/or HIV-2 and monoclonal[human or mouse origin] anti-p24 antibodies for HIV-1.
In another preferred embodiment of the invention, the diagnostic kit for the screening and detection of antibodies against HIV-1 and/or HIV-2 proteins and HIV-1 p24 antigens in the human serum and plasma comprises biotinylated anti-p24 antibodies, which comprises anti-rabbit p24-biotin in conjugate diluent along with thimerosal and gentamycin as preservatives.
In still another preferred embodiment of the invention, the diagnostic kit for the screening and detection of antibodies against HIV-1 and/or HIV-2 proteins and HIV-1 p24 antigens in the human serum and plasma comprises streptavidin-enzyme

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conjugate, which comprises streptavidin-HRPO in conjugate diluent along with thimerosal and gentamycin.
In yet another preferred embodiment of the invention, the diagnostic kit for the screening and detection of antibodies against HIV-1 and/or HIV-2 proteins and HJV-1 p24 antigens in the human serum and plasma comprises envelope antigen-enzyme conjugate, which comprises [gp41+gp36]-HRPO conjugate in conjugate diluent along with thimerosal and gentamycin.
In further preferred embodiment of the invention/ the diagnostic kit for the screening and detection of antibodies against HIV-1 and/or HIV-2 proteins and HIV-1 p24 antigens in the human serum and plasma comprises HIV-1 antibody control, HIV-2 antibody control, HIV-1 p24 antigen control, HIV negative control, sample diluent, colour reagent, stopping solution and washing solution.
In still further preferred embodiment of the invention, said HIV-1 and HIV-2 antibody control comprises inactivated anti-HIV-1 and HIV-2 containing human serum, respectively that are preserved by addition of thimerosal and gentamycin.
In yet further preferred embodiment of the invention, said HIV-1 p24 antigen control comprises inactivated HIV-1 p24 antigen containing human serum that is preserved by addition of thimerosal and gentamycin.
In more particular embodiment of the invention, said HIV negative control comprises an inactivated normal human serum preserved by addition of thimerosal and gentamycin.
In more specific embodiment of the invention, said colour reagent comprises 3,3', 5,5'-tetra methyl benzidine dimethyl sulfoxide and H2O2 preserved in thimerosal and gentamycin.


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In more preferred embodiment of the invention, said sample diluent comprises a mixture of phosphate buffer, BSA, bovine immunoglobulin, Tween-20, TritonXl00 MgCl2, preserved in thimerosal and gentamycin.
In separate preferred embodiment of the invention, said stopping solution comprises a solution of concentrated phosphoric acid and deionized water.
In yet separate preferred embodiment of the invention, said washing solution comprises a solution of TRIS buffer, NaCl and Tween-20 in deionized water.
In an essential embodiment of the invention, the diagnostic kit for the screening and detection of antibodies against HIV-1 and/or HIV-2 proteins and HIV-1 p24 antigens in the human serum and plasma is stored at temperature between 2°C and 8 °C.
In another preferred embodiment of the present invention, the method for the preparation of a diagnostic kit the screening and detection of antibodies against HIV-1 and/or HIV-2 proteins and HIV-1 p24 antigens in the human serum and plasma comprises preparing microtitre plate as a solid support in which in which recombinant antigens of HIV-1 and/or HIV-2 and anti-HIV-1 and/or HIV-2 antibodies are coated or adsorbed onto plurality of microwells; providing anti-anti-p24 antibody biotin, streptavidin-HRPO conjugate, [gp41+gp36]-enzyme conjugate; further providing HIV-1 antibody control, HIV-2 antibody control, HIV-1 p24 antigen control and HIV negative control; and preparing solutions of diagnostic reagents like sample diluent, colour reagent, stopping solution and washing solution.
In more preferred embodiment of the invention, the method for the preparation of a diagnostic kit the screening and detection of antibodies against HIV-1 and/ or HIV-2 proteins and HIV-1 p24 antigens in the human serum and plasma, which is based on solid phase immunoassay technique comprises coating or adsorbing onto plurality of microwells of the microtitre plate a mixture of recombinant antigens of HIV-1 and/or HIV-2 and anti-HIV-1 and/or HIV-2 antibodies in bicarbonate buffer using

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covalent linkage; blocking and stabilizing said synthetic peptide antigens using blocking and stabilizing solutions.
In most preferred embodiment of the invention, the method for preparing the microtitre plate comprises coating or adsorbing onto plurality of the microwells number of homogenous layers of recombinant gp41 and gp36 antigens of HIV-1 and/or HIV-2 and monoclonaI[human or mouse origin] anti-p24 antibodies for HIV-
1 in bicarbonate buffer using covalent linkage; blocking said antigens and antibodies
using blocking solution containing phosphate buffer, BSA and Trans-001; and
stabilising said blocked antigens and antibodies using stabilizing solution containing
phosphate buffer saline (PBS) of pH 7.4, Trans-002 and Tran-003 (bovine
immunoglobulin).
In another preferred embodiment of the invention, the method for the preparation of a diagnostic kit the screening and detection of antibodies against HIV-1 and/or HIV-
2 proteins and HIV-1 p24 antigens in the human serum and plasma comprises
preparmg anti-p2Ab-biotin, streptavidin-HRPO conjugate, [gp41+gp36]-enzyme
conjugate by covalently coupling said antibodies and antigens with biotin and
HRPO as a suitable enzyme label or marker.
In further preferred embodiment of the invention, the method for the preparation of a diagnostic kit the screening and detection of antibodies against HIV-1 and/ or HIV-2 proteins and HIV-1 p24 antigens in the human serum and plasma comprises preparing solutions of HIV-1 antibody control, HIV-2 antibody control, HIV-1 p24 antigen control, colour reagent, sample diluent, stopping solution and washing solution that are required for the screening and detection of antibodies against HIV-1 and/or HIV-2 proteins and HIV-1 p24 antigens in the human serum and plasma.
In more preferred embodiment of the method, it comprises preparing a solution of HIV-1 and HIV-2 antibody control, which comprises inactivated anti-HIV-1 and HIV-2 containing human serum, respectively that are preserved by addition of thimerosal and gentamycin.

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In another preferred embodiment of the method, it comprises preparing a solution of HIV-1 p24 antigen control, which comprises inactivated HIV-1 p24 antigen containing human serum that is preserved by addition of thimerosal and gentamycin.
In still another preferred embodiment of the method, it comprises preparing a solution of HIV negative control, which comprises an inactivated normal human serum preserved by addition of thimerosal and gentamycin.
In yet another preferred embodiment of the method, it comprises preparing a colour reagent, which comprises 3,3', 5,5'-tetra methyl benzidine dimethyl sulfoxide and H2O2 preserved in thimerosal and gentamycin.
In separate preferred embodiment of the method, it comprises preparing a sample diluent, which comprises a mixture of phosphate buffer, BSA, bovine immunoglobulin, Tween-20, TritonXlOO MgCl2, preserved in thimerosal and gentamycin.
In still preferred embodiment of the method, it comprises preparing a stopping solution comprises a solution of concentrated phosphoric acid and deionized water.
In yet preferred embodiment of the method, it comprises preparing a washing solution comprises a solution of TRIS buffer, NaCI and Tween-20 in deionized water.
In another preferred embodiment of the present invention, the in vitro diagnostic method for the screening and detection of antibodies against HIV-1 and/or HIV-2 proteins and HIV-1 p24 antigens in the human serum and plasma comprises adding human serum and/or plasma samples in the microwells of microtitre plate wherein mixture of recombinant HIV-1 and/or HIV-2 antigens and anti-HIV-1 and/or HIV-2 antibodies are coaled or adsorbed for forming stable complexes with HIV envelope antigens and/or p24 antibodies, if p24 antigens and/or antibodies specific for HIV-1 and/or HIV-2 are present in the sample; adding anti-p24-Ab-biotin and streptavidin-HRPO conjugates, and/or [gp41+gp36]-HRPO conjugate for identifying srid

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antigen-antibody complexes so formed; adding further colour reagent that contains peroxidase substrate for detecting anti-HIV-1 and/or HIV-2 antibodies and p24 antigens by developing a blue colour in proportion to the amount of anti-HIV-1 or HIV-2 antibodies and/ or p24 antigens bound to the wells.
In more preferred embodiment of the invention, the in vitro diagnostic method for the screening and detection of antibodies against HIV-1 and/or HIV-2 proteins and HIV-1 p24 antigens in the human serum and plasma comprises contacting human serum and/or plasma samples with a mixture of recombinant gp41 and gp36 antigens and monoclonalfhuman or mouse origin] anti-p24 antibodies, which are coated or adsorbed onto the microwells of microtitre plate, thereby forming stable complexes with HIV envelope antigens and/ or anti-p24 antibodies; contacting further with anti-p24-Ab-biotin and streptavidin-HRPO conjugates, and/or [gp41+gp36]-HRPO conjugate, thereby identifying said antigen-antibody complexes formed; reacting with colour reagent containing substrate of the peroxidase, thereby developing the blue colour in the proportion to amount of anti-HIV-1 or HIV-2 antibodies and/or p24 HIV-1 antigens.
In more preferred embodiment of the invention, the in vitro diagnostic method for the screening and detection of antibodies against HIV-1 and/or HIV-2 proteins and p24 HIV-1 antigens in the human serum and plasma comprises adding measured amount of human serum and/or plasma samples to the microwells containing sample diluent and are coated or adsorbed with recombinant gp41 and recombinant gp36 antigens and anti-p24 monoclonalfhuman or mouse origin] antibodies, thereby forming stable antigen-antibody complexes, if said antibodies and antigens specific to HIV-1 and/ or HIV-2 are present in the samples; washing the microwells by known microtitre plate washing procedure, thereby removing unbound fractions; adding further measured amounts anti-p24-Ab-biotin and streptavidin-HRPO conjugates, and/or [gp41+gp36]-HRPO conjugate to said microwells, thereby identifying said antigen-antibody complexes so formed; washing further the microwells, thereby removing the said free conjugates; adding measured amount of

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colour reagent containing substrate of HRPO, thereby developing a blue colour in the microwells containing positive control and test samples; adding further measured amount of stopping solution to the microwells, thereby stopping the catalytic reaction of conjugated enzymes; measuring an intensity of developed colour using spectrophotometer, which is directly proportional to the amount of said antibodies and antigens present therein.
In different preferred embodiment of the invention, said foregoing reagents and/or samples are employed in a predetermined amounts for screening and detection of antibodies against HIV-1 and/or HIV-2 proteins and p24 HIV-1 antigens in the human serum and plasma.
In another embodiment of the invention,. the solid support to which said recombinant HIV-1 and/or HIV-2 antigens and anti-HIV-1 and/or HIV-2 anybodies are coated or adsorbed may be any water-insoluble, water-in-suspensible, solid support. A preferred example of said solid support is microtitre plate containing plurality of microwells. The immunological components may be coated or adsorbed to the microtitre plate by covalent bonds or adsorption. The advantage of such microtitre plate is that no centrifugation step is required for the separation of solid and liquid phases. Also, the antibodies of conjugates consist of the immunological components that are covalently linked to one or more enzyme/label molecules. The Unking can be achieved either by direct condensation or using external bridging molecules known in the prior art. Thus, the reagents like carbodiimides, diisocyanates, glutaric aldehyde and bis-diazobenzidine can be used for the production of enzyme-conjugated products employing the covalent bond.
The choice of an enzyme/label, which 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 diagnostic 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

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components are involved, which can be determined spectrophotometrically. These determinations are also suitable for automation that is an additional advantage. As an enzyme suitably employed in the present invention is peroxidase, preferably Horse radish peroxidase.
Examples
The following examples serve to illustrate the use of the invention, but are not to ne regarded as limitations for the scope of the invention.
Example 1: Screening of anti-HIV-1/2 antibodies and HIV-1 antigens
A mixture of recombinant antigens of HIV-1 [gp41] and HIV- 2 [gp36] and [human or mouse origin] monoclonal antibodies against HIV-1 p24 antigens are coated or adsorbed onto the microwells of microtitre plate. When human plasma and/or serum samples added to sample diluent containing microwells, if p24 antigens and/or antibodies specific for HIV-1 and/or HIV-2 are present in the samples, they form stable antigen-antibody complexes with HIV envelop antigens and/or anti-p24 antibodies attached to the microwells. Followed by a wash step, antigen-antibody complexes are then identified through addition of biotinylated p24 antibody and streptavidin horseradish peroxidase (HRPO) conjugate; and/or envelope antigen-HRPO conjugate. The colour reagent containing the substrate of HRPO is then added to the microwells. During incubation, a blue colour develops in proportion to the amount of anti-HIV-1/2 antibodies and/or p24 antigens bound to the well, thus establishing their presence/absence in the sample.
Example 2: Test procedure for the screening and detection anti-HIV-1/2 antibodies and HIV-1 antigens
(a) Bring all the reagents and test specimens at room temperature before use; (b) in each run, there will be one negative control, one HIV 1 antibody control, one HIV 2 antibody control and one HIV p24 antigen control; (c) add 50µl sample diluent to each well and add 50µ1 sample or p24 antigen control or antibody control to each

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well, add 50µl of p24-Ab-Biotin to each well and incubate one hour at 37°C; (d) wash the plate as per known microplate washing procedure; (e) add 50µ1 of streptavidin-HRPO conjugate and 50µl HIV (gp36+gp41) HRPO conjugate to each well except the blank well, cover the plate with black cover and incubate 30 minutes at 37° C; f) wash the plate as per microplate washing procedure; (g) add 50µl of colour reagent to each well, cover the plate with black cover and incubate for 15minutes in dark at 20-30°C; (h) add lOOul of stopping solution to each well; and (i) read absorbance at 450nm (using 620/630/650nm as reference wavelength).
Example: 3 - Calculation for cut-off value determination for the detection anti-HIV-1/2 antibodies and HIV-1 antigens
Blank value: Absorbance of blank values should be less than 0.2.
Positive Control:. Absorbance of individual positive control should be grater than 1.0.
Negative Control: Absorbance of individual negative control should be less than 0.2.
NCx: Average value of negative controls.
Calculation of NCx:
For example:
NC Absorbance
1. 0.06
2. 0.063
3. 0.066 NCx: (0.06+ 0.063+ 0.066)/3 = 0.063 Cut-off value formula: 0.3 + NCx

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Cut-off vale: 0.3 + 0.0.063 = 0.36

Interpretation of test results :
If the absorbance of the test serum and/ or plasma is less than cut-off value, then the sample is considered as non-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 absorbencies 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.
Repeatedly Reactive specimens found using diagnostic of the present invention must be further confirmed with other tests e.g. western blot or indirect immunofluorescence assay.
Limitation of the test:
A non-reactive result with this test does not preclude the possibility of HIV infection. Assay performance :
It is important that time of reaction in each well is held constant for best -esults. Pipetting of samples should not extend beyond 15minutes. Addition of colour reagent solution initiates a reaction. Reading should be taken within 5 to 30 minutes after addition of stopping solution. Highly Iipaemic serum, hemolysed serum and contaminated serum with micro-organism should not be used.
Sensitivity :
The minimal detectable concentration of HIV-1 p24 antigens by this assay is estimated to be 5.0pg/ml.

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Any further modifications in and/or improvements in any aspect of the embodiments of the present invention will also fall under the scope of the present invention. In view of the foregoing description and examples, 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 the present invention. Various features of the invention hereinbefore described are set forth in the foregoing claims.


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WE CLAIM :

1. A diagnostic kit for the screening and detection of antibodies against HIV-1
and/or HIV-2 proteins and HIV-1 p24 antigens in the human serum and
plasma comprises:
a. a solid support in which recombinant antigens of HIV-1 and/or HIV-2
and anti-HIV-1 and/or HIV-2 antibodies are coated or adsorbed onto
plurality of microwells;
b. conjugates selected from the group of biotinylated anti-p24 antibodies,
streptavidin-enzyme conjugate and envelope antigen-enzyme
conjugate; and
c. immunological reagents selected form the group of HIV-1 antibody
control, HIV-2 antibody control, HIV-1 p24 antigen contrc1, HIV
negative control, sample diluent, colour reagent, stopping solution and
washing solution.
2. The diagnostic kit as claimed in claim 1, wherein the solid support comprises
microtib'e plate, wherein recombinant gp41 and recombinant gp36 antigens of HIV-1 and/or HIV-2 and monoclonal [human or mouse origin] anti-p24 antibodies for HIV-1 are coated or adsorbed onto plurality of microwells.
3. The diagnostic kit as claimed in claim 1, wherein the microwells of microtitre
plate are coated or adsorbed with recombinant gp41 of HIV-1 and recombinant gp36 antigens of HIV-2 as wells as anti-p24 antigen monoclonal antibodies for HIV-1.
4. The diagnostic kit as claimed in claim 1, wherein the microtitre plate used for
the solid phase immunoassay comprises minimum 96 and maximum 980
immunochemically coated or adsorbed microwells containing recombinant

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gp41 and gp36 antigens and anti-p24 [human or mouse origin] monoclonal antibodies.
5. The diagnostic kit as claimed in claim 1, wherein the biotinylated anti-p24 antibodies comprises anti-rabbit p24-bio tiny late d antibodies in conjugate diluent along with thimerosal and gentamycin as preservatives.
6. The diagnostic kit as claimed in claim 1, wherein the streptavidin-enzyme conjugate comprises streptavidin-HRPO in conjugate diluent along with thimerosal and gentamycin.
7. The diagnostic kit as claimed in claim 1, wherein the envelope antigen-enzyme conjugate comprises [gp41+gp36]-HRPO in conjugate diluent along with thimerosal and gentamycin.
8. The diagnostic kit as claimed in claim 1, wherein HIV-1 and HIV-2 antibodies control comprises inactivated anti-HIV-1 and HIV-2 containing human seriim preserved using thimerosal and gentamycin.
9. Tlie diagnostic kit as claimed in claim 1, wherein HIV-1 p24 antigens control comprises inactivated HIV-1 p24 antigen containing human serum preserved using thimerosal and gentamycin.
10. The diagnostic kit as claimed in claim 1, wherein HIV negative control comprises an inactivated normal human serum preserved using thimerosal and gentamycin.
11. The diagnostic kit as claimed in claim 1, wherein the colour reagent comprises 3,3/ 5,5'-tetra methyl benzidine dimethyl sulfoxide and H2O2 preserved using thimerosal and gentamycin.
12. The diagnostic kit as claimed in claim 1, wherein the sample diluent comprises
a mixture of phosphate buffer, BSA, bovine immunoglobulin, Tween-20, TrilonXl00 MgCl2 preserved using thimerosal and gentamycin.

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13. The diagnostic kit as claimed in claim 1, wherein the stopping i jlution comprises a solution of concentrated phosphoric acid and deionized water.
14. The diagnostic kit as claimed in claim 1, wherein the washing solution comprises a solution of TRIS buffer, NaCl and Tween-20 in deionized water.
15. A method for the preparation of a diagnostic kit the screening and detection of antibodies against HIV-l and/or HIV-2 proteins and HIV-l p24 antigens in the human serum and plasma comprises:
a. preparing microtitre plate as a solid support, wherein recombinant
gp41 antigens of HIV-l and recombinant gp36 antigens of HIV-2 and
anti-p24 [human or mouse origin] monoclonal antibodies of HIV-l are
coated or adsorbed onto plurality of microwells;
b. preparing conjugate solutions of anti-p24 biotinylated antibodies,
streptavidin-HRPO, [gp41+gp36]-HRPO;
c. preparing solutions of HIV-l antibody control, HIV-2 antibody control,
HIV-l p24 antigen control and HIV negative control; and
d. preparing solutions of immunological reagents like sample diluent,
colour reagent, stopping solution and washing solution.


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16. An in vitro diagnostic metliod for the screening and detection of antibodies against HIV-1 and/or HIV-2 proteins and HIV-1 p24 antigens in the human serum and plasma comprises:
a. contacting human serum and/or plasma samples with recombinant
gp41 of HIV-1 and gp36 of HIV-2 and anti-p24 monoclonaI[hu nan or
mouse origin] antibodies, which are coated or adsorbed onto the
microwells of microtitre plate, thereby forming stable complexes with
HIV envelope antigens and/or anti-p24 antibodies;
b. contacting said antibody-antigen complexes with anti-p24-biotinylated
antibodies and streptavidin-HRPO conjugate and/or [gp41+gp36]-
HRPO conjugate, thereby identifying said antigen-antibody complexes
formed;
c. reacting said complexes with colour reagent containing substrate of
HRPO, thereby developing the blue colour, which in proportion to
amount of anti-HIV-1 or HIV-2 antibodies and/or p24 HIV-1 antigens
present in the samples.

For TRANSASIA BIO-MEDICALS LTD.
^ RAVINDRA YEVLE VICE PRESIDENT - LEGAL, IP AND COMPANY SECRETAR/