Title of Invention	A DEVICE KIT FOR ANALYZING THE PRESENCE OF TUBERCULOSIS BACILLI IN A SAMPLE
Abstract	A test kit for analyzing the presence of tuberculosis bacilli in a sample comprising a test device having a cellulosic membrane enclosed in an air tight rectangular container having a circular hole opening in the center of the upper lid, a container having TB antibody solution, a container having gold conjugate and a TB antibody conjugate and a buffer solution.

Full Text

FIELD OF THE INVENTION M. tuberculosis is exceptionally easily transmitted, as it is carried in airborne particles termed "droplet nuclei," produced when a patient with active tuberculosis coughs. These particles are from 1-5.mu. in size, and are readily suspended in air currents. Infection occurs when droplet nuclei are inhaled and reach the terminal airways of the new host's lungs. Usually, the host immune response limits the multiplication and spread of the organism, although some organisms may remain dormant, but viable, for many years post-infection. Individuals infected with M. tuberculosis but without disease, usually have a positive skin test (i.e., with purified protein derivative [PPD]), but are asymptomatic and generally not infectious. However, latently infected individuals have a 10% risk for developing active tuberculosis at some point during their life; the risk is greatest within the first two years post-infection. For HIV-positive individuals, the risk is much greater, with the risk at 10-15% per year for progression to active disease (F. S. Nolte and B. Metchock, "Mycobacterium," in Manual of Clinical Microbiology, Sixth The field of diagnostic and clinical microbiology has continued to evolve, and yet, there remains a general need for systems that provide rapid and reliable detection of disease and infection due to microorganisms such as M. tuberculosis. Nontheless, the role of the clinical mycobacteriology laboratories cannot be underestimated in view of their potential contributions in controlling the spread of tuberculosis and non-tuberculosis disease through the timely detection, isolation, identification, and determination of drug susceptibility of these organisms. 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 M. tuberculosis (Nolte and Metchock, supra, at p. 400). Traditionally, tuberculosis surveillance has involved the use of preliminary skin tests (e.g., tuberculin tests), with positives being further evaluated for active disease by radiographic analysis (i.e., chest X-rays), and sputum cultures. Other samples are sometimes submitted to the laboratory for culture, including blood, bronchoalveolar lavage fluid, bronchial washings, gastric lavage fluids, urine, body fluids (e.g., cerebrospinal [CSF], pleural, peritoneal, pericardial, etc.), tissues (e.g., lymph nodes, skin, or other biopsy materials), abscess contents, aspirated fluids, The analysis of clinical specimens is important in science and medicine. A wide variety of assays to determine qualitative and/or quantitative characteristics of a specimen are known in the art. Vscan test kit is available in the market by which Rapid flow through diagnostic test can be carried out for antibody detection. However the limitation of this kit is that only serum or whole blood is used as sample. Nucleic acid PCR based genetic test: DMA detection test where a single DMA molecule is amplified in the presence of Tagpol enzyme in thermocycle. It is a sensitive technique which requires skill, knowledge and sophisticated instruments to run PCR. PRIOR ART : US Patent no. 6291178 describes a method for the preservation of bodily fluid samples. The invention describes a method for the preservation and storage of human saliva samples for use in subsequent component testing thereof. US Patent no 6383763 describes a 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 Mycobacterium tuberculosis and leprae. The proteins and nucleic acid of the present invention have applications in diagnostics and therapeutics. 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 identify 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 device and 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. It is therefore the object of the present invention to provide a test kit with a rapid and easy antigen-antibody based detection system. The other object of the present invention to provide a test kit with a direct bacilli observation method. The present invention provides kits for the detection of Mycobacterium in a test sample, comprising: a) a solid base support and b) a monoclonal or polyclonal antibody directed against a portion of antigen immobilized on the membrane of the solid support. In one embodiment, the kit further comprises a filter device to be place on the solid support. In a particularly preferred embodiment, the Mycobacterium species detected is Mycobacterium tuberculosis. It is also contemplated that the kit of the present invention will include additional components, including but not limited to such items as decontaminated solution, antibody solution, buffer and TB antibody conjugate. It is also contemplated that the pellets of the sample be prepared through the use of decontamination solution. A test kit for analyzing the presence of tuberculosis bacilli in a sample by a process as claimed in claim 1 comprising: A test device having a cellulosic membrane enclosed in an airtight rectangular container having a circular hole cavity in the center of the upper lid A nitrocellulosic membrane placed inside the container, aligned horizontally facing the said circular hole cavity A container having Tb antibody solution highly specific for mycobacterium tuberculosis bacilli A container having gold conjugate solution adapted to react with antigen antibody complex to give a red background on the said membrane And having TB antibody conjugate And a container having a buffer solution A process for making sample under test form the sputum having tuberculosis bacilli comprising the following steps: Taking the sample of sputum in a container Adding the decontamination solution twice the volume of the sample to obtain a clear solution sample Adding equal amount of phosphate buffer( pH 4-7) Centrifuging the solution in a plastic tube at the speed of at least 3000rpm for about 30-40 minutes Discarding the supernatant and collecting the pellets Dissolving the pellet in a neutralized solution Boiling 200µl of neutralized sample with 200µl of phosphate buffer of pH (4-7) at 100deg. For 10-15 minutes in a hot bath in appendorf, to obtain the processed testing sample. A process for analyzing the presence of tuberculosis bacilli in a processed sample as claimed in claim 1 comprising the steps of: pouring the said processed sample on the membrane device till its complete absorption in the membrane Adding 3 drops of buffer solution ( 4-7) pH wait till its complete absorption Adding 2 drops of TB antibody solution on the membrane device Adding 3 drops of buffer solution & wait till it is completely absorbed Adding 2 drops of TB antibody conjugate till it is completely soaked on the membrane Adding 3 drops of buffer solution, soaking it on membrane, Read the result. A process as claimed in claim 6 where in the said TB antibody conjugate is prepared by the following steps: Adding to the initial solution 0.01% to 0.50% (w/v). Gold chloride ( HAuCI4), that is at a boil sodium citrate( 1-5%) w/v. Stirring the solution rapidly, boiling till a pink purple colour develops Mixing in a buffer solution of molarity ranging from 20 mM to 50 mM and pH 6.5 to 9.0 TB monoclonal antibodies, Mixing the antibody solution with gold colloidal for 1 to 2 hr Adding 1µg to l0µg /ml of gold colloid solution non - immune animal serum( mouse or goat or rabbit or sheep) to the above solution Mixing and keeping the solution from 2 hr to overnight Centrifuging the said solution and discarding the supernatant dissolving the centrifuge in a buffer containing stabilizers such as BSA, gelatin to obtain the TB antigen conjugate. BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS Fig. 1 shows the test device Fig. 2 shows membrane having antigen / TB bacilli Fig 3. shows the membrane having antigen / TB bacilli and TB antibody Fig. 4 shows the membrane, Antigen / TB bacilli, TB antibody and TB antigen conjugate DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a test kit and rapid and easy test method for detection of the presence of mycobacterium tuberculosis bacilli by reacting with a very specific monoclonal or polyclonal antibody directed against them. This complex is made to react with TB antigen conjugate to obtain a visual pink colour on the device membrane. Before the test is carried out the sputum sample is taken in a container and decontamination solution of the double the volume of the sample is added to it (e.g. to 2.5 ml sample 5ml of decontamination solution is added). The said decontamination solution comprises of 1-4 % w/v sodium hydroxide, 1.8-3% w/v Sodium citrate and 0.9-1.5 w/v NALC (Sodium Acetyl L Cysteine). The sample is then centrifuged in a tarson tube at the speed of 3000 rpm for about 30 minutes. This cold centrifugation results into formation of pellets. The pellets obtained are neutailized with phosphate buffer having a pH between 4-7. The solution is further neutralized with a neutralizing solution comprising 1m and hydrochloric acid and 4.5-5.5m Sodium hydroxide. 200 ul of neutrilized sample with 200 ul of PB is boiled at 100°C for 10-15 minute. Now this is the processed sample. Fig. 1 shows the test device comprising a test device (1) having a cellulosic membrane (2) enclosed in an airtight rectangular container (3) having a circular hole (4) opening in the center of the upper lid. A filtering device removably fitted over the said test device (1) provided with a filter in its circular depression central cavity (5) adapted to be fitted to the upper lid (6) of the test device encircling the central cavity (5) having the cellulosic membrane (2). In the procedure the membrane device is placed in the circular cavity of the upper lid (6) of the test device (1). The treated sample (3) is poured on the membrane and is allowed to be absorbed completely in the membrane. Few drops of buffer solution are added and the filter is removed. Step-1 : Processed sample as explained above is poured on the membrane device. Wait till the sample is completely soaked on the membrane device, as illustrated in Fig. (2). Step-2 : 3 drops of buffer solution having pH 4-7 is added and wail till the solution has completely soaked on the device. Step-3 : 2 drops of TB antibody solution (7) are poured on the membrane device. Allow it to soak in the membrane. Step-4 : Add 3 drops of buffer solution on the membrane device. Wait till the sample is completely soaked on the membrane device Step-5 : 2 drops of TB antigen conjugate (8) are poured on the membrane device. Allow it to soak in the membrane. Step-6 : Add 3 drops of buffer solution on the membrane device and read results. Pink surface on membrane device shows the sample is positive while negative leaves no trace on the device. This is a very quick and easy method to validate the positivity and negativity of the sample. The molarity of buffer can range from 20 mM to 50 mM and pH 6.5 to 9.0. The antibody concentration preferably is 1 M9/ml to 10 ug/ml of Gold colloid solution The concentration of serum could be 1 ug to 10 ug / ml of gold colloid solution. This solution is mixed for 2 hr to overnight. After this the solution is centrifuged and the supernatant is discarded. The pallet is redissolved in a buffer containing stabilizers like BSA, gelatin. All pulmonary and extrapulmonary including CSF pleural fluid, pus, lymph mode aspirates, biopsies can be used as samples to confirm the mycobaterial TB identity of the isolated TB bacilli. The subject invention is a mere statement of invention, where various alterations and modifications are possible without deviating from the scope of the invention hence the same should not be construed to restrict the scope of the invention. I claim : 1. A kit for analyzing the presence of tuberculosis bacilli in a sample comprising: a test device having a cellulosic membrane enclosed in an air tight rectangular container having a circular hole cavity in the centre of the upper lid, a nitrocellulosic membrane placed inside the container, aligned horizontally facing the said circular hole cavity; a container having TB antibody solution highly specific for Mycobacterium tuberculosis bacilli, a container having TB antibody conjugate and; a container having a buffer solution. 2. A kit as claimed in claim 1 where in the said antibody are monoclonal or polyclonal. 3. A kit as claimed in claim 1, wherein said TB antibody conjugate is prepared by the following steps : adding to the initial solution 0.01 to 0.50 % (w/v), Gold chloride (HAuc14), that is at a boil sodium citrate (1-1.5% w/v); stirring the solution rapidly , boiling till a pink purple colour develops; mixing in a buffer solution of molarity ranging from 20mM to 50mM and pH 6.5 to 9.0 TB antibody; mixing the antibody solution with gold colloid for 1 to 2 hrs; adding 1 µg to 10 µg/10mI of gold colloid solution non immune animal serum (mouse or goat or rabbit or sheep) to the above solution; mixing and keeping the solution from 2 hr. to overnight; centrifuging the said solution and discarding the supernatant; dissolving the centrifuge in a buffer containing stabilizers such as BSA, gelatin to obtain the TB antibody conjugate. 4. A kit as claimed in claim 1 where in the processed testing sample is prepared by: taking the sample of sputum in a container; adding the decontamination solution twice the volume of the sample to obtain a clear solution sample; adding equal amount of phosphate buffer of pH 4 to 7; centrifuging the solution in a plastic tube at a speed of at least 3000rpm for about 30 to 40 minutes; discarding the supernatant and collecting the pellets; dissolving the pellet in a neutralized solution boiling 200µlof neutralized sample with 200µlof phosphate buffer of pH 4 to 7 at l00degree for 10 to 15 minutes in hot bath append of, to obtain the processed testing sample. 5. A sample as claimed in claim 4 where in the decontamination solution comprises 1-4% w/v sodium hydroxide 1.8 - 3% w/v Sodium citrate 0.9 - 1.5 % w/v NALC ( Sodium Acetyl L Cystine) 6. A kit for analyzing the presence of tuberculosis bacilli as substantially as herein before described.

Documents:

204-DEL-2006-Abstract-(06-11-2008).pdf

204-del-2006-abstract.pdf

204-DEL-2006-Claims-(06-11-2008).pdf

204-del-2006-claims.pdf

204-DEL-2006-Correspondence-Others-(06-11-2008).pdf

204-del-2006-correspondence-others.pdf

204-DEL-2006-Description (Complete)-(06-11-2008).pdf

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

204-del-2006-drawings.pdf

204-DEL-2006-Form-1-(06-11-2008).pdf

204-del-2006-form-1.pdf

204-DEL-2006-Form-2-(06-11-2008).pdf

204-del-2006-form-2.pdf

204-del-2006-form-3.pdf

204-del-2006-form-9.pdf

204-del-2006-post grant opposition representation.pdf

204-DEL-2006-Post Grant Opposition-(19-08-2010).pdf

204-del-2006-post grant reply.pdf

Patent-225717-Post-Grant-Opposition-(22-07-2010).pdf