Title of Invention

A PROCESS AND A DEVICE THEREOF

Abstract

The present invention relates to a microlisa HIV (Ag & Ab) device for analyzing the presence of HIV 1 and HIV 2 antibodies and HIV 1 p24 antigen to HIV 1 in human serum and plasma. Also a process for making microlisa HIV (Ag & Ab) device for analyzing the presence of HIV 1 and HIV 2 antibodies and p24 antigen to HIV 1 in human serum and plasma has been described. The present invention also relates to a process for the analyzing the presence of HIV 1 and HIV 2 antibodies and p24 antigen to HIV 1 in human serum and plasma.

Full Text

FIELD OF THE INVENTION The present invention relates to a microlisa HIV (Ag & Ab) device for analyzing the presence of HIV 1 and HIV 2 antibodies and HIV 1 p24 antigen to HIV 1 in human serum and plasma. More specifically, the present invention relates to a process for making microlisa HIV (Ag & Ab) device for analyzing the presence of HIV 1 and HIV 2 antibodies and p24 antigen to HIV 1 in human serum and plasma. The present invention also relates to a process for the analyzing the presence of HIV 1 and HIV 2 antibodies and p24 antigen to HIV 1 in human serum and plasma. The Enzyme Linked Immunosorbant Assay (ELISA) is recommended as the screening method for the detection of HIV 1 & HIV 2 antibodies and p24 antigen to HIV-1. Microlisa HIV Ag & Ab is developed to detect antibodies to HIV -1 and / HIV 2 with equal reactivity; for and also antigen to HIV-1. Antigen can generally be detected in acute phase and during symptomatic phase of AIDS and antibodies can be detected throughout the infection. It is an in-vitro qualitative enzyme immunoassay for the detection on antibodies to HIV -1 and or HIV -2 and HIV1 antigen in human serum or plasma. It is intended for clinical diagnostic testing and screening of blood donors or other individuals at the risk for HIV-1 / HIV-2infection and HIV-1 antigen . The serological events following the HIV infection are represented graphically in figure 1. In individuals infected with HIV, antigen appears first before antibody of HIV but due to the seroconversion, the antigen is lost and antibody develops within 3-6weeks after infection and thereby the level of the antibody increases. Microlisa HIV Ag & Ab is a test device for the detection of both antigen and antibody to HIV. Antigen can generally only be detected during the acute and during the symptomatic phase of AIDS. Antibodies to HIV 1 and HIV2 can be detected throughout virtually the entire infection period. When the virus enters in to the body, the earlier detection of the disease is impossible because the virus enters in to a window phase and subsequently the immune system start producing antibodies. So the use of highly sensitive antibody assay 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. So the Microlisa HIV Ag & Ab detects both HIV1, HIV2 and HIV-1 antigens preferably p24 antigen in human serum and plasma. Antigens used for HIV 1 in the microtiter wells are a complex of recombinent proteins/ peptides, representing immunodominant epitopes, gp41and C terminus of gp120 .Similarly antigens used for HIV2 are recombinent protein/ peptides of gp 36. For HIV -1 P24 antigen detection, antibodies used are HIV -1 p24 antibodies. PRIOR ART: An HIV ELISA, [also called an HIV Enzyme ImmunoAssay (EIA)] is the first and most basic test to determine if an individual is positive for HIV. The most commonly used format for ELISA is microplate ELISA commonly known as "microelisa". Today, microelisa is one of the most reliable method of choice for screening blood and blood bank products for viral infectious and some bacterial infections. 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 diameter; 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. 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 env region of HIV1, HIV1-SubO and/or of HIV2 and at least one antibody against the pol and/or gag region of HIV1, HIV1-SubO and/or HIV2, reagent kits and test strips suitable for diagnostic procedure as well as monoclonal antibodies against p24 and their use. 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 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 7115363 relates to a new class of retroviruses, designated by HIV-2, of which samples have been deposited to the ECACC under numbers 87.01.1001 and 87.01.1002 and to the NCIB under numbers 12.398 and 12.399.lt relates also to antigens capable to be obtained from this virus, particularly proteins p12, p16, p26 and gp140. These various antigens can be used for the diagnosis of the disease, especially by contacting these antigens with a serum of a patient Submitted to the diagnosis. It relates to immunogenic compositions containing more particularly the glycoprotein gp 140. Finally it concerns neucleotidic sequences, which can be used especially as hybridization probes, derived from the RNA of HIV 2. US 6265149 directed toward in vitro methods for the detection of human immunodeficiency virus type 2 ( HIV 2) antibodies employing polypeptide fragments of HIV 2 Gag. These methods have sub optimal sensitivity and specificity and abundance of false positive in low prevalence population. These patents suffer from disadvantages like not user-friendly, time consuming, laborious and costly. It doesn't provide a method of detection of HIV for a man of ordinary skill to perform. The object of the present invention is to overcome the above said disadvantages and hence the present invention came. It is the object of the present invention to provide a diagnostic method for HIV which requires minimal amounts of labour and equipment. The test procedure is quite simple and rapid, i.e. the test can be completed within 30 minutes. It does not require any instrumentation and trained person. It is very economical. Another object of the present invention is to provide an inexpensive, novel, simple, rapid diagnostic device, which detects and differentiates HIV antigen and antibodies present in human serum and plasma. Yet another object of the present invention is to provide a test device to the man of ordinary prudence, which will take a short time to perform the test. Principle of the test The test is performed in a 8X12 wells which contains an 8x12 matrix of 96 wells , each of which are about 1 cm high and 0.7 cm in diameter. Microlisa HIV Ag & Ab test is an enzyme immunoassay based on "Sandwich ELISA" Figure 2 shows the principle. Recombinant protein/ peptides gp41 and C terminus of gp 120 are for HIV-1 immunodominant epitopes of gp 36 are for HIV -2 and p24 antibodies for the detection of HIV -1 p24 antigen are coated on to microtiter wells. Specimens and controls are added to the microtiter wells followed by addition of enzyme conjugate. HIV 1 & 2 antigen and HIV -1 p24 antibodies linked with HRPO. ( HRPO is an enzyme, Horse Radish Per Oxide). A sandwich complex is formed in the well where in HIV -1 or HIV 2 antibodies or HIV 1 p24 antigen ( from serum sample) is sandwiched between the antigens and antigens HRPO and antibody and antibody HRPO conjugate. ( antibodies in the sample are sandwiched between the antigens in the microwells and antigen HRPO conjugate and HIV-1 p24 antigens are sandwiched in between the antibodies of HIV-1 p24 in the micro wells and antibodies HRPO conjugate). The plate is then washed to remove unbound material. Finally substrate solution containing chromogen and hydrogen peroxide is added to the wells and incubated. A blue colour will develop in proportion to the amount of HIV-1and HIV-2 antibodies and HIV-1 p24 antigen present in the specimen. The colour reaction is stopped by a stop solution. The enzyme substrate reaction is read by EIA reader for absorbance at a wavelength of 450 nm. If the sample does not contain HIV- 1 or HIV-2 antibodies or HIV-1 p24 antigen, then enzyme conjugate will not bind and the solution in the wells be either colourless or only a faint background colour develops. Interpretation of the result After adding the conjugate , if the sample is blue in colour then the patients' sample is reactive for HIV. If there is no colour to the solution, then the sample is not reactive to the HIV. Stop solution such as acids added to stop the reaction. After adding the stop solution, if the solution is yellow in colour, the sample is reactive for HIV and if there is no yellow colour, then the sample is not reactive for HIV. Samples Reagents and the samples should be at room temperature before beginning the assay Only human serum or plasma should be used for the test. Fresh serum or plasma samples are preferred. While preparing the samples, remove the serum from the clot as soon as possible to avoid heamolysis. Specimens should be free of microbial contamination and may be stored at 2 to 8°C for one week, or frozen at 20°Cor lower. Repeated freezing and thawing should be avoided. Frozen sample Microlisa HIV Ag& Ab test is best used with fresh samples that have not been frozen and thawed. However the most frozen samples will perform well if the following procedure suggested is followed. Allow the frozen samples to thaw in a vertical position in the rack. Don't shake the sample. This allows the particles to settle in the bottom. The sample should be centrifuged. (Preparation of working chromoqeneic substrate solution( TMB) Dilute TMB concentrate in the ratio 1:100 in substrate, i.e. TMB diluent. The 100X solution may crystallize during storage. If crystals are present, resolubilize by warming at room temperature. TMB Concentrated OOX) TMB means 3,3,5,5,Tetra Methyl Benzedene, which is dissolved in Dimethyl sulphoxide which is used as indicator. TMB Diluent Buffer solution containing substrate is the TMB Diluent. Stop solution Acids are the stop solution. Process of the test All the reagents should be dispensed in the center of the well. 1. Leave A-1 well as blank which is to test the quality of the Substrate. 2. Add 50 (it negative control in each well No. B.1 and C-1 respectively. Negative control is added to the well to perform the negative test.( 3. Add 50 (.tl positive control -1 (PC -1 ) in D-1, E-1 & F-1 wells which is to test the performance of the antibody to HIV1 and HIV 2.. 4. Add 50 ^l positive control 2 (PC-2 ) in G-1 well to test the performance of HIV-1 p24 antigen. 5. Add 50 (il sample in each well, starting from H-1 well. 6. Add 100 \JL\ of working enzyme conjugate to each well except A1. Gently shake the plate for 2-3 seconds to mix the sample & conjugate. 7. Cover the plate and allow to incubate in an incubator at 37°C +_1 °C for 60 minutes. 8. Add wash buffer solution to the well. 9. Add 100 fii chromogenic 1MB substrate to the solution. 10. Incubate it in dark at room temperature for 30 minutes 11. At the end of the incubation period, add the stop solution. 12. Add stop solution to the well to stop the reaction and read the result afterSO minutes. According to the present invention a microlisa HIV (Ag & Ab) device for analyzing the presence of HIV 1 and HIV 2 antibodies and HIV 1 p24 antigen to HIV 1 in human serum and plasma comprises 8 x 12 matrix of 96 wells about one centimeter high and 0.7 centimeter in diameter characterized in that the recombinant proteins gp41, C terminus of gp120 for HIV 1 and gp36 for HIV 2 representing immunodominant epitopes and HIV 1 p24 antibodies are added to the microtiter wells wherein specimens and controller are added to the microtiter followed by addition of enzyme conjugate In an embodiment of the present invention a process for making microlisa HIV (Ag & Ab) device for analyzing the presence of HIV 1 and HIV 2 antibodies and p24 antigen to HIV 1 in human serum and plasma comprises the following steps: a. dissolving dilute HIV 1 gp 41 and gp 120 in the ration of 200 to 800 nano grams in a buffer solution and followed by mixing it thoroughly; b. adding to the mixed solution 200-800 nano grams of HIV gp 36 and p24 antibodies; c. stirring the solution at 50-100 rpm in a magnetic stirrer to obtain a homogenous solution ; d. dispensing the solution in the wells in the range of 50 - 200 jil; e. incubating the wells overnight at 2-10 deg. C; f. washing the wells with the buffer solution of pH 7.2 to 7.6 at least 4 to 5 times g. blocking the washed wells with protein solution made in 10% BSA solution; h. removing the unutilized solution by inverting the wells and drying the wells at room temperature thus obtaining the device. In another embodiment of the present invention, the enzyme conjugate is HIV1 and HIV2 antigens and HIV1 p24 antibodies linked with an enzyme such as HRPO prepared by the following steps: a. making three different solutions of HIV1 antigen, HIV2 antigen and HIV1 p24 antibodies separately with an 20 mg of enzyme such as HRPO dissolved in 1ml of distilled water; b. taking 0.08 molar per iodide and mixing in the ration 1:10 with HRPO; c. shaking and leaving the mixture at room temperature for 60 minutes; d. mixing with the activated HRPO solution obtained with 10 mg of HIV 1 antigen or 10 mg of HIV 2 antigen or 10 mg HIV! p24 antibodies. e. allowing the solution to maturize for 2 hours at ambient temperature; f. adding 1:20 ratio of borohydride solutionwith the above solution and leaving it to maturize for 30 minutes at ambient temperature; allowing the reaction between antigens and antibodies with HRPO; g. adding 1:20 ration ethanol Amine; h. allowing the solution to maturize for 30 minutes followed by dialyzing i. mixing these three solution obtained of HIV1 antigen, HIV2 antigen and HIV1 p24 antibodies and allowing it to maturize for one week j. adding buffer solution i.e. BSA in PBS at the percentage 5% to 10% thus obtaining the enzyme conjugate In yet another embodiment of the present invention, a process for analyzing the presence of HIV 1 and HIV 2 antibodies and p24 antigen to HIV 1 in human serum and plasma comprises: a. leaving the uppermost corner well A as blank to test the quality of the substrate; b. adding 50 j^l negative control to each well adjacent to the first well vertically represented by B1 and C1 to perform the negative test; c. adding 50 |J positive control in next wells vertically below the negative control well represented by D1, E1 and F1 to test the performance of antibody to HIV 1 and HIV 2; d. adding 50 ^l positive control PC2 in the next well to the F represented by G1 to test the HIV1 p24 antigen; e. adding 50 (.il sample to rest wells starting from the well H to all wells; f. adding 100 ^l of working enzyme conjugate to each well except A1 followed by shaking the plates for 2-3 seconds to mix the sample and conjugate; g. covering the plate; h. incubating the covered plate in an incubator at 37 deg. C. ± 1 deg. C. for 55 to 65 minutes; i. adding wash buffer solution to the wells j. adding 100 |j,l chromogenic 1MB substrate to the solution; k. incubating the plates in the dark room at ambient temperature for 30 minutes; I. adding the stop solution after the completion of the incubation period to stop the reaction; m. reading the results after 30 minutes In still another embodiment of the present invention, the said negative control (-) is normal human sera diluted in saline solution which is negative for HIV, HCV and HBV and the positive control (1+) is human sera diluted in saline solution which is positive to antibodies of HIV1, and negative for HCV and HBV. In yet another embodiment of the present invention, the positive control (2+) is a recombinant HIV 1 p24 antigen diluted in saline solution. The invention will be described with reference to the following accompanying drawings wherein: 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. Figure 2 shows the various stages of coating of antigens and antibodies wherein 1: Coated antigens of HIV-1 and HIV-2 and p24 antibodies on the well 2: Antibodies of HIV and p24 antigen in the serum/ plasma 3.: Attachment of antigen and antibodies. 4: Addition of HIV Antigen and p 24 linked to HRPO ( enzyme conjugate) 5: Addition of Chromogenic Substrate and stop solution and final reading of result at 450 nm/630 nm. Figure 3 is the microtiter. A more complete appreciation of the invention and the attendant advantage will be more clearly understood by reference to the following nonlimiting example which is for illustrative purpose and the same should not be construed to restrict the scope of the invention. Example 1: Preparing the enzyme conjugate: Three different solutions of HIV1 antigen, HIV2 antigen and HIV1 p24 antibodies were made separately with an 20 mg of enzyme such as HRPO dissolved in 1ml of distilled water followed by taking 0.08 molar per iodide and mixing in the ration 1:10 with HRPO; shaking and leaving the mixture at room temperature for 60 minutes; mixing with the activated HRPO solution obtained with 10 mg of HIV 1 antigen or 10 mg of HIV 2 antigen or 10 mg HIV 1 p24 antibodies; allowing the solution to maturize for 2 hours at ambient temperature; adding 1:20 ratio of borohydride solutionwith the above solution and leaving it to maturize for 30 minutes at ambient temperature; allowing the reaction between antigens and antibodies with HRPO; adding 1:20 ration ethanol Amine; allowing the solution to maturize for 30 minutes followed by dialyzing, mixing these three solution obtained of HIV1 antigen, HIV2 antigen and HIV1 p24 antibodies and allowing it to maturize for one week adding buffer solution i.e. BSA in PBS at the percentage 9% to obtain the enzyme conjugate Example 2 The uppermost corner well A was left as blank to test the quality of the substrate; followed by adding 50 nl negative control to each well adjacent to the first well vertically represented by B1 and C1 to perform the negative test; adding 50 nJ positive control in next wells vertically below the negative control well represented by D1, E1 and F1 to test the performance of antibody to HIV 1 and HIV 2; adding 50 jal positive control PC2 in the next well to the F represented by G1 to test the HIV1 p24 antigen; adding 50 ^l sample to rest wells starting from the well H to all wells; adding 100 (.il of working enzyme conjugate to each well except A1 followed by shaking the plates for 2-3 seconds to mix the sample and conjugate; covering the plate; incubating the covered plate in an incubator at 37 deg. C. + 1 deg. C. for 58 minutes; adding wash buffer solution to the wells; adding 100 jal chromogenic 1MB substrate to the solution; incubating the plates in the dark room at ambient temperature for 30 minutes; adding the stop solution after the completion of the incubation period to stop the reaction; and the results were read. Example 3 Dilute HIV 1 gp 41 and gp 120 were dissolved in the ration of 300 nano grams in a buffer solution and followed by mixing it thoroughly and adding to the mixed solution 400 nano grams of HIV gp 36 and p24 antibodies followed by stirring the solution at 70 rpm in a magnetic stirrer to obtain a homogenous solution; dispensing the solution in the wells in the range of 100 jil; incubating the wells overnight at 6 deg. C; washing the wells with the buffer solution of pH 7.2 to 7.6 at least 4 to 5 times; blocking the washed wells with protein solution made in 10% BSA solution; removing the unutilized solution by inverting the wells and drying the wells at room temperature to obtain the device. I Claim: 1. Microlisa HIV ( Ag and Ab) Device for analyzing the presence of HIV1 and HIV 2 antibodies and p24 antigen to HIV1 in human serum and plasma comprising 8 X 12 matrix of 96 wells about one centimeter high and 0.7centimeter in diameter characterized in that the recombinant proteins gp 41, C terminus of gp 120 for HIV1 and gp 36 for HIV 2 representing immunodominant epitopes and HIV 1 p24 antibodies are added to the microtiter wells. 2. A process for making Microlisa HIV (Ag & Ab) device comprising the following steps: Dissolve HIV 1 antigen glycoprotein gp 41 and glycoprotein gp 120 in the ratio 200 to 800 nanograms/ ml in a buffer solution followed by mixing it thoroughly, to the above solution, add 200-800 ng/ ml of HIV antigen glycoprotein 36 and anti HIV p24 antibodies to HIV 1, stirring the solution at 50- 200 rpm in a magnetic stirrer to obtain a homogeneous solution, dispense the solution in the wells in the range of 50 to 200 µl, incubate the wells overnight at 2 to 10°C, wash the wells with a buffer solution of pH 7.2 to 7.6 at least 4 to 5 times, then block the washed wells with protein solution made in 10 % BSA solution and remove the unutilized solution by inverting the wells and drying the wells at room temperature thus obtaining the device. 3. A process as claimed in claim 2 where in the concentration of the antigens and the antibodies used are 200 to 800 nano grams. 4. A process as claimed in claim 2 where in the concentration of coating on the wells are 50 to 200µl. 5. A device as claimed in claim 1 where in the enzyme conjugate is HIV 1 and HIV2 antigens and HIV1 p24 antibodies linked with HRPO is prepared by the following steps: a. making three different solutions of HIV 1 antigen , HIV 2 antigen and HIV1 p24 antibodies separately with 2mg of enzyme such as HRPO dissolved in 1 ml of distilled water; b. taking 0.08molar per iodide and mixing in the ratio 1:10 with HRPO. c. shaking and leaving the mixture at room temperature for 60 minutes; d. mixing with the activated HRPO solution obtained with 10 mg of HIV 1 antigen or 10 mg of HIV 2 antigen or 10 mg of HIV 1 p24 antibodies, e. allowing the solution to maturize for 2 hrs at ambient temperature, f. adding 1:20 ratio of borohydride solution with the above solution and leaving it to maturize for 30 minutes at ambient temperature; allowing the reaction between antigens and antibodies with HRPO; g. adding 1:20 ratio Ethanol Amine; h. allowing the solution to maturize for 30 minutes followed by dialyzing-i. mixing these three solution obtained of HIV 1 antigen , HIV 2 antigen and HIV 1 p24 antibodies and allowing it to maturize for one week; j. adding buffer solution i.e. BSA in PBS at the percentage 5% to 10% thus obtaining the enzyme conjugate. 6. A microlisa HIV (Ag & Ab) device for analyzing the presence of HIV 1 and HIV 2 antibodies and HIV 1 p24 antigen to HIV in human serum and plasma as here in before described with reference to the accompanying drawings.

Documents:

2690-del-2006-1-Petition-137-(18-01-2011).pdf

2690-DEL-2006-1-Post Grant Opposition-(18-01-2011).pdf

2690-del-2006-abstract.pdf

2690-DEL-2006-Assignment-(01-11-2011).pdf

2690-DEL-2006-Assignment-(05-08-2011).pdf

2690-DEL-2006-Assignment-(10-10-2011).pdf

2690-del-2006-Assignment-(30-12-2009).pdf

2690-DEL-2006-Claims-(23-09-2008).pdf

2690-DEL-2006-Claims-(29-09-2008).pdf

2690-del-2006-claims.pdf

2690-DEL-2006-Correspondence Others-(01-11-2011).pdf

2690-DEL-2006-Correspondence Others-(05-08-2011).pdf

2690-del-2006-Correspondence-Others-(02-02-2011).pdf

2690-DEL-2006-Correspondence-Others-(18-01-2011).pdf

2690-DEL-2006-Correspondence-Others-(23-09-2008).pdf

2690-del-2006-Correspondence-Others-(30-12-2009).pdf

2690-del-2006-correspondence-others-1.pdf

2690-DEL-2006-Description (Complete)-(23-09-2008).pdf

2690-del-2006-description (complete).pdf

2690-del-2006-drawings.pdf

2690-del-2006-form-1.pdf

2690-del-2006-form-13-(29-10-2007).pdf

2690-del-2006-Form-16-(30-12-2009).pdf

2690-del-2006-form-18.pdf

2690-DEL-2006-Form-2-(23-09-2008).pdf

2690-del-2006-form-2.pdf

2690-del-2006-form-3.pdf

2690-del-2006-form-5.pdf

2690-DEL-2006-Post-Grant -(26-11-2010).pdf

2690-DEL-2006-Post-Grant Opposition-(03-12-2010)-.pdf

2690-DEL-2006-Post-Grant Opposition-(03-12-2010).pdf

2690-DEL-2006-Post-Grant-Opposition-(04-01-2011).pdf