Title of Invention

A DEVICE FOR THE RAPID DETECTION OF HIV P24 ANTIGEN AND ANTIBODIES TO HIV 1 AND HIV2 AND A PROCESS OF PREPARATION THERE OF

Abstract

The present invention relates to HIV Ag & Ab EIA comb, a test kit and a device for the early detection of the HIV-1, HIV-2 antibodies and p24 antigen in human serum or plasma. The device is a comb which comprises of four dots which are HIV -1, HIV-2 detection for the detection of HIV1 and HIV2 antibodies and P24 detection dot for the detection of p24 antigen and Control dot to test the test performance. It provides an early detection of the presence of antigen to p24 and antibodies to HIV-1 and HIV-2. The p24 detection dot is coated with monoclonal or polyclonal antibodies and HIV-1 detection dot is coated with glycoprotein gp41 and C terminus of gp 120 and detection dot for HIV-2 is coated with gycoprotein gp 36, and control dot is coated with mono/ poly antihuman IgG.

Full Text

The present invention relates to a diagnostic device for the early detection of HIV p24 antigen, HIV-1 antibodies and HIV -2 antibodies in human serum or plasma. More specifically, the subject invention relates to a process for making a test on a Comb format based on Enzyme Immuno Assay principle and an analyzing device for the detection of HIV-1 antibodies, HIV-2 antibodies and HIV p24 antigen in human serum or plasma, It is an Enzyme Immuno Assay (EIA) for the detection of HIV1 antibodies, HIV2 antibodies and p24 antigen in human serum or plasma. The respective p24 antibody and the HIV1 and HIV 2 antigens are applied on the plastic polystyrene material in the form of a dots. It is an assay leading to the differential detection of antibodies to HIV-1 and HIV-2 and p24 antigen in human serum or plasma. Back ground The Human Immunodeficiency Virus is responsible agent for the Acquired Immunodeficiency Syndrome in humans. HIV causes AIDS by attacking the immune system's CD4 cells. When the immune system loses too many CD cells, we are less able to fight off infection and can develop serious infection. AIDS is usually associated with two distinct type of HIV: HIV 1 and HIV-2. The first confirmed case of AIDS was identified in 1983 and by 1984 the etiological agent, the HTV, subsequently named HIV -1 was isolated. 2 HIV -2 viruses was first isolated from the AIDS patients in West Africa in 1986 and subsequently detected as an infectious agent for the first time in the US the following year. These two viruses belong to the retrovirus group and are slow viruses. The structure, gene organization and serological behavior of HIV -1 and HIV-2 and their complete nucleotide sequence have been determined. This knowledge has laid foundation for the development of a new assay leading to differential detection of antibodies and antigens in human serum or plasma. EIA ( Enzyme Immuno Assay) continues to be the screening test for HIV infection with high sensitivity and specificity. The standard of practice for screening blood in blood banks for HIV is EIA. EIA methods are highly accurate, and suitable for high volume testing. Prior Art US Patent No: 20060286036 teaches a method for identifying an agent that inhibits HIV infection comprising assaying a biological activity of an isopeptidase . The anti-HIV agents are identified by screening test compounds for ability to modulate a biological activity of isopeptidase T (IsoT), e.g., its isopeptidase activity or its binding to another molecule such as viral protein R (Vpr). Such IsoT modulators can be further examined for their activity in inhibiting an activity indicative of HIV infection or HIV replication. These novel anti-HIV agents are useful in the prevention or treatment of HIV infection and conditions associated with or caused by HIV infection. US Patent No: 6870045 describe the compositions and methods for synthesizing and detecting HIV-2 specific amplicons. Particularly described are oligoneucleotides that are useful as hybridization probe, and amplication primers that facilitate detection of very low levels of HIV-2 nucleic acids. US Patent No: 5641624 provide a method for determining the amount of HIV-1 p24 antigen or anti-HIV-1 p24 antibody present in a suitable bodily fluid sample from an HIV-1-infected subject. This invention also provides a kit for determining the amount of HIV-1 p24 antigen or anti-HIV-1 p24 antibody present in an HIV-1-infected subject. 3 US Patent No: 6593079 concerns a process for diagnosis of an HIV infection by means of an immunoassay using the specific detection of the p24 antigen of HIV 1, HIVl-SubO and/or the p26 antigen of HIV2, at least one antibody against the env region of HIV1, HIVl-Sub0 and/or of HIV2 and at least one antibody against the pol and/or gag region of HIV1, HIVl-SubO and/or HIV2, reagent kits and test strips suitable for diagnostic procedure as well as monoclonal antibodies against p24 and their use. Description HIV test devices are available in the known art for the detection of HIV antibodies in human beings associated with such devices. But no HIV Enzyme Immuno Assay Comb test device is available in the market with the facility for the simultaneous detection of the HIV 1 antibodies, HIV 2 antibodies and HIV p24 antigen in human beings. No Enzyme Immuno Assay Comb test device with four dots is available in the market. In this, specific antigens are coated on the comb for the detection of the HIV-1 and HIV-2 antibodies and specific monoclonal or polyclonal antibodies are coated on the comb for the detection of the p24 antigen. p24 antigen detection dot is included in the comb for the early and simultaneous detection of the p24 antigen. It is a rapid, visual EIA test for the qualitative and differential detection of antibodies to HIV -1 and HIV-2 and p24 antigen in human serum or plasma. Another object of the present invention is that this is the EIA Comb test device wherein an in built control dot for the testing of the workability of the device is included so that the workability can be tested simultaneously and separately along with the detection of the p24 antigen and antibodies to HIV. The HIV Ag & Ab EIA Comb device is a visual, rapid, sensitive and accurate immunoassay for the differential detection of HIV-1 antibodies, HIV-2 antibodies, and HIV p24 antigen in human serum or plasma by using HIV-1 antigen, HIV-2 antigen and HIV p24 antibodies immobilized on the specific dots in the Comb. 4 The serological events following the HIV infection are represented graphically in figure 1. In individuals infected with HIV, antigen appears first before antibodies of HIV but due to the seroconversion, the antigen is lost and antibody develops within 2 to 6 weeks after infection and thereby the level of the antibody increases. HIV p24 antigen test device for the detection of the specific p24 antigen is a test device for the early and rapid detection of the HIV in human body. Antigen can generally only be detected during the acute and during the symptomatic phase of AIDS. Antibodies to HIV 1 & 2 can be detected throughout virtually the entire infection period. When the HIV virus enters into the body, the earlier detection of the disease is impossible because the virus enters in a window phase and subsequently the immune system start producing antibodies. So the use of highly sensitive antibody assays is therefore an established approach in serodiagnosis of HIV infection. Progressive improvements in assay sensitivity have reduced that window phase. Further shortening of this period can be achieved by the incorporation of HIV antigen detection in such antibody tests enabling the detection of the infected individuals at the earliest possible moment. p24 antibody dot detects the specific p24 antigens of HIV. So early detection of HIV is possible. The present invention device contains a Comb which is made of polystyrene plastic, having eight individual teeth, each a separate test spaced to fit in to a standard 96 micro titer wells in which assay is to be performed. Each comb consists of 4 dots immobilized on one side of each tooth of polystyrene comb. This comb is provided with an inbuilt control dot. The lowest dot is the dot for the detection of HIV-1 antibodies, the dot above the lowest dot (HIV 1 detection dot) is for the detection of the HIV-2 antibodies, the dot above the dot for the detection of the HIV-2 is p24 antigen detection dot and the dot at the top is the inbuilt control dot. The location of all these three dots (HIV 1 Ag, HIV 2 Ag and p24 antibody) are interchangeable except the control dot. The control dot is immobilized with the monoclonal or polyclonal antihuman IgG of chicken, donkey, horse or rabbit. The detection dot for the detection of the p24 antigen is coated with the specific p24 monoclonal or polyclonal antibodies , detection dot for the detection of HIV 1 is coated with recombinant protein/ peptide or modified version in the form of biotinylated recombinant protein/ peptide such as gp 41 and C terminus of gp 120 5 and HIV-2 detection dot is coated with recombinant protein / peptide or biotinylated recombinant protein/ peptide immunodominant epitopes of gp 36. "HIV Ag& Ab EIA" Comb is based on enzyme immunoassay and employs the binding of enzyme with chromogenic substrate to visualize the immobilized immune complex. HIV -1 and HIV-2 antigens and p24 antibodies are immobilized as circular dots on the polystyerene comb as shown in the figure 2. When the comb is incubated with the specimen containing HIV-1, HIV-2 antibodies and p 24 antigen, these antibodies bind specifically to the antigen and p24 antigen bind to the p24 antibodies. The comb is washed to remove unbound antibodies and antigens. The comb is then incubated in micro wells containing enzyme conjugate. This conjugate will bind to the antigen- antibody/ antibody - antigen complex present on the comb. Then the comb is placed in the micro wells containing substrate solution and is incubated for eight minutes. The unbound conjugate will react with substrate. The results can be readable from the gray -blue coloured dots on the comb as shown in the figure 3. According to the invention, a device for the rapid detection of the HIV p24 antigen and antibodies to HIV-1 and HIV-2 in human serum or plasma comprising a polystyerene comb having four dots HIV-1, HIV-2, p24 ,and control dot, which are coated with antigen to HIV -1 and HIV-2 and p24 antibodies and control dot is coated with antihuman IgG of rabbit or chicken or goat. According to the invention, a process for making HIV Ag & Ab EIA Comb device as claimed in claim 1 wherein HIV1 detection dot is prepared as follows, adding 5 mM - l00mM carbonate buffer solution of pH 8 - 9.6,1 nano gram to 10 micro gram of each recombinanat/ synthetic proteins / Biotinylated recombinant / synthetic proteins glycoprotein gp 41 ,C terminus of Glycoprotein 120, vortexing the mixture to get a homogeneous solution. Coating lμl- 10 μl of the above mixture to the HIV 1 detection dot and allowing it to dry at 37°C for 1 hr to 20 hrs and preferably 4 hrs. According to the invention, a process for making HIV Ag & Ab EIA Comb device as claimed in claim 1 wherein HIV 2 detection dot is prepared as follows: 6 adding 5 mM - l00mM carbonate buffer solution of pH 8 - 9.6, 1 nano gram to 10 micro gram of each recombinanat/ synthetic proteins / Biotinylated recombinant / synthetic proteins glycoprotein gp 36, vortexing the mixture to get a homogeneous solution, Coating 1 JΜL- 10 ΜL of the above mixture to the HIV 2detection dot and allowing it to dry at 37°C for 1 hr to 20 hrs and preferably 4 hrs. According to the invention, a process for making HIV Ag & Ab EIA Comb device as claimed in claim 1 wherein p24 detection dot is prepared as follows:- adding 5 mM - l00mM carbonate buffer solution of pH 8 - 9.6, 1 nano gram to 10 micro gram specific biotinylated monoclonal or polyclonal or F(ab1)2 p24 antibodies and vortexing the mixture to get a homogeneous solution, Coating lμl- 10 μl of the above mixture to the HIV p24 antigen detection dot and allowing it to dry at 37°C for 1 hr to 20 hrs and preferably 4 hrs. According to the invention, a process for making HIV Ag & Ab EIA Comb device as claimed in claim 1 wherein Control dot is prepared as follows:- adding 5 mM - l00mM carbonate buffer solution of pH 8- 9.6 , 1 nano gram o 10 microgram antihuman IgG or biotinylated antihuman IgG for eg. of chicken or goat and vortexing the mixture to get a homogeneous solution, Coating lμl-10 μl of the above mixture to the control dot and allowing it to dry at 37oC for 1 to 20 hrs and preferably 4 hrs. According to the invention, a process for analyzing the presence of HIV 1 , HIV2 and p24 antigen in human serum or plasma comprising of the following steps: a. taking open and taking out the required number of the Comb from the foil. b. placing the comb in to the microcuvettes-containing sample and incubating it for 8 minutes at room temperature. During incubation, withdrawing and inserting the combing in the microcuvettes for 15 seconds. c. removing the comb from the microcuvettes containing the sample and blot the tips of the arms on absorbent material. 7 d. placing the comb in to the microcuvettes containing the wash buffer and wash the comb for one minute.by carefully mixing the comb up and down, Removing the comb from there and blot in the tips of the comb on absorbent material. e. placing the comb in to the microcuvettes containing mixture of enzyme conjugates as in format 1 and incubate for 8 minutes at room temperature. During incubation, withdraw and insert the comb in to the microcuvettes for 15 minutes. f. again washing the comb with wash buffer after taking it out from the microcuvett containing enzyme conjugate. g. placing the microcuvett containing the substrate and incubate it for 8 minutes at room temperature. During incubation, withdraw and insert the comb in the microcuvett for 15 seconds. h. taking the comb out and wash the comb with distilled water in the microcuvett for one minute. i. allowing and drying the comb in a clean surface and read the result. In an embodiment of the present invention Substrate is XXX which is available in the market. In an embodiment of the present invention the interpretation of the result are shown in the figure 3. 1. If only one dot, the control dot is visible on the comb, then the sample is non reactive to the antigen and antibodies and the workability of the device is perfectly right. 2. If the control dot and the dot for HIV-1 are visible in gray colour, then the sample is reactive for HIV -1 and the device is working perfectly. 3. If the control dot and the dot for the detection of the HIV-2 appears in colour, then the sample is reactive for HFV-2. 4. If the control dot and the dot for the detection of p24 antigen appears in colour, then the sample is reactive for HIV. 8 5. If the control dot and the HIV-1 and HIV-2 dots appear in colour, then the sample is reactive for both HIV-1 and HIV-2. 6. If the control dot and the p24 dot and HIV-1 dot appears in colour, the sample is reactive for HIV-1 and p24 antigen. 7. If the control dot and p24 dot and the HIV-2 detection dot appears in colour, then the sample is reactive for HIV-2 and p 24 antigen. 8. If all the four dots appear in colour, the sample is reactive for both HIV antigen and HIV1 and HIV 2 antibodies and the device is working perfectly. The invention will be described with reference to the following accompanying drawings wherein. Figure 1 shows the fourth generation of the HIV Ag and Ab EIA comb. The figure clearly shows the difference in the various devices developed from time to time. In the first generation, the antibodies were detected only after 6 weeks. In the second generation, the antibodies were detected before 6 weeks but after 4 weeks. In the third generation, the antibodies were detected after three weeks but before 4 weeks. In the fourth generation, the antigens were detected before three weeks but after 2 weeks. So this is an important invention in the history of HIV. Figure 2 shows the polystyrene comb. Figure 3 shows the interpretation of the test results The sample should be only human serum or plasma. Collect blood in a sterile vial and allow to clot and separate the serum by centrifugation at room temperature. If serum or plasma is not assayed immediately it should be stored at 2-8 °C. Sample preparation Add 250μl of sample diluent using dropper provided. Squeeze the dropper and fill the diluent up to the mark. And then transfer the complete solution to the required number of the microcuvettes. Add 1 drop of the sample to the to the above respective microcuvettes 9 with the help of the sample dropper. Mix the sample with the sample diluent repeatedly and make sure that no bubbles are formed while making the sample. In an embodiment of the present invention, the Comb can be prepared as follows. There are four formats in which the test can be performed. In the three formats the comb can be coated as follows. Preparation of Comb Preparation of HIV 1 detection dot Adding 5mM to 100 mM carbonate buffer solution of pH 8- 9.6 , 1 nanogram to 10 microgram of each recombinant proteins/ synthetic peptides/ biotinylated recombinant /synthetic protein/ peptides glycoprotein gp 41 , C terminus of Glycoprotein gp 120, vortexing the mixture to get a homogeneous solution, Coating 1 μl- 10 ul of the above solution to the HIV 2 detection dot and allowing it to dry at 37°C for 1 hr to 5 hrs preferably 4 hrs. Preparation of HIV 2 detection dot Adding 5 mM - l00mM carbonate buffer solution of pH 8 - 9.6, 1 nano gram to 10 micro gram of each recombinanat/ synthetic proteins / Biotinylated recombinant / synthetic proteins glycoprotein gp 36, vortexing the mixture to get a homogeneous solution, Coating lμl- 10 ul of the above mixture to the HIV2 detection dot and allowing it to dry at 37°C for 1 hr to 20 hrs and preferably 4 hrs. Preparation of HIV 2 detection dot Adding 5mM to 100 mM carbonate buffer solution of pH 8- 9.6 , 10 nanogram to 800 nnogram of each recombinant proteins/ synthetic peptides/ biotinylated recombinant synthetic protein/ pep [tides glycoprotein gp 36 , vortexing the mixture to get a 10 homogeneous solution, Coating 1 μl- 10 μl of the above solution to the HIV 2 detection dot and allowing it to dry at 37oC for 1 hr to 5 hrs preferably 4 hrs. Preparation of p24 detection dot Adding 5mM to l00mM carbonate buffer solution of pH 8- 9.6, 10 nano gram to 1 microgram specific biotinylated monoclonal or polyclonal or F(ab1)2 p24 antibodies vortexing the mixture to get a homogeneous solution, Coating 1 μl- 10 μl of the above solution to the HIV 1 p24 detection dot and allowing it to dry at 37oC for 1 hr to 6 hrs preferably 4 hrs in the open atmosphere. In format 4, the control dot is coated with biotinylated protein, p24 detection dot is coated with monoclonal or polyclonal or F(ab1)2 monoclonal or polyclonal p24 antibodies, and HIV 1 and HIV2 is coated with recombinant or synthetic proteins. Format 1 Preparation of Enzyme conjugate Alkaline phosphatase- p 24 mnoclonal or polyclonal antibody conjugate, Alkaline Phosphatase HIV 1 antigen conjugate, Alkaline Phosphatase HIV 2 antigen conjugate and Alkaline phoshatase - antihuman IgG conjugate is used as the enzyme for the detection of the p24 antigen and antibodies to HIV1 and 2. These three conjugates are mixed together and the final concentration of each conjugate in the mixture should be between 1: 5 to 1: 20. Stepwise addition of each conjugate is also possible. Format 2 In format 2, Alkaline phosphatase - p24 monoclonal or polyclonal antibody conjugate and Alkaline Phosphatase - antihuman IgG and anti human IgM conjugate is used as the conjugate. This also can be used together as a mixture or separately. 11 Format 3 In Format 3 Alkaline Phosphatase - p24 antibody conjugate and Alkaline phosphatase - Protein A conjugate or antihuman IgG conjugate is used as the enzyme. In an embodiment of the present invention, test procedure for the detection of HIV 1 & HIV 2 antibodies and p24 antigen, is as follows. a. Taking open and taking out the required number of the Comb from the foil. a. Placing the comb in to the microcuvettes-containing sample and incubating it for 8 minutes at room temperature. During incubation, withdrawing and inserting the comb in the microcuvettes for 15 seconds. b. Removing the comb from the microcuvettes containing the sample and blot the tips of the arms on absorbent material. c. Placing the comb in to the microcuvettes containing the wash buffer and washing the comb for one minute by carefully mixing the comb up and down. Removing the comb from there and blot in the tips of the comb on absorbent material. d. Placing the comb in to the microcuvettes containing mixture of enzyme conjugates as in format 1 and incubating for 8 minutes at room temperature. During incubation, withdrawing and inserting the comb in to the microcuvettes for 15 minutes. e. Again washing the comb with wash buffer after taking it out from the microcuvette containing enzyme conjugate. f. Placing the microcuvette containing the substrate and incubating it for 8 minutes at room temperature. During incubation, withdrawing and inserting the comb in the microcuvette for 15 seconds. g. Taking the comb out and wash the comb with distilled water in the microcuvette for one minute. j. Allow drying the comb in a clean surface and read the result 12 Format 4 AP - streptavidin conjugate is used . Biotinylated antigens or antibodies are added followed by addition of conjugate. In Format 4, the test procedure is as follows: a. Taking open and taking out the required number of the Comb from the foil. b. Placing the comb in to the microcuvettes-containing sample and incubating it for 8 minutes at room temperature. During incubation, withdrawing and inserting the comb in the microcuvettes for 15 seconds. c. Removing the comb from the microcuvettes containing the sample and blot the tips of the arms on absorbent material. d. Placing the comb in to the microcuvettes containing the wash buffer and washing the comb for one minute by carefully mixing the comb up and down. Removing the comb from there and blot in the tips of the comb on absorbent material. e. Placing the comb in the microcuvettes containing the biotinylated antigen and antibody. Incubating it for 8 minutes. f. Placing the comb in to the microcuvettes containing the Wash buffer and washing the comb for one minute by carefully mixing the comb up and down. Removing the comb from there and blot in the tips of the comb on absorbent material. g. Placing the comb in to the microcuvettes containing AP - Streptavidin conjugate and incubating for 8 minutes at room temperature. During incubation, withdrawing and inserting the comb in to the microcuvettes for 15 minutes. h. Again washing the comb with wash buffer after taking it out from the microcuvette containing enzyme conjugate. 13 i. Placing the microcuvette containing the substrate and incubating it for 8 minutes at room temperature. During incubation, withdrawing and inserting the comb in the microcuvette for 15 seconds. j. Taking the comb out and washing the comb with distilled water in the microcuvette for one minute. k. Allowing drying the comb in a clean surface and read the result 14 I Claim:- 1. A device for the rapid detection of the HIV p24 antigen and antibodies to HIV-1 and HIV-2 in human serum or plasma comprising a polystyerene comb having four dots HIV-1, HIV-2, p24 ,and control dot, which are coated with antigen to HIV -1 and HIV-2 and p24 antibodies and control dot is coated with antihuman IgG of rabbit or chicken or goat. 3. A device as claimed in claim 1 where in the HIV1 and HIV-2 detection spot for the detection of HIV 1 and HIV2 antibodies are coated with recombinant antigens/ synthetic peptides/ biotinylated HIV-1 and HIV-2 antigens. 4. A device as claimed in claim 1 wherein HIV 1 detection dot is coated with the recombinant antigens gp 41 and C terminus of gp 120. 5. A device as claimed in claim 1 wherein the HIV2 detection dot is coated with gp 36 recombinant antigen. 6. A device as claimed in claim 1 wherein the p24 antigen detection dot is coated with monoclonal or polyclonal p 24 antibodies/F(ab1)2 p24 antibodies. 7. A device as claimed in claim 1 where in the control dot is coated with monoclonal or polyclonal antihuman IgG. 8. A process for making HIV Ag & Ab EIA Comb device as claimed in claim 1 wherein HFV1 detection dot is prepared as follows. adding 5 mM - l00mM carbonate buffer solution of pH 8 - 9.6,1 nano gram to 10 micro gram of each recombinanat/ synthetic proteins / Biotinylated recombinant / synthetic proteins glycoprotein gp 41 ,C terminus of Glycoprotein 120, vortexing the mixture to get a homogeneous solution. Coating lμl- 10 μl of the above mixture to the HIV 1 detection dot and allowing it to dry at 37°C for 1 hr to 20 hrs and preferably 4 hrs. 9. A process for making HIV Ag & Ab EIA Comb device as claimed in claim 1 wherein HIV 2 detection dot is prepared as follows:- adding 5 mM - l00mM carbonate buffer solution of pH 8 - 9.6, 1 nano gram to 10 micro gram of each recombinanat/ synthetic proteins / Biotinylated recombinant / synthetic proteins glycoprotein gp 36, vortexing the mixture to get a homogeneous 15 solution, Coating lμl- 10 μl of the above mixture to the HIV 2detection dot and allowing it to dry at 37°C for 1 hr to 20 hrs and preferably 4 hrs. 10. A process for making HIV Ag & Ab EIA Comb device as claimed in claim 1 wherein p24 detection dot is prepared as follows:- adding 5 mM - l00mM carbonate buffer solution of pH 8 - 9.6, 1 nano gram to 10 micro gram specific biotinylated monoclonal or polyclonal or F(ab1)2 p24 antibodies and vortexing the mixture to get a homogeneous solution, Coating lμl- 10 μl of the above mixture to the HIV p24 antigen detection dot and allowing it to dry at 37°C for 1 hr to 20 hrs and preferably 4 hrs. 11. A process for making HIV Ag & Ab EIA Comb device as claimed in claim 1 wherein Control dot is prepared as follows:- adding 5 mM - l00mM carbonate buffer solution of pH 8- 9.6 , 1 nano gram o 10 microgram antihuman IgG or biotinylated antihuman IgG for eg. of chicken or goat and vortexing the mixture to get a homogeneous solution, Coating lμl- 10 μl of the above mixture to the control dot and allowing it to dry at 37oC for 1 to 20 hrs and preferably 4 hrs. 11. A process for analyzing the presence of HIV 1 , HIV2 and p24 antigen in human serum or plasma comprising of the following steps: a. taking open and taking out the required number of the Comb from the foil. b. placing the comb in to the microcuvettes-containing sample and incubating it for 8 minutes at room temperature. During incubation, withdrawing and inserting the combing in the microcuvettes for 15 seconds. c. removing the comb from the microcuvettes containing the sample and blot the tips of the arms on absorbent material. 16 d. placing the comb in to the microcuvettes containing the wash buffer and wash the comb for one minute by carefully mixing the comb up and down, Removing the comb from there and blot in the tips of the comb on absorbent material. e. placing the comb in to the microcuvettes containing mixture of enzyme conjugates as in format 1 and incubate for 8 minutes at room temperature. During incubation, withdraw and insert the comb in to the microcuvettes for 15 minutes. f. again washing the comb with wash buffer after taking it out from the microcuvett containing enzyme conjugate. g. placing the microcuvett containing the substrate and incubate it for 8 minutes at room temperature. During incubation, withdraw and insert the comb in the microcuvett for 15 seconds. h. taking the comb out and wash the comb with distilled water in the microcuvett for one minute. i. allowing and drying the comb in a clean surface and read the result. 12. A HIV Ag & Ab El A Comb device for analyzing the presence of HIV1 and HIV2 anbtibodies and p24 antigen in human serum or plasma as herein before described with reference to the accompanying drawings. 13. A process for analyzing the presence of HIV 1 and HIV2antibodies and p24 antigen to HIV in human serum or plasma as herein before described with reference to the accompanying drawings. Dated this 4th day of May, 2007. 17 The present invention relates to HIV Ag & Ab EIA comb, a test kit and a device for the early detection of the HIV-1, HIV-2 antibodies and p24 antigen in human serum or plasma. The device is a comb which comprises of four dots which are HIV -1, HIV-2 detection for the detection of HIV1 and HIV2 antibodies and P24 detection dot for the detection of p24 antigen and Control dot to test the test performance. It provides an early detection of the presence of antigen to p24 and antibodies to HIV-1 and HIV-2. The p24 detection dot is coated with monoclonal or polyclonal antibodies and HIV-1 detection dot is coated with glycoprotein gp41 and C terminus of gp 120 and detection dot for HIV-2 is coated with gycoprotein gp 36, and control dot is coated with mono/ poly antihuman IgG.

Documents:

00696-kol-2007-abstract.pdf

00696-kol-2007-claims.pdf

00696-kol-2007-correspondence others 1.1.pdf

00696-kol-2007-correspondence others 1.2.pdf

00696-kol-2007-correspondence others 1.3.pdf

00696-kol-2007-correspondence others.pdf

00696-kol-2007-correspondence.pdf

00696-kol-2007-description complete.pdf

00696-kol-2007-drawings.pdf

00696-kol-2007-form 1.pdf

00696-kol-2007-form 18.pdf

00696-kol-2007-form 2.pdf

00696-kol-2007-form 3.pdf

00696-kol-2007-form 5.pdf

00696-kol-2007-form-9.pdf

00696-kol-2007-gpa.pdf

696-KOL-2007-(02-12-2013)-REPLY STATEMENT OF PRE-GRANT OPPOSITION-.pdf

696-KOL-2007-(02-12-2013)-REPLY STATEMENT OF PRE-GRANT OPPOSITION.pdf

696-KOL-2007-(12-10-2012)-CORRESPONDENCE.pdf

696-KOL-2007-(12-10-2012)-OTHERS.pdf

696-KOL-2007-(19-10-2012)-CORRESPONDENCE.pdf

696-KOL-2007-(19-10-2012)-PRE GRANT REPLY RECIEVED.pdf

696-KOL-2007-(20-07-2012)-PRE GRANT OPPOSITION.pdf

696-KOL-2007-ABSTRACT 1.1.pdf

696-KOL-2007-CANCELLED PAGES.pdf

696-KOL-2007-CLAIMS 1.1.pdf

696-KOL-2007-CORRESPONDENCE 1.1.pdf

696-KOL-2007-CORRESPONDENCE 1.6.pdf

696-KOL-2007-CORRESPONDENCE 1.7.pdf

696-KOL-2007-CORRESPONDENCE OTHERS 1.5.pdf

696-KOL-2007-CORRESPONDENCE-1.8.pdf

696-KOL-2007-DESCRIPTION COMPLETE 1.1.pdf

696-KOL-2007-FORM 1-1.1.pdf

696-KOL-2007-FORM 13.1.1.pdf

696-kol-2007-form 13.pdf

696-KOL-2007-FORM 2.1.pdf

696-KOL-2007-FORM 26.pdf

696-KOL-2007-FORM 3-1.1.pdf

696-KOL-2007-FORM 5-1.1.pdf

696-KOL-2007-GRANTED-SPECIFICATION (COMPLETE).pdf

696-KOL-2007-OTHERS-1.1.pdf

696-KOL-2007-OTHERS-1.2.pdf

696-KOL-2007-OTHERS.pdf

696-KOL-2007-PA.pdf

696-KOL-2007-POST GRANT OPPOSITION.pdf

696-KOL-2007-REPLY F.E.R.pdf

696-KOL-2007-REPLY TO EXAMINATION REPORT.pdf