FORM 2
THE PATENT ACT 1970 (39 Of 1970)
&
The Patents Rules, 2003 COMPELETG/PROVISIONAL SPECIFICATION See Section 10, and rule 13)
1. TITLE OF INVENTION
A SOLID PHASE IMMUNOASSAY KIT FOR DETECTION OF ANTIBODIES TO HUMAN IMMUNO DEFICIENCY VIRUSES AND METHOD OF PREPARING THE SAME;
2. APPLICANT(S)

a) Name
b) Nationality
c) Address

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

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

A solid phase immunoassay kit for detection of antibodies to human immuno deficiency viruses and method of preparing the same
FIELD OF THE INVENTION
The present invention relates to solid phase immunoassay kit for detection of antibodies to human immunodeficiency virus (HIV) in serum and plasma. It particularly relates to a specific and sensitive diagnostic kit for detection of antibodies to the human immunodeficiency virus (HIV-1 and HIV-2) using safe recombinant proteins. It also relates to a method of producing said diagnostic kit and an in vitro method of detecting HIV -1 and/or HIV -2 using said kit.
BACKGROUND OF THE INVENTION
Significant number of population in the world is infected with HIV. Patients suffering from HIV infection show presence of different anti-HIV antibodies in serum and plasma. Among the various antigens, antibodies to glycoprotein 41 of HIV-I (gp41) and glycoprotein 36 of HIV -2 (gp36) antigens have been found specific and sensitive. Anti-HIV -1 and Anti HIV -2 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 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 HIY -1 is widespread and has been reported 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 antibodies against the glycoproteins antigens gp41 and gp36 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 antibodies to HIV -1 and/or HIV -2 viral proteins. Therefore, a kit based on solid phase immunoassay for the detection of antibodies to etiological agents, HIV-1 and/or HIV-2, in human serum and/or plasma and a simple and costeffective method of preparing the same without special equipment(s) and expertise is highly desirable.
PRIOR ART OF THE INVENTION
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 (SOS 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 ehmination 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 nonspecific reactions
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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., Froc. 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 parls 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
Accordingly, object invention is to provide a solid phase immunoassay kit/or detection of HIV -1 and/or HIV -2 in biological fluids under question.
Accordingly, further object of the present invention is to provide a solid phase immunoassay kit for rapid detection of antibodies to HIV -1 and/or HIV -2 in the biological samples under question.
Accordingly, yet further object of the present invention is to develop a reliable and cost-effective immunoassay diagnostic kit/br screening of HIV -1 and/or HIV-2 antibodies in human serum and/or plasma.
Accordingly, another specific object of the present invention is to provide a solid phase immunoassay kit for serological detection of antibodies against glycoprotein antigens gp41 and gp36 of HIV -1 and/or HIV -2.

Accordingly, still another specific object of the present invention is to provide a method For preparation of an solid phase immunoassay kit for screening and detection of antibodies to HIV -1 and/or HIV -2.
Accordingly, another object of the present invention is to provide an in vitro method for serological analysis of biological fluids under question.
Accordingly, another object of the present invention is to provide a serological analysis method for detection in human serum and/or plasma the antibodies to HIV-1 and/or HIV-2
SUMMARY OF THE INVENTION
Accordingly, present invention provides a solid phase immunoassay kit for detection of antibodies to HIV -1 and/or HIV -2 in biological samples in question. The biological sample in question is more particularly blood product, such as human serum and plasma of HIV-1 and/or HIV-2 infected persons. The solid phase immunoassay kit comprises spotted on a solid support a mixture of recombinant and/or synthetic proteins for screening and detection of antibodies to HIV -1 and/ or HIV -2 in human serum and/or plasma. The said kit also comprises other immunological or serological reagents, such as conjugate, positive control, negative control, signal reagent, sample diluent and washing solution.
Also, provided is a method for preparation of a solid phase immunoassay kit for detection and screening of antibodies to HIV -1 and/or HIV -2 in human serum and/or plasma. The method comprises coating on to a solid support a mixture of recombinant and/or synthetic proteins as antigens of HIV -1 and/or HIV -2. The said method also comprises preparing other immunological or serological reagents, such as a conjugate, a positive control, a negative control, a signal reagent, a sample diluent and a washing solution.
Further, also provided is an in vitro method for serological analysis of biological fluids under question using a solid phase immunoassay kit of the present invention for
screening and detection of anti-HIV -1 and/or HIV -2 antibodies. The said method
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comprises contacting the to a solid spotted support with a mixture of recombinant and/or synthetic proteins antigens of HIV -1 and/or HIV -2 biological fluids under question. The said biological fluids are particularly blood products, such as human serum and plasma and more are particularly human serum and/or plasma of HIV-1 and/ or HIV-2 infected persons.
DETAILED DESCRIPTION OF THE INVENTION
In a preferred embodiment of the invention, the solid phase immunoassay kit provided for detection of antibodies to HIV -1 and/or HIV -2 in human serum and/or plasma comprises spotted on said solid support a mixture of recombinant and/or synthetic glycoproteins for screening and detection of anti-HIV -1 and/or HIV-2 antibodies in human serum and/or plasma. The said recombinant and/or synthetic glycoproteins particularly comprise at least one immunogenic sequence of envelope proteins of HIV -1 and/or HIV -2. More particularly said immunogenic sequences of recombinant and/or synthetic envelope glycoproteins are gp41 and gp36 of HIV-1 and/or HIV-2.
In a more preferred embodiment of the invention, said solid phase immunoassay kit comprises: spotted on a solid support,, at least one recombinant and/or synthetic glycoprotein, comprising at least one immunogenic glycoprotein gp41 of HIV -1 envelope protein that reacts with antibodies of HIV -1 in seropositive samples; spotted on a solid support, at least one recombinant and/or synthetic glycoprotein, comprising at least one immunogenic glycoprotein gp36 of HIV-2 envelope protein that reacts with antibodies of HIV -2 in seropositive samples.
In a further embodiment of the present invention, the solid phase immunoassay kit also comprises other immunological or serological reagents like a conjugate, a positive control, a negative control, a signal reagent, a sample diluent and a washing solution (20X) along with a plastic boat, a microdilution plate well and adhesive strips.
In another embodiment of the present invention, said solid support of the solid phase immunoassay kit is a multi-welled antigen-coating polystyrene comb to which the mixture of recombinant and/or synthetic glycoproteins such as herein before described, each containing immunogenic sequence of the envelope protein gp41 and

gp36 of HIV-1 and/or HIV-2 is fixed.
In a preferred embodiment, the solid phase immunoassay kit comprises the ampules containing Goat-Anti-human IgG-HRPOa as a conjugate for serological analysis of human serum and/ or plasma.
In another preferred embodiment, the solid phase immunoassay kit comprises the ampules containing inactivated Anti-HIV containing human serum as an anti-HIV positive control along with thimerosal and gentamycin.
In yet another preferred embodiment, the solid phase immunoassay kit comprises the ampules containing inactivated normal human serum as a HIV negative control along with tlumerosal and gentamycin.
In other preferred embodiment, the solid phase immunoassay kit comprises the ampules containing TRIS buffer, animal serum and Tween-20 along with thimerosal and gentamycin as a sample diluent.
In separate preferred embodiment, the solid phase immunoassay kit comprises the ampules containing 3,3', 5,5] -tetra methyl benzidine, dimethyl sulfoxide and H2O2 along with thimerosal and gentamycin as colour reagent.
In distinct preferred embodiment, the solid phase immunoassay kit comprises the ampules containing TRIS buffer, sodium chloride, Tween-20 and deionized water as a washing solution concentrate (20 X).
In a more specific embodiment, a multi-welled antigen-coating polystyrene comb is first-spotted with a recombinant and/or synthetic glycoprotein, gp41, comprising at least one immunogenic region of HIV -1 envelope protein, thereby enhancing reaction with anti-HIV -1 antibodies of seropositive samples. Similarly, a multi-welled antigen-coating polystyrene comb is spotted with a recombinant and/or synthetic glycoprotein, gp36, comprising at least one immunogenic region of HIV-2 envelope protein, thereby enhancing reaction with anti-HIV -2 antibody of seropositive samples.

In another preferred embodiment, the recombinant and/or synthetic glycoproteins gp41 and gp36 may be present on the same or different multi-welled antigen-coating polystyrene comb. For example, the spotted solid support (containing gp41 and gp36) is contacted with the biological sample, and then unbound sample is removed. Next, labelled anti-immunoglobulin specific for the immunoglobulin in the sample is added to the solid support. After removal of unbound labelled anti immunoglobulin reagent, the label is detected to determine presence of HIV antibodies in the sample.
As per another object of the invention, the method for manufacturing solid phase immunoassay kit for screening and detection of HIV-1 and/or HIV-2 antibodies in human serum and/or plasma is provided. The method comprises coating on to a solid support a mixture of recombinant and/or synthetic glycoproteins as antigens against anhbodies of HIV -1 and/or HIV -2.
In another embodiment of the present invention, the method also comprises preparing the solutions of other immunological or serological reagents, such as a conjugate, a positive control, a negative control, a signal reagent, a sample diluent and a washing solution.
In a preferred embodiment, the method comprises coating on the same different solid support the either separately or a mixture of recombinant and/or, synthetic glycoproteins, gp41 and gp36, each comprising at least one immunogenic region of HIV -1 and/or HIV -2 envelope proteins.
In the most preferred embodiment, the method comprises coating on a multiwelled antigen-coating polystyrene comb the mixture of the recombinant and/or synthetic glycoproteins peptide-gp41 and gp36, each having at least one immunogenic region of HIV -1 and/or HIV -2 envelope proteins.
In another the most preferred embodiment, the process of preparing HIV -1 and HIV -2
synthetic antigen spotted comb comprises coating the a multi-welled antigencoating
polystyrene comb with coating solutions contaiiling aforesaid proteins, thereby
obtaining the comb spotted with the recombinant and/or synthetic glycoproteins, gp41
and gp36; blocking the wells of the said comb with blocking solution and finally
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stabilising the spotted peptides on said comb with stabilizing solution.
In another embodiment of the invention, the process comprises separately preparing two coating solutions, namely solution-1, comprising recombinant and/or synthetic glycoprotein-gp41 (1 mg/ml) and solution-2, comprising recombinant and/or synthetic 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 embodiment, a process of preparing the solution of conjugate comprises mixing of goat anti-human-IgG-HRPO in diluent with thimerosal and gentamycin in an appropriate or a suitable proportion.
In yet another embodiment, a process of preparing the solution of anti-HIVpositive control comprises mixing of anti-HIV -positive human serum in diluent with stabilizer, thimerosal and gentamycin in an appropriate or suitable proportion.
In yet further embodiment, the process of preparing a solution of an anti - HIVnegative control comprises mixing of normal human serum in diluent with stabilizer, thimerosal and gentamycin in an appropriate or a suitable proportion.
In yet further embodiment, the process of preparing a colour reagent comprises mixing of 3,3', 5,5' tetra methyl benzidine in dimethyl sulfoxide with H2O2, thimerosal and gentamycin in an appropriate or a suitable proportion.
In another embodiment, the process of preparing a sample diluent comprises mixing TRIS buffer in animal serum and Tween-20 with thimerosal and gentamycin in an appropriate or a suitable proportion.
In yet another embodiment, the process of preparing a washing solution concentrate comprises mixing of TRIS buffer, sodium chloride, Tween-20 in deionized water in an appropriate or a suitable proportion.
According to another object of the invention, an in vitro serological method for screening and detection of anti HIV -1 and/or HIV -2 antibodies in human serum

and/or plasma is provided. The process comprises adding human blood serum and/or plasma to the wells of polystyrene comb spotted with the recombinant and/or synthetic glycoprotein antigens of HIV -1 and/or HIV -2 with sample diluent, thereby forming stable complex of anti-HIV antibodies with bound HIV-1 and/or HIV-2 antigens, if anti-HIV -1 and/ or HIV -2 antibodies present in human serum and/or plasma; after washing step, adding to the wells the conjugate containing goat anti-human-IgG-HRPO; after second washing step, adding to the wells the colour reagent containing the substrate of HRPO, thereby developing blue colour in the wells of positive controls and test specimen; and adding to the wells the stopping solution, thereby changing the blue colour to yellow.
In preferred embodiment of the process for serological analysis, when the human blood serum and/or plasma is added to the wells of polystyrene comb containing spotted recombinant and/or synthetic glycoprotein antigens gp41 and gp36 of HIV -1 and/or HIV -2 with diluent, the bound gp41 and gp36 of HIV -1 and/or HIV2 antigens will form a stable complex, if anti-HTV-1 and/or HIV-2 antibodies are present in human blood serum and/or plasma under testing. Followed by the washing step, the goat anti-human-IgG-HRPO is added to the wells. In a second the washing step, free goat anti-human-IgG-HRPO will 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-HIV-1 and/or HIV-2 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.
Any further modifications in and/or improvements in any aspect of the embodiments of this invention will also fall under the scope of this invention. In view of the foregoing description and example, it will become apparent to those of ordinary skill in the art that equivalent modification thereof may be made without departing from the spirit and scope of this invention. Various features of the invention hereinbefore described are set forth in the foregoing claims.
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WE CLAIM:
1. A solid phase immunoassay kit for detection of antibodies to HIV -1 and/or HIV-2 in human blood serum and/or plasma consisting of immunological or serological reagents, such as conjugate, positive control, negative control, signal reagent, sample diluent and washing solution required to perform the assay characterized in that spotted on either on same or different multi-welled antigen-coating polystyrene comb either separately and/or a mixture of recombinant and/or synthetic glycoprotein antigens of HIV -1 and/or HIV -2, each comprising at least one immunogenic region of HIV-1 and/or HIV-2 envelope protein, thereby enhancing reaction with anti-HIV-1 and /or HIV -2 antibodies of seropositive samples.
2. The solid phase immunoassay kit as claimed in claim 1, wherein the spotted recombinant and/or synthetic glycoprotein antigen comprises at least one immunogenic region of envelope protein gp41 of HIV -1 and/or HIV -2.
3. The solid phase immunoassay kit as claimed in claim 1 and 2, wherein the spotted recombinant and/or synthetic glycoprotein antigen comprises at least one immunogenic region envelope protein gp36 of HIV-1 and/ or HIV-2.
4. The solid phase immunoassay kit as claimed in claim 1, wherein the spotted solid support .is a multi-welled antigen coating polystyrene comb to which the mixture of recombinant and/or synthetic glycoproteins gp41 and gp36 of HIV-1 and/or HIV-2 are fixed.
5. The solid phase immunoassay kit as claimed in claim 1, wherein the goat-anti-human IgG-HRPO as a conjugate, inactivated anti-HIV containing human serum as an anti-HIV positive control, inactivated normal human serum as a HIV negative control, colour reagent, sample diluent, stopping solution and washing solution as herein described are provided in the ampules.
6. A method for manufacturing a solid phase immunoassay kit for detection and screening of antibodies to HIV -1 and/or HIV -2 in human blood serum and/or

plasma comprises spotting either on same or different multi-welled antigen-coating polystyrene comb either separately and/or a mixture of recombinant and/or synthetic glycoproteins gp41 and gp36, each comprising at least one immunogenic region envelope protein of HIV -1 and/or HIV -2 and preparing immunological or serological reagents, such as a conjugate, a positive control a negative control, a signal reagent, a sample diluent and a washing solution.
7 The method for manufacturing the solid phase immunoassay kit as claimed in claim 6, wherein the spotting of the recombinant and/or synthetic glycoproteins antigens on polystyrene comb comprises coating with solutions containing said glycoprotein antigens, thereby obtaining the comb spotted with the recombinant and/or synthetic antigens, gp41 and gp36, and blocking the wells of the said comb with blocking solution and finally stabilising the spotted peptides on said comb with stabilizing solution.
8 The method for manufacturing a solid phase immunoassay kit as claimed in claim 6, wherein the solutions of the conjugate, anti-HIV positive control, HIV negative control, colour reagent, sample diluent, stopping solution, washing solution are prepared such as herein described.
9 An in vitro method of serological analysis of human blood serum and/or plasma for detection and screening of anti-HIV -1 and/or HIV -2 antibodies using the solid phase immunoassay kit of claims 1 to 5 comprises contacting human blood serum and/or plasma with either separately and/or a mixture of recombinant and/or synthetic glycoproteins gp41 and gp36, each comprising at least one immunogenic region envelope protein of HIV -1 and/or HIV -2 spotted on same or different multi-welled antigen-coating polystyrene comb and thereby detecting a stable complex of bound HIV-1 and/or HIV-2 antigens with anti-HIV-I and/or HIV-2 antibodies such as herein described.

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10. The method as claimed in claim 9, wherein contacting the human blood serum and/or plasma with recombinant and/or synthetic glycoproteins antigens comprises adding to the wells of multi-welled antigen-coating polystyrene comb, spotted with an immunogenic recombinant and/or synthetic glycoprotein antigens, the human blood plasma and/or serum with sample diluent, thereby forming stable complex of anti HIV -1 and/or HIV -2 antibodies with said bound antigens and detecting the said complex such as herein described.
Dated this 12th day of December, 2005.

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