FORM 2
THE PATENT ACT 1970 (39 of 1970)
&
The Patents Rules, 2003 COMPLETE SPECIFICATION
See Section 10, and rule 13)
1. TITLE OF INVENTION
A RAPID DIAGNOSTIC TEST KIT

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

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

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FIELD OF INVENTION
Present invention relates to a rapid diagnostic test kit for detection human immunodeficiency virus (HIV). It more particularly relates to flow through rapid diagnostic test kit in portable cassette for rapid serological assays of HIV-1 and HIV-2 antibodies in human blood serum or plasma within no time/ that means on the same day.
BACKGROUND OF THE INVENTION
Significant number of population in the world is being infected regularly with HIV. Patients suffering from HIV infection show the presence of different anti-HIV antibodies in blood serum and plasma. Among the various antigens, antibodies to glycoprotein 41 of HIV-1 (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.
PRIOR ART OF THE INVENTION
At present, different detection methods described herein after are used and mainly based on presence of HIV antibodies in blood human serum or plasma as subsequent to viral infection, serological analysis ensures the presence of said antibodies in the blood.
The said viral protein are currently detected or screened using a Enzyme Linked Immunoassays (ELISAs) and requires further confirmation of its results by Western-Blot method. However, continued concerns about false-positive screening results led to the implementation of a sequential two-test algorithm, comprising an ELISA screening test followed by Western-Blot or immunofluorescence assay as a supplemental test, to confirm HIV positively. The recommended tests such as ELISA and Western-blot require specialized equipment and technical expertise, and they cannot be completed in less than 24 hours. In practice, given the time necessary
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to transport specimens to a laboratory, perform the tests in batches, and transmit test results typically must requires 1-2 weeks before to learn test results.
ELISA and Western-Blot were not feasible for small laboratories in many developing countries like India where resources are limited and electricity may not be consistently available. As discussed above, these tests require many hours to perform, refrigeration, and sophisticated, expensive equipments. In view of above requirements, time constraint, and shortfalls, the tests described herein before require considerable investment to set up laboratory and thereby proved to be cost-intensive.
Compared to that a rapid HIV tests using a rapid test kits demonstrate sensitivities and specificities comparable to those of ELISA, currently used for screening and detection of HIV antibodies. Algorithms comprised of a combination of two or more rapid test methods produce HIV test results with predictive values comparable to those of the ELISA-Western-Blot combination. The rapid HIV tests offer additional advantages of low cost and same-day results and are likely to gain increasing acceptance for HIV screening and diagnosis in both developed and developing countries.
After due consideration of drawbacks of earlier test methods and advantages offered by the rapid test methods, number of simple, cost-effective rapid assays kits emerged to meet the demand in such countries those can not offered cost-intensive methods such as herein before described, both for blood screening and diagnosis. Numerous studies demonstrated that alternative confirmatory strategies using algorithms with combinations of screening tests produced reliable results, comparable to those of the standard ELISA and Western blot.
Screening and detection with combinations of rapid HIV tests proved to be less expensive than the ELISA/Western-Blot algorithm and also made it possible to offer same-day test results. The lower cost made these rapid assay methods and kits more acceptable and feasible for developing countries such as India for knowing serostatus of individuals.
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Although more than 60 rapid HIV tests and kits have been developed and used in various countries/ only 2 have received approval from the Food and Drug Administration (FDA) for use in the United States. The first is Recombigen HIV-1 LA (a latex agglutination test) and second one is SUDS (Single Use Diagnostic System for HIV-1) . The Recombigen was withdrawn from the U.S. market because of poor performance due to difficulty distinguishing reactive test results from the background granularity of the latex particles. The SUDS remains commercially available in the United States and few other developed countries.
Although the said rapid assay kit was available in US and other developed countries, it was almost not available in the markets of developing countries such as India due to its higher cost. Another reason could be lack of required sensitivity for HIV-1 (Group M and 0) and HIV-2 predominantly being detected in most of the Asian countries.
In India, the rapid test kits are being manufactured by J. Mitra & Co. , SD Diagnostics, Embee Diagnostics, etc. These test kits are based on detection of IgG antibodies specific to a set of HIV antigens. All of them are too costly and lacking required sensitivity for HIV-1 (Group M and 0) antigens. Therefore, it is felt necessary to develop a improved serological test kit for the detection and diagnosis of, HIV-1 (Group M and 0) and HIV-2 antibodies and HIV-1 and HIV-2 infection, respectively.
OBJECT OF THE INVENTION
In view of above, it was object of the present invention to prepare the rapid diagnostic test kit for detection HIV.
Further, object of the invention is to provide a flow-through rapid diagnostic test kit in portable cassette for the rapid serological assay of HIV-1 and/or HIV-2 antibodies.
Another, object of the invention is to specifically design and develop a reliable and cost-effective flow-through rapid diagnostic test kit for screening and detection of HIV-1 (Group M and 0) and/or HIV-2 antibodies in blood serum and/or plasma of HIV infected patients.

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Still, another object of the present invention is a cost-effective process for manufacturing a improved quality rapid diagnostic test kit for screening and detection of HIV-1 and/or HIV-2 antibodies in the blood serum and/or plasma.
Still/ further object of the present invention is to propose a serological laboratory method of detection of HIV-1 and/or HIV-2 antibodies in blood samples of HIV-1 and HIV infected patients.
SUMMARY OF THE INVENTION
Accordingly, in the present invention the rapid diagnostic test kit in potable cassette based on membrane immune-concentration technique employing solid-phase capture technology for the rapid detection of HIV-1 and/or HIV-2 antibodies in blood serum and/or plasma of the infected person is provided. In the present invention also provided are the method of manufacturing the said rapid kit and detection of HIV-1 and/or HIV-2 infection using the said kit.
DESCRIPTION OF THE INVENTION
The rapid diagnostic test kit in potable cassette having a absorbent pad paced in square-shape base and a top cover detachably fitted in such manner that the overall cassette is air tight, wherein the top cover has central situated square-shape window in exact confirmation to the that of square-shape immuni-filtration membrane of the said kit, so as to easily load the biological sample characterized in that the rapid test kit comprises an immune-absorbent matrix that is designed to immobilize plurality dot coatings of recombinant glycoprotein antigens of HIV-1 and HIV-2 and also goat anti-human IgG, wherein three circular coatings are loaded to immobilize recombinant glycoproteins of
HIV-1 and HIV-2 and goat anti-human IgG for the detection of antibodies raised against the said HIV antigens in the blood serum and/or plasma of the infected person.
The said rapid diagnostic kit of the invention further comprises assay buffer, anti-HIV-positive control, anti-HIV-negative control and colloidal conjugate.
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DETAILED DESCRIPTION OF THE INVENTION
In the preferred embodiment of the invention, the said immuno-adsorbent membrane is immuno-filtration membrane.
In the most preferred embodiment of the invention, the said immuno-filtration membrane is porous matrix to allow flows of specimen through and absorbed into it.
In still most preferred embodiment of the invention, the said immuno-filtration membrane is nitrocellulose membrane of pore size 0.45 urn.
In another embodiment of the invention, the said recombinant glycoproteins are HIV-1, Group M, HIV-1, Group 0 and HIV-2 antigens.
In the most preferred embodiment of the invention, the said recombinant antigens are glycoprotein 41 of HIV-1 (gp41) and glycoprotein 36 of HIV-2 (gp36).
In another embodiment of the invention, the said goat anti-human IgG is spotted and immobilized as a control to confirm the working condition of the said kit.
In another embodiment of the invention, the said assay buffer comprises TRIS buffer along with animal protein, urea, salt, amino acids, Tween-20 and antibiotics. (Please check these ingredients)
In another embodiment of the invention, the said anti-HIV-positive control is anti-HIV-positive human serum with serum stabilizer in appropriate or suitable proportion.
In another embodiment of the invention, the said anti-HIV-negative control is normal human serum with serum stabilizer in appropriate or suitable proportion.
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In another embodiment of the invention, the said colloidal conjugate is concentrated Protein A colloidal gold conjugate with conjugate stabilizer in appropriate or suitable proportion.
The rapid test kit involves a flow of fluid containing the analyte through a porous membrane and into an absorbent membrane pad. A second layer, or sub-membrane, inhibits the immediate back-flow of fluids, which can obscure results. These tests can be used to detect both antibodies and antigens. To detect antibodies and antigens, the corresponding analyte is bound or immobilized as a dot or line on the membrane. This reagent "captures" the analyte as it flows through the membrane. To perform the test, a sample is applied to the membrane and allowed to wick through by capillary action. Thereafter, sequentially, there is a wash step, addition of the signal reagent, and a second wash to clear the membrane. The solutions can be added as rapidly as the previous liquids are absorbed into the cassette's membrane.
Most of the rapid assay kits not require reagent and other specialized equipment. The three most common generic assay formats uses so far are: (i) membrane immuno-concentration assay (flow-through) (ii) immuno-chromatographic (lateral-flow) assay and (iii) particle agglutination assay.
The present invention is mainly based on the first principle of membrane immuno-concentration assays, which employs solid-phase capture technology. It involves the immobilization of HIV antigens on a porous matrix. The specimen flows through the said matrix or membrane and is absorbed into an absorbent pad. A dot or a line visibly forms on the membrane when developed with a signal reagent (usually a colloidal gold or selenium conjugate). The flow-through tests require steps for the addition of specimen, wash buffers, and signal reagent, and they can usually be completed in 5 to 15 minutes. Most are used with serum or plasma and some are equipped with a filter to allow the use of whole-blood specimens.
Brief description of other two assay principles, namely, the immuno-chromatographic (lateral-flow) and particle agglutination is disclosed herein after as references in its entirety. The immuno-chromatographic assay, the recent development, potentially requires only one step and incorporates both antigen and signal reagent into a nitrocellulose strip. The specimen is applied to
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an absorbent pad from which it is wicked/ combines with signal reagent/ and migrates through the strip. A positive reaction results in a visual line on the membrane where HIV antigen has been applied.
The particle agglutination assays typically requires 10 to 60 minutes or more and must be used with serum or plasma. When a patient specimen containing HIV antibodies is mixed with minute HIV antigen-coated latex particles, cross linking occurs and results in agglutination.
In the case of the present invention, more Particularly, in the form of three circular dots/coating, the recombinant gp41 of HIV-1, the recombinant gp36 of HIV-2 and goat anti-human IgG are immobilized separately on the said nitrocellulose membrane as a matrix placed over absorbent pad fitted in portable cassette. First addition of assay buffer is required to block the free site of the membrane. When human blood serum and/or plasma is added to the membrane, the bound HIV-1 and/or HIV-2 antigens will form a stable immune complex if, anti-HIV-1 and/or HIV-2 antibodies are present in the specimen. However, goat anti-human IgG:Human IgG complex will appear in all the cases to checking the working condition of the said kit as a control spot. Followed by wash step, Protein A colloidal gold conjugate is added to the membrane. A third wash step will remove free unbound Protein A colloidal gold reagent from the membrane. The nitrocellulose membrane containing anti-HIV-negative control sample will show one pungent/wine red colour spot and the test specimen containing antibodies to gp41 of HIV-1 and/or gp36 of HIV-2 will show additional pungent/wine red colour spot(s). The intensity of pungent-red colour is directly proportional to the amount of bound anti-HIV antibody to the membrane.
According to another object of the present invention/ the method for manufacturing the flow-through rapid diagnostic test kit for rapid serological screening and detection of HIV-1 and HIV-2 antibodies in human blood serum and/or plasma for HIV infection is provided. The method comprises immobilizing the recombinant glycoprotein HIV antigens, gp41 of HIV-1 (Group M and 0) and gp36 of HIV-2 and goat anti-human IgG in three circular dot coatings on porous immuno-filtration matrix such as nitrocellulose to employ solid-phase capture technique for immuno-concentration membrane.
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For the said process, it comprises, preparing separately three coating solutions/ namely Solution I/containing gp41 of HIV-1/ Group M and 0 (1 mg/ml) , Solution 2, containing gp36 of HIV-2 (1 mg/ml) and Solution 3, containing goat anti-human IgG (0.5 mg/ml) and thereafter 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 of the invention, the process comprises preparing solution of Protein A colloidal gold conjugate with conjugate stabilizer and thereafter dilute the conjugate in stabilizer in appropriate or suitable proportion.
In another embodiment of the invention, the process comprises preparing the solution of anti-HIV-positive control by mixing the anti-HIV-positive human serum with serum stabilizer in appropriate or suitable proportion.
In another embodiment of the invention, the process comprise preparing the solution of anti-HIV-negative control by mixing the serum normal human with serum stabilizer in appropriate or suitable proportion.
In another embodiment of the invention, the process comprise providing TRIS buffer, animal protein, urea, salts amino acids, Tween-20 and antibiotics.
According to another object of the present invention, the serological method for in vitro detection of antibodies of antigens gp41 of HIV-1 and/or gp36 of HIV-2 in human blood serum and/or plasma is provided. The process comprise: adding first the assay buffer to block the free site of the membrane, loading the human blood serum and/or plasma on to the immuno-filtration membrane thereby detecting the bound HIV-1 and/or HIV-2 antigens by forming a stable antigen-antibody complex, if anti-HIV-1 and/or HIV-2 antibodies are present, checking working condition of said kit by detecting goat anti-human IgG:Human IgG complex as a control, washing the immuno-filtration membrane containing immune complex(s)with Protein A colloidal gold conjugate, washing further with buffer to remove free unbound Protein A colloidal gold
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reagent and detecting the pungent/wine red colour spot. If, the nitrocellulose membrane containing negative control sample, shows only one pungent/wine red colour spot and if, test specimen containing antibodies to gp41 of HIV-1 and/or gp36 of HIV-2 shows one and/or two additional pungent/wine red colour spot(s). The intensity of pungent red colour is directly proportional to the amount of bound anti-HIV antibody.
The schematic presentation of the immuno-filtration matrix of the said kit is illustrated in figure 1 of the accompanying drawing, wherein the immuno-filtration matrix (1) is paced over absorbent pad (2) and over the said immuno-filtration matrix (1), the anti-human IgG and recombinant antigens gp41 of HIV-1 and gp36 of HIV-2 is immobilised as coatings (3), (4) and (4).
Advantages of the present invention include a very rapid test procedure/ with results available in as few as 3 to 5 minutes/ but the tests need to be performed individually or in small batches of 5 and require constant attention.
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.

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WE CLAIM:
1. A rapid diagnostic kit in portable cassette for detecting HIV infection having a absorbent pad paced in square-shape base and a top cover detachably fitted in such manner that the overall cassette is air tight/wherein the top cover has central situated square-shape window in exact confirmation to the that of square-shape immuno-filtration membrane of the kit characterized in that the kit comprises an immuno-absorbent matrix that is designed to immobilize plurality dot coatings of recombinant glycoprotein antigens of HIV-I/ and HIV-2 and goat anti-human IgG/ wherein three circular coatings loaded to immobilize recombinant glycoproteins of HIV-I/and HIV-2 and goat anti-human IgG for detection of antibodies raised against the said HIV antigens in the blood serum and/or plasma of infected person; and further comprises assay buffer/ anti-HIV-positive control, anti-HIV-negative control and colloidal conjugate.
2. The rapid test kit as claimed in claim 1/ wherein the immuno-adsorbent matrix is an immune-filtration membrane.
3. The rapid test kit as claimed in claim 1 to 1, wherein the immuno-filtration membrane is a porous.
4. The rapid test kit as claimed in claim 1 to 3, wherein the immuno-filtration membrane is a nitrocellulose membrane of pore size 0.45 urn.
5. The rapid test kit as claimed in claim 1, wherein the recombinant lypoprotein is HIV-1 antigen of Group M and Group 0.
6. The rapid test kit as claimed in claim 1, wherein the recombinant lypoprotein is HIV-2 antigens.
7. The rapid test kit as claimed in claim 1 and 5, wherein the recombinant antigens is glycoprotein 41 of HIV-1 (gp41).
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8. The rapid test kit as claimed in claim 1 and 6, wherein the recombinant antigens is glycoprotein 36 of HIV-2 (gp36).
9. The rapid test kit as claimed in claim 1, wherein the assay buffer comprises TRIS buffer along with animal protein, urea, salt, amino acids, Tween-20 and antibiotics.
10. The rapid test kit as claimed in claim 1, wherein the anti-HIV-positive control is anti-HIV human serum with serum stabilizer in appropriate proportion.
11. The rapid test kit as claimed in claim 1, wherein the anti-HIV-negative control is normal human serum with serum stabilizer in appropriate proportion.
12. The rapid test kit as claimed in claim 1, wherein the colloidal conjugate is concentrated Protein A colloidal gold conjugate with conjugate stabilizer in appropriate proportion.
13. A method for manufacturing flow-through rapid diagnostic test kit for rapid detection of HIV infection comprises immobilizing recombinant glycoprotein HIV antigens, gp41 of HIV-1 (Group M and 0), and gp36 of HIV-2 and goat anti-human IgG in three circular dot coatings on porous nitrocellulose immune-concentration membrane/-and providing solutions of Protein A colloidal gold conjugate, anti-HIV-positive control, anti-HIV-negative control and assay buffer.
14. The method as claimed in claim 13/ comprises preparing coating solutions of gp41 and gp36 by separately mixing 1 mg in 1 ml solvent.
15. The method as claimed in claim 13, comprises preparing coating solution of goat anti-human IgG by mixing 0.5 mg in solvent.
16. The method as claimed in claim 13, comprises preparing solution of Protein A colloidal gold conjugate with conjugate stabilizer in appropriate proportion.
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17. The method as claimed in claim 13, comprises preparing solution of anti-HIV-positive control by mixing anti-HIV human serum with serum stabilizer in appropriate proportion.
18. The method as claimed in claim 13/ comprises preparing solution of anti-HIV-negative control by mixing normal human serum with serum stabilizer in appropriate proportion.
19. The method as claimed in claim 13/ comprises providing TRIS buffer, animal protein urea, salts amino acids, Tween-20 and antibiotics.
20. A method for in vitro detection of antibodies of gp41 of HIV-1 and/or gp36 of HIV-2 comprise adding first assay buffer to block the free site of the membrane; loading human blood serum and/or plasma on to the immuno-filtration membrane thereby detecting the bound HIV-1 and/or HIV-2 antigens by forming a stable antigen-antibody complex; checking working condition of said kit by detecting goat anti-human IgG : Human IgG complex as a control; washing immuno-filtration membrane containing immune complex(s)with Protein A colloidal gold conjugate;washing further with buffer, to remove free unbound Protein A colloidal gold reagent and detecting the pungent/wine red colour spot.
Dated this 4th day of July, 2005.

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