Title of Invention

"A DEVICE FOR RAPID DETECTION OF HIV ANTIGEN"

Abstract

The present invention relates to HIV TRIDOT Ag test device for an early detection of HIV antigen (fig 1), HIV-1 antibodies and HIV-2 antibodies( fig 1). HIV TRIDOT Ag test device which 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 or whole blood using HIV-1 antigen, HIV-2 and HIV P24 antibodies immobilized on an immunofiltration membrane.

Full Text

FIELD OF THE INVENTION The present invention relates to a device for an early detection of HIV antigen, HIV-1 antibodies and HIV-2 antibodies. More specifically, the subject invention relates to a diagnostic kit herein after referred as fourth generation diagnostic kit for the detection of HIV P24 antigen, HIV-1 antibodies and HIV-2 antibodies. HIV- TRIDOT Ag test device which 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 or whole blood using HIV-1 antigen, HIV-2 antigen and HIV P24 antibodies immobilized on an immunofiltration membrane. BACKGROUND OF THE INVENTION HIV test kits are available in the known art for the detection of HIV antibodies in human beings associated with such kits. But no rapid flow through of immunochromatography test kit is available with the facility the simultaneous detection of the HIV1 antibodies, HIV 2 antibodies and HIV P24 antigen in human beings. US Patent No. 5134864 describes that retroviruses associated with Acquired immune deficiency syndrome (AIDS), including Lymphadenopathy Associated Virus (LAV) are isolated from the sera of patients afflicted with Leymphadenopathy syndrome (LAS) or AIDS. LAV is a human immunodeficiency virus (HIV) .viral extract, structural proteins and other fractions of the retrovirus immunologically recognize the sera of such patients. Immunological reaction is used to detect antibodies that specifically bind to antigenic sites of the retrovirus in samples of body fluids from patients with AIDS or risk of AIDS. A kit for in vitro assay of LAS or AIDS is provided. US Patent No.5695930 discusses a rapid and accurate test kit for the detection of antibodies to HIV in saliva. The identification of antibodies to HIV in the saliva of sero positive individuals is shown using a test kit that requires no special machinery or skill and can be conducted by a single person in the privacy of their own home. US Patent No. 5721095 discloses a method for producing an improved solid phase antigenic reagent useful in an immunoassay for detecting antibodies specific for a virus, such as the human immunodeficiency virus, which comprising the addition to a natural viral lysate a synthetic or recombinant viral protein or peptide. Also provided is an improved immunoassay utilizing the solid phase antigenic reagent. US Patent No.5169753 describes a novel retrovirus isolated from human lymphoma cells. The retrovirus is characterized by a c-type retroviral particle of approximately 100 nm diameters and an approximately 27,000 molecular weight p24 core protein. Also disclosed are cell lines from which the virus can be obtained and screening methods for detecting the presence of the virus in human sera. US Patent No. 5055391 relates to a new variety of retroviruses designated human immunodeficiency virus type II, HIV-2, samples of which have been deposited at CNCM as I-502 and I-532. IT also concerns purified forms of the antigens which can be obtained from this virus, in particular from the gp 36 and gp 130-140 proteins. These various antigens are useful in medical diagnosis and kits, in particular by being placed in contact with serum of the patient to be diagnosed. Lastly, the invention relates to immunizing compositions, in particular containing at least one of glycoprotein gp36 and gp 130-140. US Patent No. 6,593,079 concerns a process for diagnosis of an HIV infection by means of an immunoassay using the specific detection of the p24 antigen of HIV1, HIV1 subO and /or the p26 antigen of HIV2 at least one antibody against the pol/or gag region of HIV1, HIV 1-Sub 0 and /or HIV 2, reagent kits and test strips suitable for diagnostic procedure as well as monoclonal antibodies against p24 and their use. US Patent No.6818392 relates to novel monoclonal antibodies which may be used in the detection of human immunodeficiency virus (HIV). These antibodies exhibit an unusually high degree of sensitivity, a remarkably broad range of specificity and bind to novel shared, non cross reactive epitopes. In particular, the monoclonal antibodies of the present invention may be utilized to detect HIV-1 antigen and HIV-2 core antigen in a patient sample. US Patent No.5641624 provides 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. US Patent No. 6242174 describes a method of discriminating between specific antibodies in samples of sera or other body fluids from humans or other primates containing antibodies arising from infection with HTLV-1 .containing antibodies arising from infection with HTLV-II or containing antibodies arising from infection with related retroviruses. In said method, the sample to be analyzed is subjected to at least four immunoassays, each using a different diagnostic antigen selected from four defined groups of peptides. Additionally, an immunoassay kit adapted for said method of discrimination, new peptides and a method of detecting antibodies with said peptides are described. The present device is having four dots instead of two or three dots and where in the dots are meant for control dot HIV-1 antibodies dot, HIV-2 antibodies dots and the fourth one for HIV P24 antigen. The HIV P24 antigen is a break through in the technology as no device is available which can detect the HIV antigen that is detecting the HIV at a very early stage that is within 4 weeks from the infection of HIV where only the antigens of HIV are present and antibodies have not been generated. The combination of different conjugates are used for early detection of HIV-1 and HIV-2 antibodies in the human sample and HIV P24 antigen detection is included for the earlier detection of HIV P24 antigens. Object of the invention The object of this invention is to provide an HIV test device wherein the presence of antibodies to HIV-1 & HIV-2, presence of p24 antigen and the workability of the test device can be detected separately and simultaneously. In order to achieve the 100% reliable result, the antigen and antibody detection test using antigen and antibodies is devised thus providing greater sensitivity and better specificity. Summary of the invention The subject invention relates to a HIV TRIDOT Ag test device which is 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 or whole blood using HIV-1 antigen, HIV-2 antigen AND HIV p24 antibodies immobilized on an immunofiltration membrane. The user benefits because running one test is more time and cost efficient than running two. Moreover the early HIV detection is extremely important for all the patients regardless of lifestyle in reducing further transmission. The study confirms that the combined HIV Ab/Ag assays are able to detect HIV infection earlier than an antibody only assay. Detailed Description of the invention 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 diseases is impossible because the virus enters in a window phase and subsequently the immune system starts 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 test 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 device of the present invention includes three dots/lines and one built control dot/line wherein at least one test dot/line uses antigens. The function of the control dot is to indicate the proper functioning of the device. Accordingly the present invention relates to a rapid, visual, sensitive and qualitative in vitro diagnostic kit for the detection of the antibodies and antigens of a HIV in human serum and plasma and whole blood comprising a test device, a buffer solution, a conjugate and p24 conjugate. The device comprises of a plastic base, an absorbent pad, made of cellulosic material for e.g. cellulose nitrate. The membrane is cut from the cellulosic material and is mounted on an absorbent pad which is housed in the casing. The casing comprises of top cover and bottom cover air tightly pressed with each other. A hole is provided in the top cover at the center of the device through which the membrane is exposed. The membrane is mounted on a laminated or unlaminated absorbent. The recombinant protein or biotinylated recombinant proteins or synthetic peptides or biotinylated synthetic peptides or a mixture thereof are immobilized in the membrane. The control dot meant to test the workability of the device is coated with antihuman IgG e.g. donkey or chicken or any other non cross reacting antibody. After adding the conjugate reagent, it gives a pink colour which determines that the device is workable. HIV1 detection dot for the detection of HIV1 antibody is coated with glycoprotein 41 and C terminus of glycoprotein 120 in the ratio 45:55 respectively and vice versa. Preferably the ratio of glycoprotein 41 and C terminus of glycoprotein 120 is in the ratio 50:50. Similarly the HIV 2 detection dot for the detection of HIV 2 antibodies is coated with glycoprotein 36 immobilized on the said membrane in the form of dot or line. HIV P24 detection dot for the detection of the HIV P24 antigen is coated is 1 nanogram to 10 microgram. All these four spots are within the circumference of the immunofiltration membrane. Working example Sample: The sample should be only human serum or plasma or whole blood. Collect blood in a clean dry vial and allow to clot or separate the serum by centrifugation at room temperature. For using whole blood as sample a separate filter device is provided to fit into the nest of existing testing device. It separates the RBCs from the blood and allows plasma to filter and pass on the testing device. Preparation of buffer solution: A liquid dispensing buffer is used to dissolve the glycoprotein HIV1 and HIV2 and dispensing on the device. Preparation of protein A conjugate: 1. To an initial solution of 0.01%-0.05 %(w/v) Gold chloride (HAuCL4) that is at boiling temperature sodium citrate solution (1-5%) is added. The solution is rapidly stirred and boiled until a pink purple colour develops. Protein A is added to the same. 2. Any non immune animal serum (Mouse /goat /rabbit/sheep) is added to the above solution. The concentration of serum could be 1 microgram to 10 microgram/ml of gold colloid solution. This solution is mixed for 2 hrs to over night. After this the solution is centrifuged and the supernatant is discarded .dissolve the pellet in buffer containing stabilizers such as BSA, Gelatin to obtain the protein A conjugate. Preparation of p24 conjugate: Preparation of p24 antibody conjugate in the form of liquid lyophilized comprises adding sodium citrate solution (1-5%) to 0.01% - 0.05% (w/v) gold chloride (HauCI4) at a boiling temperature; rapidly stirring and boiling the solution until a pink purple colour develops: adding p24 antibody to the solution; adding any non immune animal serum such as mouse/goat/sheep/rabbit 1 microgram to 10 microgram/ml of gold colloidal solution; mixing the solution for 2 hours to overnight; centrifuging the solution to discard the supernatant. Preparation of streptavidin gold conjugate: 1. To an initial solution of 0.01 %- 0.05% (w/v) gold chloride (HAuCI4) that is at a boiling temperature, sodium citrate solution (15%) is added. The solution is rapidly stirred and boiled until a pink purple colour develops Streptavidin is added to the same. 2. Any non-immune animal serum (mouse/goat/rabbit/sheep) is added to the above solution. Solution is centrifuged and the supernatant is discarded. Dissolve the pellet in buffer containing stabilizers such as BSA, Gelatin to obtain the streptavidin gold conjugate. Test procedure 1. Adding 5 drops of wash buffer to the device and waiting till the solution is completely absorbed by the membrane. All solution and sample should be added to the center of the membrane. 2. Adding 1 drop of sample. The sample should be only human serum or plasma. When the patient's sample passes through the membrane, HIV antibodies and p24 antigens if present in the sample binds to the immobilized HIV antigens and p24 antibody respectively. 3. Adding 5 drops of buffer solution. 4. Adding 2 drops of protein A conjugate. 5. Adding 2 drops of wash buffer. 6. Adding 2 drops of p24 conjugate. P24 conjugate binds to the p24 antibody-antigen complex and protein A conjugate binds to the Fc portion of the HIV antibodies to give pinkish purple color dots against whitish background. 7. Adding 5 drops of wash buffer solution to wash off other impurities present. 8. Reading the results. There are four formats by which the test can be performed. Interpretation of results for all the four formats will be same as given below: On performing the test, if only the control dot appears which is in pink colour, then the result is interpreted as negative and the procedure carried out for the detection are perfectly right. If both the control dot and hivl antibody detection spot appears in pink colour, then the result is that the patient is reactive for HIV-1 antibody. If the control dot and the HIV appear in pink colour, then the patient is reactive for HIV-2 antibodies, if both HIV1 and HIV2 antibody detection dot and the control dot appear in pink colour, then the patient is reactive for both HIV1 & HIV2 antibodies . if both control dot and p24 detection spot appear in pink color, indicating the early stage of HIV infection. If the control dot, HIV p24 and the HIV 1 dot appear pink in colour, then the patient is reactive for HIV-1 antibody and HIV P24 antigen. If the control dot p24 antigen dot and the HIV-2 DOT appear pink in colour, then the patient is reactive for HIV-2 antibody and HIV P24 antigen. If all the four dots appear pink in colour, then the patient is reactive for HIV-1 & HIV-2 antibodies and HIV P24 antigen. According to the present invention, a device for the rapid detection of HIV antigen and antibodies to HIV-1 and HIV-2 in the human beings comprises an airtight container having an absorbent pad, a nitrocellulose membrane mounted on the absorbent pad provided with a circular hole in the top cover through which the membrane is exposed. Four dots HIV1, HIV2, P24 and C are provided on the membrane wherein the dots HIV1 and HIV2 are coated with a homogenous solution of HIV antigens, the control dot is coated with IgG and the fourth dot is coated with p24 antibodies wherein the antibodies detect the HIV antigens in human body while T1 and T2 detect the antibodies of HIV 1 and HIV2. The invention will be described with reference to the following accompanying drawings wherein: Figure 1 shows the fourth generation HIV Tridot and Ag. Figure 2,3,4,5,6,7,8 and 9 shows the functioning of the device. Figure 1 shows clearly the difference in the various devices developed from time to time. In the first generation, the antibodies were detected after more than six weeks of getting the HIV infection. In the second generation the antibodies were detected after about six weeks of getting the HIV infection after about four weeks. In the third generation, the antibodies were detected round about four weeks of getting the HIV infection. In the fourth generation the antigens are detected in less than three weeks of the infection. So, it is a breakthrough in the technology as the infection is detected before the growth of the antibodies. Figures 2 shows that all the four dots are reactive indicating that the device is working and the presence of antigens and HIV1 and HIV2 antibodies. Figures 3 shows that the device is working and the presence of p24 antigen and HIV 1 antibodies. Figure 4 shows that the device is working and the presence of HIV 1 antibodies. Figure 5 shows that the device is working but is not reactive to any antigen or antibodies. Figure 6 shows that the device is working and the presence of p24 antigens. Figure 7 shows that the device is working and the presence of p24 antigen and HIV antibodies. Figure 8 shows that the device is working and the presence of hiv-2. Figure 9 shows that the device is not working. I claim : 1. A Device for the rapid detection of HIV antigen and antibodies to HIV-1 HIV-2 in the human beings comprising an airtight container having an absorbent pad, a nitrocellulose membrane mounted on the absorbent pad, provided with a circular hole in the top cover through which the membrane is exposed, four dots HIV1,HIV2,P24 and C are provided on the membrane wherein the dots HIV1 and HIV2 are coated with a homogeneous solution of HIV antigens, the control dot is coated with IgG and the fourth dot is coated with p24 antibodies wherein the antibodies are capable to detect the HIV antigens in human body while HIV-1 and HIV-2 are capable to detect the antibodies of HIV 1 and HIV 2. 2. A Device as claimed in claim 1, wherein the glycoprotein 41 and C terminus of glycoprotein 120 are in the ratio of 45:55:55:45. 3. A Device as claimed in claim 1, wherein the amount of coating used is 1 nanogram to 10 microgram. 4. A Device as claimed in claim 1, wherein preparation of p24 antibody conjugate in the form of liquid lyophilized comprises; a) adding sodium citrate solution (1-5%) to 0.01% -0.05% (w/v) gold chloride (HauCL4) at a boiling temperature; b) rapidly stirring and boiling the solution until a pink purple colour develops: c) adding p24 antibody to the solution; d) adding any non-immune animal serum such as mouse/goat/sheep/rabbit Imicrogram to 10 microgram/ml of gold colloidal solution:; e) mixing the solution for 2 hours to overnight; f) centrifuging the solution to discard the supernatant; g) Dissolving the pellet in buffer containing stabilizers like BSA, Gelatin or the like to obtain p24 antibody gold conjugate. 5. A Device as herein before described with reference to the accompanying drawings.

Documents:

2654-DEL-2006-Abstract-(26-09-2008).pdf

2654-del-2006-abstract.pdf

2654-DEL-2006-Assignment-(12-12-2011).pdf

2654-DEL-2006-Claims-(26-09-2008).pdf

2654-del-2006-claims.pdf

2654-DEL-2006-Correspondence Others-(12-12-2011).pdf

2654-DEL-2006-Correspondence-Others-(06-08-2008).pdf

2654-DEL-2006-Correspondence-Others-(26-09-2008).pdf

2654-del-2006-correspondence-others-1.pdf

2654-DEL-2006-Description (Complete)-(26-09-2008).pdf

2654-del-2006-description (complete).pdf

2654-del-2006-drawings.pdf

2654-del-2006-form-1.pdf

2654-del-2006-form-13-(06-08-2008).pdf

2654-DEL-2006-Form-16-(12-12-2011).pdf

2654-del-2006-form-18.pdf

2654-DEL-2006-Form-2-(26-09-2008).pdf

2654-del-2006-form-2.pdf

2654-DEL-2006-Form-26-(06-08-2008).pdf

2654-DEL-2006-Form-26-(26-09-2008).pdf

2654-del-2006-form-3.pdf

2654-del-2006-form-5.pdf

2654-DEL-2006-GPA-(09-02-2011).pdf

2654-del-2006-petition-137-(18-01-2011).pdf

2654-DEL-2006-Post Grant Opposition-(18-01-2011).pdf

2654-DEL-2006-Post-Grant -(26-11-2010).pdf

2654-DEL-2006-Post-Grant Opposition-(03-12-2010).pdf

2654-DEL-2006-Post-Grant Opposition-(09-02-2011).pdf

2654-DEL-2006-Post-Grant-Opposition-(04-01-2011).pdf

Patent-No-225308-1-Post-Grant Opposition-(09-02-2011).pdf