Title of Invention

A KIT FOR ANALYZING THE PRESENCE OF MYCOBACTERIUM TUBERCULOSIS BACILLI ANTIGEN.

Abstract

The present invention relates to a kit for analyzing the presence of Mycobacterium Tuberculosis bacilli antigen in a sample and a device for rapid and easy test method for detection of presence of Mycobacterium Tuberculosis bacilli antigen by reaction with very specific monoclonal or polyclonal antibody directed against the device membrane. A built in control dot is provided to validate the efficacy of the reagents used in the test and to validate the compliance of the procedural steps.

Full Text

FIELD OF THE INVENTION The present invention relates to a process for analyzing the presence of Mycobacterium Tuberculosis bacilli in a sample and a device for rapid, and easy test method for detection of presence of Mycobacterium Tuberculosis bacilli by reaction with very specific monoclonal or polyclonal antibody directed against them. This complex is made to react with conjugate to obtain a visual colour on the device membrane. A built in control dot is provided to validate the efficacy of the reagents used in the test and to validate the compliance of the procedural steps. BACKGROUND OF THE INVENTION Tuberculosis is a persistent problem in the developing world and biggest cause of morality. Mycobacterium tuberculosis is the causative agent of tuberculosis. Mycobacterium tuberculosis infections often results in pulmonary disease but can cause skin infections, lymphadenitis, meningitis, and other manifestations beside advent of AIDS has made the disease a major public health problem which has recently been exacerbated by increasing numbers of high-risk patients. For more than a Century detection of Mycobacterium tuberculosis generally relieves on a combination of staining of acid fast bacilli (AFB) and culture. AFB staining is unable to identity the species of different mycobacterium while culture needs 6 weeks time for result interpretation. So, inadequacies of the sputum smear test and the rise of TB epidemic have renewed efforts to develop new and innovative approaches to TB diagnosis. Diagnos TB Ag kit has been developed as a rapid direct antigen diagnostic tests which demonstrate a superior performance against the widely used sputum smear test and the current gold standard culture method.. As Diagnos TB Ag kit is an antigen based test, it has several advantages over antibody based test. Firstly, they don't rely on the host immune system to detect disease - often problematic when the host is immune comprised. Secondly, they can measure concentrations of pathogen in real time in the host, which can then be directly related to disease progression. Also, the field of the diagnostic and clinical microbiology has continued to evolve and yet there remains a general need for systems that provide rapid and reliable detection of the diseases and infection due to microorganisms such as M. Tuberculosis. There is a new sense of urgency regarding the reporting of acid fast smear cultures and drug susceptibility results to physicians, prompted in large part to the emergence of multi-drug resistant strains of tuberculosis (Nolte and Metchock supra at p. 400). Traditionally tuberculosis surveillance has involved the use of preliminary skin tests (e.g. tuberculin tests) with positive beings further evaluated for active diseases by radiographic analysis and sputum cultures. Other samples are sometimes submitted to the laboratory for culture including blood, bronchoalveolar lavage fluids (e.g. cerebrospinal (CSF), pleural peritoneal pericardial etc.), tissues (e.g. lymph nodes, skin, or other biopsy materials), abscess, aspirated fluids. Culture methods: Once the specimen is received by the laboratory suspected of containing mycobacteria, the specimen will generally be stained and examined for the pres nee of AFB. Sputum and other dirty specimens are decontaminated and concentrated prior to the staining, culturing and immunobiological methods. Methods for diagnosis of tuberculosis based on immunologic methods such as detection of delayed type sensitivity skin responses as well as the detection of an i-mycobacteria antibodies or mycobacterial antigens have been studied. The tuberculin skin test, still commonly used, was the first immuno diagnostic test developed for detection of tuberculosis. Vscan test kit is available in the market by which rapid flow through diagnostic test can be carried out for the antibody detection being the antibody detection kit. Only serum or blood can be used for the antibody detection test. Moreover antibody detection tests are unreliable because antibody formation in case of MTB is delayed and slow. In comparison, antibody detection suffers from the problem that host immunoglobulin levels can remain high many years after the pathogen has been cleared from the body. Thus Rapid diagnosis of TB bacilli in patients can lead to fewer ultimate complications and avoidance of unnecessary treatment. Nucleic acid PCR based genetic test: DNA detection test where a single DNA molecule is amplified in the presence of Taqpol enzyme in thermocycler. It is sensitive technique which requires skill, knowledge and sophisticated instruments to run PCR. US Patent NO: 6291178 describes a method and apparatus for the preservation of a saliva sample for use in subsequent quantitative chemical assays. The method involves collecting a saliva sample at a location, directly into a specimen cup. The specimen cup contains a predetermined volume of aqueous solution of pH buffered saline and enzymatic inhibitor and is optionally adapted with a constituent compound specific, qualitative test unit. U3 Patent No. 6383763 describes methods and compositions for the detection of infection and disease due to members of the genus Mycobacterium. In particular, the present invention is well-suited to the detection and identification of patients with disease or infection due to M. tuberculosis or MAC. US Patent No. 6583266 features nucleic acid and proteins derived from K'ycobacterium tuberculosis and leprae. The proteins and nucleic acid of the p asent invention have applications in diagnostics and therapeutics. The main disadvantage of the culture is the need for more sophisticated rt sources and more qualified technicians because specificity can suffer due to careless technique. The total test time may take 3-4 weeks for confirmation of results. Sometimes the slide preparation is not perfect. Diagnostic delay of culture kills time and clinical interpretations. In US Patent No. 6506384, a number of protein and glycoprotein antigens secreted by Mycobacterium tuberculosis (Mt) have been identified as "early" Mt antigens on the basis early antibodies present in subjects infected with Mt prior to the development of detectable clinical disease. These early Mt antigens, in particular an 88 kDa secreted protein having a pi of about 5.2 and the sequence of SEQ ID NO: 106, which is present in Mt lipoarabinomannan-free culture filtrate, a protein characterized as Mt antigen 85C; a protein characterized as Mt antigen MPT51, a glycoprotein characterized as Mt antigen MPT32; and a 49 kDa protein having a pi of about 5.1, are useful in immunoassay methods for early, rapid detection of TB in a subject. Preferred immunoassays detect the antibodies in the subject's urine. Also provided are antigenic compositions, kits and methods to useful for detecting an early Mt antigen, an early Mt antibody, and immune complexes thereof. US Patent No. 6599691 describes a rapid, non-invasive, semi-quantitative immunoassay of saliva to aid in the diagnosis of diseases, e.g., using saliva to detect subjects actively or previously infected with Mycobacterium tuberculosis, a causative organism of tuberculosis. The semi-quantitative assay comprises spotting disease-related antigens on the surface of a solid substrate; contacting the solid substrate with a saliva sample which, in positive subjects, contains primary antibodies to the disease-related antigens; contacting the primary antibodies with a label capable of being detected; and detecting and reading the label whereby exposure to the antigens is determined. The device for conducting these assays is a frame or support which holds a solid substrate capable of immobilizing the antigens of interest while permitting drainage of other materials or fluids away from the immobilized antigens, A less rapid, quantitative assay has also been developed by adapting the rapid, semi-quantitative assay to an enzyme linked immunosorbant assay thereby providing a quantitative assay capable of assessing multiple saliva samples simultaneously. The main object of the present invention is to provide a process for direct bacilli detection / observation using antigen antibody complex formation system. SUMMARY OF THE INVENTION The present invention relates to a process for the detection of Mycobacterium in a test sample comprising ) a solid base support and b) a polyclonal monoclonal antibody directed against a portion of antigen immobilized on the membrane of the solid support. The present invention also relates to a device for the rapid and easy test method for the detection of presence of Mycobacterium Tuberculosis bacilli by reaction with very specific monoclonal or polyclonal antibody directed against them. This complex is made to react with conjugate to obtain a visual colour on the device membrane. A built in control dot is provided to validate the efficacy of the reagents used in the test and to validate the compliance of the procedural steps. According, the present invention provided the process for analyzing the presents of Mycobacterium Tuberculosis in a sample comprising the following steps- a. preparing the sample in MTB solution as herein described; b. pouring the sample at least 400-800 µl on the pre-coated membrane of the test device by means of a dropper; c. allowing the sample to be absorbed completely on the membrane, enabling the antigens in the processed sample to adhere in the membrane; d. adding 3-4 drops of buffer solution of pH (4 to 7), and allowing it to be absorbed completely; e. adding 2-3 drops of TB antibody solution as herein described on the membrane, forming a antigen-antibody complex; f. adding 2-4 drops of wash buffer; g. adding 2 drops of protein A conjugate; h. adding 3 drops of wash buffer solution; i. reading the result wherein the pink colour of the membrane of the device indicates that the sample is positive The present invention also provide a device for rapid and easy method for analyzing the present of Mycobacterium Tuberculosis bacilli in a sample. The said device comprises of an airtight container having a circular opening at the top lid, a nitrocellulose membrane having a pore size of p. f. 0.2 to 2.0 µ attached to the said base, a control dot provided on the cell membrane comprising antibodies of mouse/rabbit/goat/sheep. In an embodiment of the present invention, the sample is prepared by making a homogenous solution of TRIS (white crystalline powder C4H11NO3) in a double distilled water to obtain an alkaline pH above 8, adjusting the pH with HCI concentrate to pH 6.5 to 7.5, mixing thoroughly with 25 millimolar EDTA (Ethylene Diamiene Tetra Acetic Acid) and a surfactant 0.05% to 0.25% w/v and allowing it till a cleat solution is obtained, adding Urea 4.5 to 9.5 molar, magnetically stirring the solution at an ambient temperature and allowing it to maturise for 2 to 3 days to obtain a clear MTB extraction solution, adding 10 ml of the MTB solution to 200-400 µl of sample either pulmonary or extra pulmonary, agitating the mixture in a cyclomixer at room temperature, allowing it to settle and repeating the steps allowing the sample to decontiminate, inactivate and lysed, obtaining a clear liquid solution. In another embodiment of the present invention, the initial buffer solution is Phosphate buffer having the pH (7.5 to 8.5). In yet another embodiment of the present invention, the extra-pulmonary sample comprises cerebrospinal fluid, plurel fluid, gastric aspirate, pus, endomaetridium tissue, bone-marrow and the like In still another embodiment of the present invention, the protein A conjugate is prepared by taking a solution of 0.01% to 0.05% w/v Gold Chloride (HauCI4), adding Sodium citrate solution (1-5%), stirring the solution rapidly and boiling until a pink purple colour develops, adding Protein A to the mixture, adding any non-immune animal serum (mouse or goat or rabbit or sheep) keeping the concentration of serum to 1µg to 10 µg /ml of gold colloid solution, mixing the solution for 2 hours to over night and centrifuging to discard the supernatant, dissolving the centrifugate in buffer containing stabilizers such as BSA, Gelatin to obtain the Protein A Gold conjugate In still another embodiment of present invention, the TB antibody solution comprises monoclonal and polyclonal antibodies. In yet another embodiment of present invention, the monoclonal and polyclonal TB antibodies are in the ratio of 1:1-3. The invention will be described with reference to the following accompanying drawings wherein: Figure 1 shows the test device having a cellulosic membrane enclosed in an airtight rectangular container having a circular hole opening in the center of the upper lid. In the procedure the membrane device is placed in the circular cavity of the upper lid of the test device. Figure 2 shows the bacilli antigen retained on the surface of the membrane after pouring the sample, adding three drops of buffer solution. Figure 3 shows the TB antibody - antigen complex after adding 2 drops of TB antibody solution. Figure 4 shows the conjugate Fc portion of the complex after carrying out all the six steps as described herein.. 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: Preparation of the sample: A homogenous solution of TRIS (white crystalline powder C4H11NO3) in a double distilled water was made to obtain an alkaline pH above 8. The pH was adjusted with HCI concentrate to 7. The solution was mixed thoroughly with 25 millimolar EDTA (Ethylene Diamiene Tetra Acetic Acid) and a surfactant 0.15% w/v and it was allowed till a clean solution was obtained. Urea was added 6 molar and the solution was magnetically stirred at an ambient temperature and it was allowed to maturise for 2 to 3 days to obtain a clear MTB extraction solution. 10 ml of the MTB solution were added to 300 µl of sample. The mixture was agitated in a cyclomixer at room temperature, allowing it to settle and the step was repeat to allow the sample to decontiminate, inactivate and lysed. Thus a clear liquid solution of sample was obtained. Preparation of Protein A conjugate: A solution of 0.03% w/v Gold Chloride (HauCI4) was taken and 3% Sodium citrate solution was added to it and the solution was stirred rapidly and was boiled until a pink purple colour was developed. Protein A was added to the mixture followed by adding non-immune animal serum of mouse keeping the concentration of serum to 5µg /ml of gold colloid solution. The solution was mixed for 6 hours and was centrifuged to discard the supernatant. The centrifugate was dissolved in buffer containing stabilizer BSA and the Protein A Gold conjugate was obtained. Analyzing the presence of Mycobacterium Tuberculosis Bacilli: 600 µl of the sample were poured on the pre-coated membrane of the test device by means of a dropper and the sample was allowed to be absorbed completely on the membrane, enabling the antigens in the processed sample to adhere in the membrane. 3 drops of buffer solution of pH 6 were added and it was allowed to be absorbed completely. 2 drops of TB antibody solution were added on the membrane, forming a antigen-antibody complex. 3 drops of wash buffer were added and 2 drops of protein A conjugate were added followed by adding 3 drops of wash buffer solution. The results were read wherein the pink colour of the membrane of the device indicated that the sample is positive While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of the invention. I Claim: 1. A kit for analyzing the presence of Mycobacterium tuberculosis bacilli in a sample comprising a test device having a cellulosic membrane enclosed in an airtight rectangular container having a circular hole opening in the centre of the upper lid wherein the membrane device is placed in the circular cavity of the upper lid of the test device and pore size of membrane is 0.2 to 2.0µ; a container having TB antibody solution highly specific for Mycobacterium tuberculosis bacilli antigen; a container having Protein A conjugate solution adapted to react with antigen antibody complex to give a pink background on the said membrane; MTB extraction solution for the dilution of the sample; and a container having buffer solution. 2. A kit as claimed in claim 1 where in the MTB extraction solution for the dilution of the sample is prepared by: a. making a homogeneous solution of TRIS( C4HnNO3) in a double distilled water to obtain an alkaline pH above 8; b. adjusting the pH with HC1 concentrate to pH 6.5 to 7.5; c. mixing thoroughly with 25 millimolar EDTA( Ethylene Diamiene Tetra Acetic Acid) and a surfactant 0.05% to 0.25% w/v and allowing it till a clear solution is obtained. d. adding 4.5 to 9.5 molar urea; e. magnetically stirring the solution at an ambient temperature and allowing it to maturize for 2 to 3 days to obtain a clear MTB extraction solution. 3. A kit as claimed in claim 1 where in the device comprises a nitrocellulose membrane having a pore size of 0.2 to 2.0µ. 4. A kit as claimed in claim 1 where in the sample is prepared by: a. adding 10ml of the MTB extraction solution to 200- 400µl of sample either pulmonary or extra pulmonary; b. agitating the mixture in a cyclomixer at room temperature, allowing it to settle and repeating the steps allowing the sample to decontaminate, inactivate and lysed, obtaining a clear liquid solution. 4. A device and a kit as herein before described with reference to the accompanying drawings.

Documents:

2653-DEL-2006-2-Post-Grant Opposition-(09-02-2011).pdf

2653-del-2006-abstract.pdf

2653-del-2006-claims.pdf

2653-del-2006-correspondence post-grant opposition.pdf

2653-del-2006-correspondence-others.pdf

2653-del-2006-correspondence-po.pdf

2653-del-2006-description (complete).pdf

2653-del-2006-drawings.pdf

2653-del-2006-form-1.pdf

2653-del-2006-form-18.pdf

2653-del-2006-form-2.pdf

2653-del-2006-form-26.pdf

2653-del-2006-form-3.pdf

2653-del-2006-form-5.pdf

2653-del-2006-form-9.pdf

2653-DEL-2006-GPA-(09-02-2011).pdf

2653-del-2006-grounds of opposition by opponent.pdf

2653-del-2006-Petition-137-(18-01-2011).pdf

2653-del-2006-reply evidence by patentee.pdf

2653-del-2006-reply statement by patentee.pdf

2653-del-2006-reply with evidence u-r 59.pdf