Title of Invention	"A KIT FOR ANALYZING THE PRESENCE OF IgM ANTIBODIES OF MYCOBACTERIUM TUBERCULOSIS"
Abstract	The invention relates to a process for analyzing the presence of IgM antibodies of Mycobacterium tuberculosis in human serum or plasma The antibody which is present in sample, is capable of reacting with a mycobacterium antigen analyzing the presence of antigen-antibody complexes and identifying the mycobacterium tuberculosis present in sample like serum, plasma and body fluids.

Full Text

FIELD OF THE INVENTION The present invention relates to a process for analyzing the presence of IgM antibodies of Mycobacterium Tuberculosis. 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 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. Tuberculosis (TB) is caused by repeated exposure to airborne droplets contaminated with a rod-shape bacterium, Mycobacterium tuberculosis. The TB bacterium is also known as the tubercle bacillus. A person with active pulmonary tuberculosis can spread the disease by coughing and sneezing. Once the person is infected with Mycobacterium tuberculosis, the infection will slowly progress to disease. More than 8 million new cases of Tuberculosis for more than three million deaths per year. Almost two and three quarter billion people (2.75 billion) or 33% of population are latently infected with TB. Among screening infectious diseases, a accurate and fast diagnostic method of tuberculosis is very important for human health maintain and the disease control. To improve the tuberculosis diagnosis, we search and select the specific antigens and prepare diagnostic kits for the detection of antibodies in serum / plasma and other body fluids. With the selection of appropriate antigen for use in serology test, it is important to increase the sensitivity without losing specificity. BACKGROUND OF THE INVENTION Organisms within the genus Mycobacterium include obligate parasites, saprophytes, and opportunistic pathogens. Most species are free-living in soil and water, but for species such as M. tuberculosis the causative agents of tuberculosis, the major ecological niche is the tissue of humans and other warm blooded animals. Despite the fact that most mycobacteria do not cause disease, a relatively small group of organisms within the genus is responsible for a large percentage of human morbidity and mortality worldwide. Tuberculosis remains a major global health problem, with nearly one third of the world's population infected. Indeed, tuberculosis is the leading cause of death due to a single infectious agent. In addition, the World Health Organization estimates that worldwide, there are 8-10 million new cases and over 3 million deaths directly attributed to this disease reported worldwide 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 micro meter 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 (Le., with purified protein derivative [PRO]), 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 of 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 micro-organisms such as M. tuberculosis. Nonetheless, 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. 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 (Le., 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. Culture Methods: Once a specimen has been received in the laboratory suspected of containing mycobacteria, the specimen will generally be stained and examined for the presence of AFB. Sputum and other i'dirty specimens are decontaminated and concentrated prior to staining, culturing & Immunological methods. Methods for diagnosis of tuberculosis based on immunological methods such as detection of delayed type hypersensitivity (DTH) skin responses, as well as the detection of anti-mycobacteria antibodies, or mycobacterial antigens have been studied. Historically, skin tests have been commonly used as indicators of infection with M. tuberculosis. The tuberculin skin test, still commonly in use, was the first immuno diagnostic test developed for detection of tuberculosis. Problems with this test include its inability to distinguish active disease from past sensitization, as well as its unknown predictive accuracy. 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. PRIOR ART: Various conventional processes are available which have limitations to carry out tests either by serum or whole blood, but no device is available which can be used and give high sensitivity with either serum or sputum or plasma. Nucleic acid PCR based genetic test: DMA detection test where a single DNA molecule is amplified in the presence of Taqpol enzyme in thermocycle. It is a sensitive technique which requires skill, knowledge and sophisticated instruments to run PCR. 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. The main disadvantage of the culture is the need- for more sophisticated resources and more qualified technicians because specificity can suffer due to care less techniques. 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. 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 manifeslations 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 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. TB-G Microlisa developed as a indirect elisa test for the detection of IgM antibodies which demonstrate a superior performance against serum test. As this test can be done with serum also, so in case of paediatric patient, it is difficult to get their sputum whereas serum is easy to get. The present invention relates to a process for analyzing the presence of IgM antibodies of Mycobacterium Tuberculosis in sera or plasma by ELISA and easy test method. Specific antigen is coated in the Microwell of Elisa plate & then serum added to the well. The Tuberculosis antibodies which are present in the serum will attach ( bind) to antigen present in the well and'make a complex. Unbound material is washed with wash buffer. Then conjugate added in the well ( Anti human IgM linked to HRPO). Then secondary antibody will bind to the complex. Unbound material is washed and followed by the addition of substrate and colour complex is formed, which is read at 450 nm. TB-M Microlisa developed as a indirect Elisa test for the detection' of IgM antibodies which demonstrate a superior performance against serum test. As this test can be done with serum also, so in case of pediatric patient, it is difficult to get their sputum whereas serum is easy to get. Microwell Elisa test for detection of IgM antibodies of M. Tuberculosis in Human serum or plasma wherein TB-M Microlisa is a solid phase enzyme linked immuno adsorbent assay (ELISA) based on the "Indirect ELISA principal". The Microwells are coated with TB antigens. The samples are added in the wells followed by addition of enzyme conjugate ( anti human IgM linked with HRPO). Unbound conjugate is then washed off with wash buffer. On addition of the substrate buffer and chromogen, a blue colour develops and its intensity is directly proportional to their concentration of TB antibodies in a sample. The reaction stopped by adding stop solution and yellow colour develops which is finally read at 450 nm. In short the device used in the test have the following features: 1. Breakaway wells with minimal wastage 2. Easy interpretation of results 3. Shelf life 12 months at 2-8°C. 4. Available in convenient pack sizes, 48 & 96 Tests. 5. Excellent sensitivity for pulmonary and extra pulmonary samples. 6. More- sensitive than AFB smear tests & culture for extra pulmonary samples. 7. Specificity: Increased power of discrimination between negative & positive samples. According to the present invention, a process for analyzing the presence of IgM antibodies of Mycobacterium Tuberculosis in human serum or plasma comprises the following steps: a) diluting the sera to be tested in the ratio of 1:100 in the Sample diluent b) leaving the A-1 well of the microwell coated with TB antibody as substrate blank c) adding 100 ml of negative control and positive control in 2 wells each B-1, C-1andD-1,E-1 d) adding 100 ml of diluted samples in the microwell e) covering aid 30 minutes at 37°C 0 washing the plates for 5- 7 times with working wash buffer g) adding 100 ml of working enzyme conjugate solution in each well including blank well and incubating for 60 minutes at 35 deg. C. to 39 deg. C. h) washing the plates again 5-7 times with working wash buffer i) adding 100 ml of working substrate solution in each well including blank well j) incubating at room temperature for 30 minutes in dark k) adding 50 ml of stop solution I) reading absorbance at 405 nm. In an embodiment of the present invention the microwell are prepared by a process comprising the steps of: a) taking 50nm phosphate buffer pH of at least 8.0, b) adding TB antigen in (1:10000) dilution in coating buffer c) dispensing 100 micro liter of antigen coating solution on the microwell plates for at least 2 hours at 36 deg. C to 40 deg. C. d) washing the plates and blocking with 1% BSA solution for 24 hours at 2 deg. C. to 8 deg. C. e) drying the plates at 37 deg. C. for 8 hours f) storing the test plate in dessicant puches at 4 deg. C. In another embodiment of the present invention, the sample specimen diluent is prepared by preparing 100 mM phosphate buffer saline pH 7.4 and adding 0.5 % BSA, 0.1 % Sodium Azide and dye followed by storing at a temperature from 2 deg. C. to 8 deg. C. In yet another embodiment of the present invention, the sample specimen is prepared by taking serum/plasma and diluting it in the sample diluent and body fluid like sputum, CSF, Plural fluid and diluting it 5 to 50 times in sample diluent. In still embodiment of the present invention, the working enzyme conjugate is prepared by linking rabbit human IgM to the HRP by following linking method: a) dissolving the 15 mg HRP in 100 mM PBS pH 7.2 at a concentration of 20 mg /ml b) adding 5 mg of Sulfo-SMCC [Sulfo Succinimidyl 4-(N- Maleimidomethyl) Cyclohexane-1-Carbooxylate] to the HRP solution followed mixing, dissolving and reacting for 30 minutes at room temperature. c) purifying the Sulfo-SMCC activated HRP using dialysis against 100 mM phosphate buffer saline pH 7.2 for 2 hour followed by adjusting the HRP concentration to 10 mg/ml for the conjugation. d) dissolving the Rabbit anti Human IgM in 100 mM PBS pH 7.2 and 10mM EDTA in 5 mg / ml ratio. e) adding 5 mg of 2-mercaptoethylamine to each ml of antibody and mixing to dissolve. f) incubating at 37°C. g) purifying the reduced Rabbit anti human IgM using dialysis against 100 mM PBS pH 7.2 for 2 hours. h) Mixing the Rabbit anti Human IgM with the Sulfo SMCC activated HRP and reacting for 60 minutes at 37°C and followed by dialysis and storing at a temperature from 2 deg. C. to 8 deg. C. In still another embodiment of the present invention, the wash buffer is prepared by mixing 20 ml of 25 X wash buffer concentrate with 450 - 500 ml of distilled water. In yet another embodiment of the present invention, the substrate solution is 1MB concentrate diluted in 1:100 in 1MB diluent. The invention will be described with reference to the following drawings wherein Fig. 1. is the systematic flow chart and procedure indicating the testing procedure and the various steps in brief. 1) Adding sample diluent to A-1 well as blank. 2) Adding 100 micro liter of negative and positive control in 2 wells each i.e. B-1, C-1 and D-1, E-1. Then transfer 100 micro liter of the diluted samples in the microwells. Incubate at 37 deg. C. forSO minutes. 3) Washing the plate 5 times with working wash solution. 4) Adding 100 micro liter of working conjugate solution in each well including blank well. Incubate at 37 deg. C. for 30 minutes. 5) Washing the plate 5 times with working wash solution. 6) Adding 100 micro liter of working substrate solution in each well including blank well. 7) Incubating at room temperature for 30 min in dark. 8) Adding 50 micro liter of stop solution 9) Reading absorbance at 450 nm. 10 PREPARATION OF PLATES: A) Microwell Preparation: We take 50 mM phosphate buffer Ph 6.0- 8.0 and add TB antigen in ( 1: 10,000 ) dilution in coating buffer and dispense 100 J..II of antigen coating solution in microwell plates for 2 hours at 3r C, after washing, the plates are blocked with 1 % BSA solution for 24 hours at 2-8 cc dried at 37 deg. C for 8 hours and stored in dessicant pouches at 4 deg. C. B) Sample specimen diluent Prepare 100 mM phosphate buffer saline pH 7.4 and add 0.5 % BSA, 0.1 % Sodium Azide and dye and store at 2-8 cc. C) Preparation of Enzyme conjugate: Rabbit anti human IgM purchased from out source and linked with the HRPO, linking method is as below: i) Dissolve the 15 mg HRP in 100 mM PBS pH 7.2 at a concentration of 20 mg / mi. ii) Add 5 mg of Sulfo-SMCC [ Sulfo Succinimidyl 4-(N- Maleimidomethyl) Cyclohexane-1-Carbooxylate ] to the HRP solution. Mix and dissolve and react for 30 minutes at room temperature, iii) Immediately purify the Sulfo-SMCC activated HRP from dialysis against 100 mM phosphate buffer saline pH 7.2 for 2 hour. After dialysis, adjust the HRP concentration to 10 mg/ml for the conjugation, iv) Dissolve the Rabbit anti Human IgM in 100 mM PBS pH7.2 + 10mM EDTA in 5 mg / ml. v) Add 5 mg of 2-mercaptoethylamine to each ml of antibody and mix to dissolve. vi) Incubate for 37°C for 1-2 hours, vii) Purify the reduced Rabbit anti human IgM using dialysis against 100 mM PBS pH 7.2 for 12.-18 hours. viii) Rabbit anti Human IgM mix with the Sulfo SMCC activated HRP immediately and react for 60 minutes at 37°C and further purified by dialysis and store at 2-8 °c. A test kit utilizing the present process comprises (A) TB-M Microlisa having the following components: 1. Microwell Plate 2. Sample dilute 3. Enzyme conjugate (100X) 4. Conjugate diluent 5. Wash buffer concentrate (25 X) 6. TMB concentrate (100 X) 7. Negative control 8. Positive control 9. Stop solution The invention also resides in preparation of the sample, wash buffer, desired conjugate, optimum substrate solution. 1) SAMPLE PREPARATION Sample like serum, plasma 10-100 times is diluted in sample diluent and body fluid like sputum, CSF, Plural fluid is diluted 5* to 50 times in sample diluent. 2) PREPARATION OF WASH BUFFER Mix 20 ml of 25 X wash buffer concentrate with 450 to 500 ml of distilled water. 3) WORKING CONJUGATE Dilute the Rabbit anti human IgM linked to HRPO enzyme conjugate!: 100 in conjugate diluent. Mixing the solution thoroughly. 4) PREPARTION OF WORKING SUBSTRATE SOLUTION Dilute the TMB concentrate 1:100 in 1MB diluent. Where IMS (Tetramethyl benzidene) diluent is prepared by mixing 100 mM Acetate 0.005% Hydrogen peroxide. TEST PROCEDURE: 1) Add sample diluent to A-1 well as blank. 2) Add 100 micro liter of negative and positive control in 2 wells each i.e. B-1, C- 1 and D-1, E-1. Then transfer 100 micro liter of the diluted samples in the microwells. Incubate at 37 deg. C. for 30 minutes. 3) Wash the plate 5 times with working wash solution. 4) Add 100 micro liter of working conjugate solution in each well including blank well. Incubate at 37°C for 30 minutes. 5) Wash the plate 5 times with working wash solution. 6) Add 100 micro liter of working substrate solution in each well including blank well. 7) Incubate at room temperature for 30 min in dark. 8) Add 50 micro liter of stop solution 9) Read absorbance at 450 nm. CALCULATION OF THE RESULT: Test validity: Blank must be Negative control (NC) must be ~ 0.150 Positive control (PC) must be ~ 0.500 Cut off value: NC + 0.500 INTERPRETATION OF RESULTS: 1. Test samples with absorbance value less than the Cut off value are non reactive and may be considered as negative for anti TB IgM. 2. Test specimens with absorbance value greater than or equal to the Cut off value are reactive for anti TB-lgM. 3. It seems reasonable to define arrange of +/- 20% around the value of the Cut off as a gray zone. In such case the repetition of the test with the same serum or with a new sample of the same patient taken after 2 to 4 week is recommended both samples should be measured in parallel in the same run. 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. We claim: 1. A process for analyzing the presence of IgM antibodies of Mycobacterium Tuberculosis in human serum or plasma comprising the following steps: a) diluting the sera to be tested in the ratio of 1:100 in the sample diluent b) leaving the A-1 well of the microwell coated with TB antibody as substrate blank c) adding 100 ml of negative control and positive control in 2 wells each B-1, C-1 andD-1,E-1 d) adding 100 ml of diluted samples in the microwell e) incubating for 60-65 minutes at 36 deg. C. to 40 deg. C. f) washing the plates for 5- 7 times with working wash buffer g) adding 100 ml of working enzyme conjugate solution in each well including blank well and incubating for 60 minutes at 35 deg. C. to 39 deg. C. h) washing the plates again 5-7 times with working wash buffer i) adding 100 ml of working substrate solution in each well including blank well j) incubating at room temperature for 30 minutes in dark k) adding 50 ml of stop solution I) reading absorbance at 405 nm. 2. A process for analyzing the presence of IgM antibodies of Mycobacterium Tuberculosis in human serum or plasma as claimed in claim 1, wherein the said microwell are prepared by a process comprising: a) taking 50nm phosphate buffer pH of 8.0, b) adding TB antigen in (1:10000) dilution in coating buffer c) dispensing 100 micro liter of antigen coating solution on the microwell plates for at least 2 hours at 37 deg. C to 40 deg. C. d) washing the plates and blocking with 1% BSA solution for 24 hours at 2 deg. C. to 8 deg. C. e) drying the plates at 37 deg. C. for 8 hours f) storing the test plate in desiccant puches at 4 deg. C. 3. A process for analyzing the presence of IgM antibodies of Mycobacterium Tuberculosis in human serum or plasmas claimed in claim 1, wherein the said sample specimen diluent is prepared by preparing 100 mM phosphate buffer saline pH 7.4 and adding 0.5 % BSA, 0.1 % Sodium Azide and dye followed by storing at a temperature from 2 deg. C. to 8 deg. C. 4. A process for analyzing the presence of IgM antibodies of Mycobacterium Tuberculosis in human serum or plasmas claimed in claim 1, wherein sample specimen is prepared by taking serum/plasma and diluting it in the sample diluent and body fluid like sputum, CSF, Plural fluid and diluting it 5 to 50 times in sample diluent. 5. A process for analyzing the presence of IgM antibodies of Mycobacterium Tuberculosis in human serum or plasmas claimed in claim 1, wherein the said working enzyme conjugate is prepared by linking rabbit human IgG to the HRP by following linking method: a) dissolving the 15 mg HRP in 100 mM PBS pH 7.2 at a concentration of 20 mg /ml b) adding 5 mg of Sulfo-SMCC [Sulfo Succinimidyl 4-(N- Maleimidomethyl) Cyclohexane-1-Carbooxylate] to the HRP solution followed mixing, dissolving and reacting for 30 minutes at room temperature. c) purifying the Sulfo-SMCC activated HRP using dialysis against 100 mM phosphate buffer saline pH 7.2 for 2 hour followed by adjusting the HRP concentration to 10 mg/ml for the conjugation. d) dissolving the Rabbit anti Human IgM in 100 mM PBS pH 7.2 and 10mM EDTA in 5 mg / ml ratio. e) adding 5 mg of 2-mercaptoethylamine to each ml of antibody and mixing to dissolve. f) incubating at 37°C. g) purifying the reduced Rabbit anti human IgM using dialysis against 100 mM PBS pH 7.2 for 2 hours. h) Mixing the Rabbit anti Human IgM with the Sulfo SMCC activated HRP and reacting for 60 minutes at 37°C and followed by dialysis and storing at a temperature from 2 deg. C. to 8 deg. C. 6. A process for analyzing the presence of IgM antibodies of Mycobacterium Tuberculosis in human serum or plasmas claimed in claim 1 , wherein the wash buffer is prepared by mixing 20 ml of 25 X wash buffer concentrate with 450 - 500 ml of distilled water. 7. A process for analyzing the presence of IgM antibodies of Mycobacterium Tuberculosis in human serum or plasmas claimed in claim 1, wherein the substrate solution is TMB concentrate diluted in 1:100 in TMB diluent. 8. A process for analyzing the presence of IgM antibodies of Mycobacterium Tuberculosis in human serum or plasmas as herein substantially described with the help of accompanying drawing.

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448-del-2006-abstract.pdf

448-del-2006-claims.pdf

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

448-del-2006-correspondence-others.pdf

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

448-del-2006-description(provisional).pdf

448-del-2006-drawings.pdf

448-del-2006-form-1.pdf

448-del-2006-form-18.pdf

448-del-2006-form-2.pdf

448-del-2006-form-26.pdf

448-del-2006-form-3.pdf

448-del-2006-form-5.pdf

448-del-2006-form-9.pdf