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
An immunodiagnosis kit for detecting anti-gp41 and/or anti
gp36 antibodies to HIV proteins;

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 : -

An immunodiagnosis kit for detecting anti-gp41 and/or anti-gp36 antibodies to HIV proteins
FIELD OF THE INVENTION
Present invention relates to an immunodiagnosis kit for detecting antibodies to antigens of human immunodeficiency virus (HIV). More particularly, it relates to an immunodiagnosis kit for detecting anti-glycoprotein antigen antibodies of HIV-1 and HIV-2 in human serum and/or plasma that comprises recombinant glycoprotein antigens.
BACKGROUND AND PRIOR ART OF THE INVENTION
Significant population in the world is infected with HIV. Human serum and/or plasma of patients suffering from HIV infection show presence of different anti-HIV antibodies. Among the various antigens, antibodies to glycoprotein 41 (gp41) of HIV-1 and glycoprotein 36 (gp36) of HIV-2 antigens have been found specific and sensitive. Anti-gp41 and Anti-gp36 antibodies are absent in non-infected healthy individuals. Patients suffering from HIV infection show presence of IgG 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 results in death of the infected person. Even today there is no cure for 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.
AIDS has already reached epidemic proportion in the United States, Europe, Africa and Asia. This is a disorder of the immune system associated with opportunistic infections. 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
2

almost in all corners of the world as compared to the effects of HIV-2, which 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 potential blood donors who are being infected with HIV-1 and/or 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 anti-gp36 antibodies against gp41 and gp36 antigens of HIV-1 and HIV-2, respectively, can do it possible.
Virus-based, enzyme-linked immuno-sorbent assays (ELISA) require special equipment and also expertise and are very expensive to perform. In addition, they produce a significant number of false positive results. Immuno-fluorescence (IF) tests are less expensive and easier to perform than the above said method that requires the use of a fluorescence microscope. Subsequent to viral infection, serological analysis ensured the presence of anti-gp41 and /or anti-gp36 antibodies to HIV-1 and/or HIV-2 viral proteins. Therefore, an immunodiagnosis kit based on solid phase immunoassay technique for detecting anti-gp41 and/or anti-gp36 antibodies in human serum and/or plasma without special equipment(s) and expertise is highly desirable.
Based on the nucleotide analysis of the HIV genome, beginning at the 5 min end, the HIV genomic RNA encodes: (i) a gag gene extending between nucleotides 310 to 1,869 that encodes for the internal structural core or nucleocapsid proteins including p24, which is the most antigenic core protein; (ii) a pol gene extending between nucleotides 1,629 to 4,673 that encodes for the enzyme, reverse transcriptase; and (iii) an env gene extending between nucleotides 5,781 to 8,369 that encodes for the envelope glycoproteins gp41 and gp36, which are the most antigenic envelope proteins [Ratner et al, Nature, 313, 277-284 (1985)].
HIV-1 and/or HIV-2 isolated from cultured T-cells of patients with AIDS and determined to be the probable etiological agent, a search was launched for an assay

method and test kit required for the same that would be useful as a diagnostic indicator of this fatal disease and also useful for effective screening blood products. Immunoassays were the likely choice because of their sensitivity.
U.S. Patent No. 4,520,113 (Gallo et al) describes three assays for anti-HIV. They comprise a strip radioimmunoassay based on the Western Blot technique, an enzyme-linked immunosorbent assay (ELISA), and an indirect immunofluorescence assay. The viral protein reagent used in these immunoassays consists of inactivated whole virus isolated from cell cultures of the immortalized neoplastic human T-cell line, HT.
Current immunoassays designed to detect the presence of antibodies to the HIV-1 and/or HIV-2 viruses in human serum and/or plasma use an ELISA method employing inactivated whole virus cultured in a cell line capable of virus replication as the antigen reagent. This method is quite sensitive, and there is a high likelihood that a truly infected person will be detected. However, uninfected persons may occasionally give positive results.
The detection of false positives in screening of blood products is one of the most pressing problems with the commonly used assay kits and methods. Presently, specimen samples are screened by an ELISA method and if the result is positive, two more tests are run on the same sample. If either is positive, the specimen is said to be repeatedly reactive. The Western Blot is the method commonly used to determine whether repeatedly reactive specimens contain antibodies to HIV-1 and/or HIV-2. However, low value repeatedly reactive specimens often do not give a positive result on the Western Blot. [Dassey et al., JAMA, 255, 743-745 (1986)]. Nevertheless, the blood product must be disposed of because doubt exists regarding its infectivity. [Chalmers et al, JAMA, 256,1778-1783 (1986)]. These false positives are often due to non-specific binding of immunoglobulins to cellular protein in the viral isolates.
Another area of concern with the presently available assays is the lack of a convenient confirmatory assay to help resolve false positives. The currently used procedure, the Western Blot, generally described by Towbin et al., Proc. Nat'l Acad.

Sci. (USA), 76, 4350 (1979), requires sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS PAGE) to separate HIV-1 and HIV-2 proteins based on their molecular weight. The separated HIV-1 and HIV-2 proteins are transferred electrophoretically from the gel to a nitrocellulose sheet. The nitrocellulose sheet is cut into strips and reacted with the test sample and then with an anti-human IgG labeled antibody. A positive result is the appearance of label at the area on the strip to which the viral proteins have migrated. Among the disadvantages of the procedure for detecting anti-HIV are the requirements for subjective interpretation, the complexity and laborious work associated with the techniques, and the inherent difficulty in controlling variations in the composition of HIV-1 and HIV-2 proteins.
The report of a positive anti-HIV assay can be devastating to a patient. Further, a false positive in blood product specimens is undesirable because the donated blood must be destroyed. Therefore, the elimination of false positives is highly appreciated.
Safety of the person performing the assay is another concern associated with the present assay method because the procedures entail culturing live virus in vitro with subsequent isolation and deactivation to provide the whole virus reagent. Both the culturing and isolation processes are inherently dangerous since HIV infection is potentially fatal.
Although the most specific test for HIV infection remains virus isolation, this is an impractical method for large-scale use because of the complexity and difficulty of isolating HIV in culture. Some form of the ELISA method with enhanced sensitivity and specificity remains the most practical and feasible procedure for large-scale screening of the blood samples.
Utilizing a mixture of recombinant proteins for the detection of HIV-1 and/or HIV-2 antibodies in solid phase immunoassay kits and methods could solve the problems highlighted in preceding paragraphs described herein before. These recombinant proteins are non-infectious; therefore, their production and isolation would be safer than the culturing of whole virus. Also, the use of pure viral proteins obtained by recombinant methods eliminate some of the false positives due to non-specific

reactions with contaminating proteins. Besides, standardization of reagents improves the specificity and predictive value of the assay.
Expression of HIV gag gene proteins, including p24, in E. coli have indicated that the HIV gag proteins produced by rDNA technology could have potential diagnostic value. [Wood et al., Cold Spring Harbor Symposium on RNA Tumor Viruses, Cold Spring Harbor, New York, May 22-26, 1985; Dowbekno et al., Proc. Natl. Acad. Sci. U.S.A., 82, 7748-7752, (1985); Ghrayeb et al., DNA, 5, 93-99 (1986); Steimer et al, Virology, 150, 283-290 (1986)]. Similarly, the expression of gp41 or parts of gp41 has also demonstrated the utility of rDNA derived HIV envelope sequences in diagnostic assays. [Wood et al., Cold Spring Harbor Symposium on RNA Tumor Viruses, Cold Spring Harbor, New York, May 22-26, 1985; Chang et al, Biotechnology, 3, 905-909 (1985); Crowl et al. Cell, 41, 979-986 (1985); Cabradilla et al, Biotechnology, 4, 128-133 (1986)]. The viral proteins expressed in E. coli have potential utility in diagnostic assays and development of solid phase immunoassays kits that have the specificity and sensitivity equal to or greater than the native viral peptides derived from the cultured cell.
OBJECT OF THE INVENTION
In view of above, one embodiment of present invention is to provide an immunodiagnosis kit based on solid phase immunoassay technique for detecting antibodies to glycoproteins of HIV-1 and/or HIV-2 in human serum and/or plasma.
Another embodiment of present invention is an immunodiagnosis kit for rapidly detecting anti-gp41 and/or anti-gp36 antibodies to HIV-1 and/or HIV-2 in human serum and/or plasma.
Yet another embodiment of present invention is an immunodiagnosis kit, which is simple, cost effective and reliable for screening of antibodies to gp41 and/or gp36
antigens of HIV-1 and/or HIV-2.

Still another embodiment of present invention is an immunodiagnosis kit that comprises recombinant antigens gp41 and/or gp36 for detecting antibodies against gp41 and gp36.
Another embodiment of present invention is an immunodiagnosis kit that comprises recombinant antigens gp41 and/or gp36 for detecting anti-gp41 and/or anti-gp36 antibodies.
In different embodiment of present invention there is also provided a process for preparing an immunodiagnosis kit for detecting said antibodies in human serum and/or plasma.
In still different embodiment of present invention there is also provided an in vitro method for detecting said antibodies in human serum and/or plasma using the kit of the present invention.
SUMMARY OF THE INVENTION
Pursuant to object of the present invention there is provided an immunodiagnosis kit for detecting antibodies to glycoproteins antigens of HIV-1 and/or HIV-2 in human serum and/or plasma, which comprises immunochemically coated onto a solid support a mixture of recombinant glycoprotein antigens The immunodiagnosis kit further comprises immunochemically acceptable reagents, such as conjugate, anti-HIV positive control, HIV negative control, colour reagent, sample diluent, stopping solution and washing solution. There is also provided a process for preparing the immunodiagnosis kit for detecting antibodies to glycoproteins antigens of HIV-1 and/or HIV-2 in human serum and/or plasma, which comprises coating on to a solid support a mixture of recombinant glycoprotein antigens of HIV-1 and/or HIV-2 and providing the solutions of immunochemically acceptable reagents. Further, there is also provided an in vitro method for detecting antibodies directed to recombinant glycoprotein antigens of anti-HIV-1 and/or HIV-2 in human serum and/ or plasma, which comprises bringing human serum and/or plasma in contact with solid support that comprises a mixture of recombinant glycoprotein

antigens of HIV-1 and/or HIV-2 and detecting antigen-antibody complex using immunochemically acceptable reagents.
STATEMENT OF THE INVENTION
In one embodiment the invention relates to an immunodiagnosis kit for detecting antibodies, anti-gp41 and/or anti-gp36, of glycoproteins antigens, gp41 and/or gp36, in human serum and/or plasma comprises:
(i) a solid support, which has plurality of microwells immunochemically coated with a mixture of recombinant glycoprotein antigens, wherein said antigens are selected form recombinant gp41 and/or recombinant gp36 antigens, which specifically bind with anti-gp41 and/or anti-gp36 antibodies of HIV-1 and/or HIV-2 present in positive samples;
(ii) an enzyme conjugate, which comprises recombinant [gp41+gp36]-HRPO in conjugate diluent; and
(iii) immunochemical reagents, which are required for detecting said antibodies.
In another embodiment the relates to a process for preparing an immunodiagnosis kit for detecting antibodies, anti-gp41 and/ or anti-gp36, to glycoproteins antigens, gp41 and/or gp36, in human serum and/or plasma comprises:
(i) preparing a coated solid support by immunochemically coating onto a solid support with plurality of microwells a mixture of recombinant glycoproteins antigens, which comprises recombinant gp41 and/or gp36 antigens that bind with anti-gp41 and/ or anti-gp36 antibodies of HIV-1 and/or HIV-2;
(ii) preparing an enzyme conjugate by covalently linking recombinant [gp41+gp36] antigens with HRPO in conjugate diluent;

(iii) preparing solutions of: an anti-HIV positive control, comprising an inactivated anti-HIV-1 human serum; a HIV negative control, comprising an inactivated normal human serum; a colour reagent comprising 3,3', 5,5'-tetramethyl benzidine, dimethyl sulfoxide and hydrogen peroxide; a sample diluent comprising TRIS buffer, animal serum Tween-20 and urea; a stopping solution comprising concentrated phosphoric and deionized water; and a washing solution comprising TRIS buffer, sodium chloride, Tween-20 in deionized water.
In still another embodiment the invention relates to an in intro method for detecting antibodies, anti-gp41 and/or anti-gp36, to glycoproteins antigens, gp41 and/or gp36, in human serum and/or plasma comprises:
(i) bringing human serum and/or plasma in contact with solid support with plurality of microwells immunochemically coated with a mixture of recombinant glycoprotein antigens, gp41 and/or gp36, that specifically bind to antibodies, anti-gp41 and/or anti-gp36, of HIV-1 and/or HIV-2, thereby forming stable antigen- antibody complex; and
(ii) detecting said antigen-antibody complex using immunochemical reagents.
DESCRIPTION OF THE INVENTION
The immunodiagnosis kit for detecting anti-gp41 and/or anti-gp36 antibodies, to glycoproteins antigens gp41 and/or gp36 in human serum and/or plasma, comprises a solid support, which has multiplicity of microwells immunochemically coated with a mixture of recombinant glycoprotein antigens, comprising recombinant gp41 and/or gp36 antigens that specifically bind to anti-gp41 and/or anti-gp36 antibodies of HIV-1 and/or HIV-2; an enzyme conjugate, which comprises recombinant [gp41+gp36]-HRPO in conjugate diluent; an anti-HIV positive control, which comprises an inactivated anti-HIV-1 human serum; a HIV negative control, which comprises an inactivated normal human serum; a colour reagent, which comprises 3,3', 5,5'-tetramethyl benzidine, dimethyl sulfoxide and hydrogen peroxide; a sample diluent, which comprises TRIS buffer, animal serum Tween-20

and urea; a stopping solution, which comprise concentrated phosphoric and deionized water; and a washing solution, which comprises TRIS buffer, sodium chloride, Tween-20 in deionized water.
The process for preparing an immunodiagnosis kit for detecting anti-gp41 and/or anti-gp36 antibodies, to glycoproteins antigens gp41 and/ or gp36 in human serum and/or plasma comprises coating immunochemically onto a solid support, which has multiplicity of microwells a mixture of recombinant glycoproteins antigens, comprising recombinant gp41 and/or gp36 antigens that specifically bind to anti-gp41 and/or anti-gp36 antibodies of HIV-1 and/or HIV-2; and preparing solutions of an enzyme conjugate comprising recombinant [gp41+gp36]-HRPO in conjugate diluent; an anti-HIV positive control comprising an inactivated anti-HIV-1 human serum; a HIV negative control comprising an inactivated normal human serum; a colour reagent comprising 3,3', 5,5'-tetramethyl benzidine, dimethyl sulfoxide and hydrogen peroxide; a sample diluent comprising TRIS buffer, animal serum Tween-20 and urea; a stopping solution comprising concentrated phosphoric and deionized water; and a washing solution comprising TRIS buffer, sodium chloride, Tween-20 in deionized water.
The in vitro method for detecting anti-gp41 and/ or anti-gp36 antibodies, to glycoproteins antigens gp41 and/or gp36 in human serum and/or plasma comprises bringing human serum and/or plasma in contact with solid support, which has multiplicity of microwells coated immunochemically with a mixture of recombinant glycoprotein antigens comprising recombinant gp41 and/or gp36 antigens that specifically bind to anti-gp41 and/or anti-gp36 antibodies of HIV-1 and/ or HIV-2, thereby forming stable antigen- antibody complex of anti-gp41 and/or anti-gp36 with recombinant gp41 and/or gp36 antigens; and detecting said antigen-antibody complex using immunochemically acceptable reagents.
DETAILED DESCRIPTION OF THE INVENTION
The instant invention can be understood more readily by reference to following detailed description of specific embodiments. Although, the present invention has

been described with reference to particular and specific details of certain embodiments, it is not intended that such details should be regarded as limitations to the scope of the invention. Unless otherwise depicted, 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 one embodiment of the present invention, the immunodiagnosis kit for detecting anti-gp41 and/or gp36 antibodies to HIV-1 and/or HIV-2 glycoprotein antigens in human serum and/ or plasma comprises coated onto a microplate plate, which has multiplicity of microwells, a mixture of recombinant glycoproteins comprising recombinant gp41 and/or gp36.
In another embodiment, the immunodiagnosis kit comprises the microplate with plurality of microwells, wherein the microwells of the plate are coated with a mixture of recombinant glycoprotein antigen, comprising recombinant gp41 and/or gp36 that bind with anti-gp41 and/or anti-gp36 antibodies directed to glycoprotein antigens gp41 and/or gp36 of HIV-1 and/or HIV-2 of seropositive samples.
In further embodiment of the present invention, the immunodiagnosis kit comprises an enzyme conjugate comprising recombinant [gp41+gp36]-HRPO in conjugate diluent with gentamycin as a preservative; an anti-HIV positive control comprising an inactivated anti-HIV-1 human serum with gentamycin and thimerosal as a preservative; a HIV negative control comprising an inactivated normal human serum with gentamycin and thimerosal; a colour reagent comprising 3,3', 5,5'-tetramethyl benzidine, dimethyl sulfoxide and hydrogen peroxide with gentamycin and thimerosal; a sample diluent comprising TRIS buffer, animal serum Tween-20 and urea with gentamycin and thimerosal; a stopping solution comprising concentrated phosphoric and deionized water; and a washing solution comprising TRIS buffer, sodium chloride, Tween-20 in deionized water.
In one preferred embodiment, the enzyme linked to the recombinant conjugated antigen is horse reddish peroxidase.

In specific embodiment of the present invention, the multi-welled antigen coated microplate is first coated with the recombinant glycoprotein, gp41, and thereby enhancing reaction with anti-gp41 antibody of HIV-1 seropositive samples. Similarly, a multi-welled antigen coated microplate is coated with the recombinant glycoprotein, gp36, and thereby enhancing reaction with anti-gp36 antibody of HIV-2 seropositive samples.
In an important embodiment, the immunodiagnosis kit of the present invention is stored at temperature between 2°C and 8 °C.
In different embodiment of the present invention, the process for preparing immunodiagnosis kit for detecting antibodies to glycoprotein antigens of HIV-1 and/or HIV-2 comprises coating onto the microwells of microplate a mixture of recombinant glycoprotein antigens comprising recombinant gp41 and/or gp36 antigens of HIV-1 and/or HIV-2.
In another different embodiment, the process for preparing the kit comprises preparing an enzyme conjugate by covalently linking an enzyme with combination of recombinant glycoprotein antigens gp41 and gp36 of HIV-1 and/or HIV-2; and preparing immunochemically acceptable reagents by preparing the solutions of said reagents, which are required for detecting antigen-antibody complex.
In preferred embodiment of the said process, the solution of a mixture of the said recombinant antigens of HIV-1 and/or HIV-2 are dissolved in bicarbonate buffer, which is used for coating on to the microwells of the plate.
In further preferred embodiment, the coated mixture of recombinant antigens is blocked using a blocking solution, which comprises phosphate buffer, BSA and Trans-001.
In still further embodiment, the blocked recombinant antigens are stabilised using a stabilising solution, which comprises PBS, Trans-002 and Tran-003 (Bovine IgG).

In another embodiment, the method comprises coating onto the same and/or different microwells of solid support either separately or a mixture of recombinant glycoproteins, gp41 and gp36.
In preferred embodiment of the invention, the process comprises separately preparing two coating solutions, namely solution-1 that comprises recombinant glycoprotein-gp41 (1 mg/ml) and solution-2 that comprises recombinant glycoprotein-gp36 (1 mg/ml) 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 another preferred embodiment, the process for preparing solution of enzyme conjugate comprises mixing of recombinant [gp41+gp36]-HRPO in conjugate diluent with gentamycin in a suitable proportion.
In yet another embodiment, the process for preparing solution of anti-HIV-positive control comprises mixing of inactivated anti-HIV-1 human serum in diluent with stabilizer and thimerosal, and gentamycin in a suitable proportion.
In still another embodiment, the process for preparing solution of HIV-negative control comprises mixing of inactivated normal human serum in diluent with stabilizer and thimerosal, and gentamycin in a suitable proportion.
In different embodiment, the process for preparing colour reagent comprises mixing of 3,3', 5,5' tetramethyl benzidine in dimethyl sulfoxide with H2O2, and thimerosal, and gentamycin in a suitable proportion.
In still different embodiment, the process for preparing sample diluent comprises mixing of TRIS buffer in animal serum, Tween-20 and urea with thimerosal and gentamycin in a suitable proportion.
In yet different embodiment, the process for preparing washing solution concentrate comprises mixing of TRIS buffer, sodium chloride, Tween-20 in deionized water in a suitable proportion.

In another different embodiment of the present invention, the in vitro method for detecting anti-gp41 and/or gp36 antibodies in human serum and/or plasma comprises bringing human serum and/or plasma in contact with mixture of recombinant antigens comprising recombinant gp41 and/or gp36 antigens coated onto the plurality of microwells, thereby forming stable complex of anti-gp41 and/or gp36 antibodies with recombinant gp41 and/ or gp36 antigens of HIV-1 and/or HIV-2, if anti-gp41 and/or anti-gp36 antibodies are present in human serum and/or plasma; adding to said microwells an enzyme conjugate, which contains recombinant [gp41+gp36]-HRPO; after washing, further adding to said microwells a colour reagent, which contains substrate of HRPO, thereby developing blue colour in the wells of positive controls and test specimen; further adding to said wells a stopping solution, thereby changing the blue colour to yellow and spectrophotometrically reading the colour intensity for the detection of presence of said antibodies in the sample.
In preferred embodiment of the process, when the human blood serum and/or plasma is added to the microwells containing coated recombinant glycoprotein antigens gp41 and/or gp36 of HIV-1 and/or HIV-2, the coated antigens will form a stable complex with anti-gp41 and/or gp36 antibodies, if present in human blood serum and/or plasma under testing. Followed by the washing step, the recombinant [gp41+gp36]-HRPO is added to the wells. In a second the washing step, free recombinant [gp41+gp36]-HRPO will be 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 will be developed in wells containing positive controls and test specimen containing anti-gp41 and/or anti-gp36 antibodies. Upon addition of stopping solution, the blue colour changes to yellow. The intensity of yellow colour is directly proportional to the amount of bound anti-HIV antibodies to the well.
In yet another preferred embodiment of the present invention, the immunological components are bound to the microwells containing plate by covalent bonds or by

adsorption. An advantage of using said microwells containing solid plate is that no centrifugation required for separation of solid and liquid phases.
In still another preferred embodiment of the present invention, the enzyme conjugate consists of immunological components that covalently linked to the enzyme molecule, which is achieved either by direct condensation or by using external bridging molecules. Thus, the enzyme coupling products is produced by employing the covalent bond, which is effected by using the reagents, for example, carbodiimides, diisocyanates, glutaric aldehyde and bis-diazobenzidine. The choice of the enzyme that is to form a part of the coupling product is determined by properties, such as, specific binding activity, high conversion rate and simplicity of detection. The determination of the enzyme activity in which coloured reaction components are involved is simple, which can be determined spectrophotometrically or fluorimetrically. These determinations are also suitable for automation that is an additional advantage. The suitable enzyme used in the kit and methods hereinbefore described is horseradish peroxidase.
EXAMPLES
The following examples serve to illustrate the invention by way of best method of performing and not to be regarded as limitations.
EXAMPLE: 1-DETECTION OF ANTI-GLYCOPROTEIN ANTIBODIES
Adding positive control or anti-gp41 and/or gp36 antibodies containing human serum and/or plasma to microwells of microplate, incubating the plate containing said mixture for 0.5 to 25 hours at temperature between 4°C and 5°C, thereby forming stable antigen-antibody complex with recombinant gp41 and/or gp36 of HIV-1 and/ or HIV-2 that are bound to the microwells and with gp41 and gp36 antigens that are attached to HRPO of enzyme conjugate; washing the plate using washing solution, thereby removing unbound fraction of enzyme conjugate; adding colour reagent, which contains substrate of HRPO, to the microwells containing said

antigen-antibody complex, thereby developing a blue colour in the wells containing positive control and anti-gp41 and/or gp36 antibodies; adding stopping solution to the wells, thereby changing the blue colour to yellow and finally measuring an intensity of the yellow colour spectrophotometrically for detecting anti-gp41 and/or gp36 antibodies to gp41 and/or gp36 antigens of HIV-and/or HIV-2.
EXAMPLE: 2 - TEST PROCEDURE FOR DETECTION OF ANTIBODIES
(a) Bring all the reagents and test specimens at room temperature before
use. (b) Add 100ml of sample diluent to each well. In each run, there will be one blank [150ml sample diluent + 50ml conjugate], three negative controls and one positive control. Add 50ml of control or test specimens to each well. Add 50ml of conjugate to each well. Cover the plate with black cover and incubate 45 minutes at room 20 - 37°C. (c) Wash the plate as per known microplate washing procedure, (d) Add 50ml of colour reagent to each well. Cover the plate with black cover and incubate for 15 minutes in dark at 20 - 37°C. (e) Add 100ml of stopping buffer to each well, (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 0.1.
NCx: Average value of negative controls.
Calculation of NCx:
For example:

NC

Absorbance

1 0.022
2 0.026
3 0.024
NCx: (0.022 + 0.026 + 0.024)/3 = 0.024
Cut-off value formula: 0.1 + NCx
Cut-off vale: 0.1 + 0.024 = 0.124
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 immunodiagnosis kit of the present invention, must be further confirmed with the tests, such as western blot or indirect immunofluorescent 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 infection.

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WE CLAIM :
1. An immunodiagnosis kit for detecting antibodies, anti-gp41 and/or anti-
gp36, of glycoproteins antigens, gp41 and/ or gp36, in human serum and/or
plasma comprises:
(i) a solid support, which has plurality of microwells immunochemically coated with a mixture of recombinant glycoprotein antigens, wherein said antigens are selected form recombinant gp41 and/or recombinant gp36 antigens, which specifically bind with anti-gp41 and/or anti-gp36 antibodies of HIV-1 and/or HIV-2 present in positive samples;
(ii) an enzyme conjugate, which comprises recombinant [gp41+gp36]-HRPO in conjugate diluent; and
(iii) immunochemical reagents, which are required for detecting said antibodies.
2. The immunodiagnosis kit according to claim 1, in which the mixture of recombinant glycoprotein antigens comprises at least one recombinant glycoprotein antigen selected form gp41 and gp36 of HIV-1 and HIV-2.
3. The immunodiagnosis kit according to claim 1, in which the mixture of recombinant glycoprotein antigens comprises recombinant glycoprotein antigens gp41 and/or gp36 of HIV-1 and/or HIV-2.
4. The immunodiagnosis kit according to claim 1, in which the solid support is a microplate with plurality of microwells coated with said recombinant glycoprotein antigens.
5. The immunodiagnosis kit according to claim 1, in which the enzyme conjugate comprises recombinant glycoprotein antigens gp41 and gp36 immunochemically linked with reddish horse peroxidase.

6. The immunodiagnosis kit according to any one of proceeding claims, in which the other immunochemical reagents are anti-HIV positive control, HIV negative control, colour reagent, sample diluent, stopping solution and washing solution.
7. The immunodiagnosis kit according to claims 1 and 6, in which the anti-HIV positive control comprises an inactivated anti-HIV-1 human serum and the HIV negative control comprises an inactivated normal human serum.
8. The immunodiagnosis kit according to claims 1 and 6, in which the colour reagent comprises a mixture of 3,3', 5,5'-tetramethyl benzidine, dimethyl sulfoxide and hydrogen peroxide.
9. The immunodiagnosis kit according to claims 1 and 6, in which the sample diluent comprises a mixture of TRIS buffer, animal serum Tween-20 and urea.
10. The immunodiagnosis kit according to claims 1 and 6, in which the stopping solution comprises concentrated phosphoric and deionized water and the washing solution comprises a mixture of TRIS buffer, sodium chloride, Tween-20 in deionized water.
11. A process for preparing an immunodiagnosis kit for detecting antibodies, anti-gp41 and/or anti-gp36, to glycoproteins antigens, gp41 and/or gp36, in human serum and/or plasma comprises:
(i) preparing a coated solid support by immunochemically coating onto a solid support with plurality of microwells a mixture of recombinant glycoproteins antigens, which comprises recombinant gp41 and/or gp36 antigens that bind with anti-gp41 and/or anti-gp36 antibodies of HIV-1 and/or HIV-2;
(ii) preparing an enzyme conjugate by covalently linking recombinant [gp41+gp36] antigens with HRPO in conjugate diluent;

(iii) preparing solutions of: an anti-HIV positive control, comprising an inactivated anti-HIV-1 human serum; a HIV negative control, comprising an inactivated normal human serum; a colour reagent comprising 3,3', 5,5'-tetramethyl benzidine, dimethyl sulfoxide and hydrogen peroxide; a sample diluent comprising TRIS buffer, animal serum Tween-20 and urea; a stopping solution comprising concentrated phosphoric and deionized water; and a washing solution comprising TRIS buffer, sodium chloride, Tween-20 in deionized water.
12. An in vitro method for detecting antibodies, anti-gp41 and/or anti-gp36, to glycoproteins antigens, gp41 and/ or gp36, in human serum and/or plasma comprises:
(i) bringing human serum and/or plasma in contact with solid support with plurality of microwells immunochemically coated with a mixture of recombinant glycoprotein antigens, gp41 and/or gp36, that specifically bind to antibodies, anti-gp41 and/or anti-gp36, of HIV-1 and/or HIV-2, thereby forming stable antigen- antibody complex; and
(ii) detecting said antigen-antibody complex using immunochemical reagents.
Dated this 24th day of March, 2007.

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