Title of Invention	A KIT FOR ANALYZING THE PRESENCE OF IgM, IgA, IgG ANTIBODIES OF MYCOBACTERIUM TUBERCULOSIS IN HUMAN SERUM OR PLASMA
Abstract	The invention relates to a process for analyzing the presence of lgM,lgA,lgG 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 serurn, plasma and body fluids.

Full Text

FIELD OF THE INVENTION The present invention relates to a process for analyzing the presence of igM,igA,lgG 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 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. 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. Such as detection of delayed typed hypersesnsitivity (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: DNA 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 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. TB-MAG Microlisa developed as a indirect elisa test for the detection of lgM,lgA,lgG 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 lgM,lgA,lgG 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 lgM,lgA,lgG 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-MAG Microlisa developed as a indirect Elisa test for the detection1 of lgM,lgA,lgG 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 lgM,lgA,lgG 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 "SANDWICH ELISA PRINCIPAL". The Microwells are coated with TB antigens. The samples are added in the wells followed by addition of enzyme conjugate ( anti human lgM,lgA,lgG 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 lgM,lgA,lgG antibodies of Mycobacterium Tuberculosis in human serum or plasma comprises the following steps: a) leaving A-1 well as substrate blank. b) adding 50 ml of negative control and positive control in 2 wells each B-1, C-1 andD-1,E-1 c) adding 50 ml of the sample in the microwell d) adding 50ml of working conjugate solution. e) incubating at 37° C for 1 hour. f) washing the plates for 5 times with working wash solution. g) adding 100 ml of working substrate solution in each well including blank well h) incubating the well at room temperature for 30 minutes in dark i) adding 50 ml of stop solution j) reading absorbance at 450 nm. In another embodiment of the present invention the microwell are prepared by a process comprising the steps of: a) taking 50 mM phosphate buffer Ph 6.0- 8.0 b) adding TB antigen or their combination (1-254) in as particular range, preferably 10 nanogram to 1 micro gram per microwell. c) dispensing 100 ml of antigen solution in micro well plate. d) incubating for 1 to 5 hours at 25°C- 37°C . e) washing the coated plates with 10 mM soldium phosphate buffer pH 6.0 - 8.0. f) blocking the plates with 0.5 - 2.0 % BSA solution . g) incubating for 12- 30 hours at 2-8°C . h) dryng at 37°C for 2 to 8 hours . i) storing in descents pouches at 2-3°C. in another embodiment of the present invention , wash buffer comprises 250 mM sodium phosphate buffer 0.15 M saline having pH .6.0 - 8.0 and 0.01% Tween -20. In another embodiment of the present invention the enzyme conjugate is prepared by linking TB antigen with HRPO by linking method as giving below: a) Dissolving the 15mg HRP in mM PBS pH 7.0 - 7.5 at a concentration of 20mg/ml. b) Adding 5 mg of Sulfo-SMCC [Sulfo Succinimidyl 4-(N-Maleimidomethyl) Cyclohexane-1-Carbooxylate] to the HRP solution. Mix and react for 30 minutes at room temperature. c) Immediately purifying the Sulfo-SMCC activated HRP from dialysis or desalting column adjust the HRP concentration to 10mg/ml for the conjugation. d) Dissolving above TB antigen and ambination (same antigen which is coated on the plate) in 100mM PBS pH 7.2+ 10mM EDTA in 1 - 5 mg / ml. e) Adding 10 molar excess of Traut Reopent to each mg of antigen and mix. f) Incubating for 37°C for 1-2 hours. g) Purifying the reduced Rabbit anti combination linked HRPO using dialysis against s against 100mM PBS pH 7.2 for 12-18 hours. h) Mixing the activated TB antigen with activated HRP incubate for 1 hrs at 37°C and further purified by dialysis and store at 2-8°C. In another embodiment of the present invention, the 1MB concentrate comprising 1 -3 gm of 1MB (Tetramehyl benzidene ) powder dissolved in 100 ml of Dimethyl Sulfoxide In another embodiment of the present invention, the substrate solution is 1MB concentrate diluted in 1:100 in 1MB diluent. In another embodiment of the present invention, 1MB diluent comprises 100 mM acetate buffer and 0.005% Hydrogen peroxide. In another embodiment of the present invention, the negative control is Dilute normal human sera (1:100 - 1: 1000) in PBS. In another embodiment of the present invention, the positive control is dilute TB positive human sera (1:100-1: 1000) in PBS. 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. a) leaving A-1 well as substrate blank. b) adding 50 ml of negative control and positive control in 2 wells each B-1, C-1 andD-1,E-1 c) adding 50 ml of the sample in the microwell d) adding 50ml of working conjugate solution. e) incubating at 37° C for 1 hour. f) washing the plates for 5 times with working wash solution. g) adding 100 ml of working substrate solution in each well including blank well h) incubating the well at room temperature for 30 minutes in dark i) adding 50 ml of stop solution j) reading absorbance at 450 nm. 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 ml of antigen solution in microwell plates incubating for 2 hours at 37°C, after washing, the plates are blocked with 1 % BSA solution for 24 hours at 2-8 °C and stored in dessicant pouches. B) Preparation of Enzyme conjugate: Rabbit anti human lgM,lgA,lgG 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 lgM,lgA,lgG using dialysis against 100 mM PBS pH 7.2 for 12.-18 hours, viii) Rabbit anti Human lgM,lgA,lgG 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. The invention is also resides in preparation of the sample, wash buffer, desired conjugate, optimum substrate solution: PREPARATION OF THE REAGENT:- 1) PREPARTION OF WASH BUFFER: Mix 20 ml of 25 X wash buffer concentrate with 480 ml of distilled water. 2) WORKING CONJUGATE: Dilute TB antigen linked to HRPO enzyme conjugate 1: 500 in conjugate diluent. Mixing the solution thoroughly. 3) PREPARTION OF WORKING SUBSTRATE SOLUTION: Dilute the 1MB concentrate 1:100 in 1MB diluent. TEST PROCEDURE: 1. Leaving A-1 well as substrate blank. 2. Add 1A50 ul of negative and positive control in 2 wells each i.e. B-l, C-l, and D-l, E-l. Then adding 50 ul of the samples in the microwells. Add 100 ul of working conjugate solution. Incubate at 37°C for 1 hour. 3. Washing the plate 5 times with working wash solution. 4. Add 100 ul of working conjugate solution in each well including blank well. 5. Washing the plate 5 times with working wash solution. 6. Incubate at room temperature for 30 min. in dark. 7. Add 50 ul of stop solution. 8. Read absorbance at 450 nm. CALCULATION OF THE RESULT: Test Validity : Blank must be Negative Control (NC) must be O.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 TB antibodies (IgM, IgA, IgG) 2. Test specimens with absorbance value greater than or equal to the Cut off value are reactive TB antibodies (IgM, IgA, IgG). 3. It seems reasonable to define arrange of +1-20% around the value of the Cut off as gray zone.In such case the repetition of the test with the same serum of 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. While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in are 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 this invention. I Claim 1. A kit for analyzing the presence of IgM, IgA, IgG antibodies of Mycobacterium Tuberculosis in human serum or plasma comprising microwells coated with TB antigens; enzyme conjugate ( TB antigen linked with HRPO); substrate buffer and chromogen. 2. A kit for analyzing the presence of IgM, IgA, IgG antibodies of Mycobacterium Tuberculosis in human serum or plasma as claimed in claim 1 where in the said microwell are prepared by a process comphsing: a. taking 50mM phoshate buffer pH 6.0 to 8.0; b. adding TB antigen or their combination (1-254) preferably 10 nanogram to 1 micro gram per microwell; c. dispensing 100ml of antigen solution in microwell plate; d. incubating for 1 to 5 hrs at 25°C - 37°C; e. washing the coated plates with 10mM sodium phosphate buffer pH 6.0 - 8.0; f. blocking the plates with 0.5-2.0% BSA Solution; g. incubating for 12- 30 hours at 2-8°C; h. drying at 37°C for 2 to 8 hours; i. storing in descents pouches at 2-3°C. 3. A kit for analyzing the presence of IgM, IgA, IgG antibodies of Mycobacterium Tuberculosis in human serum or plasma as claimed in claim 1 where in buffer comprises 250mM sodium phosphate buffer, 0.15M saline having pH 6.0- 8.0 and0.01%Tween-20. 4. A kit for analyzing the presence of IgM, IgA, IgG antibodies of Mycobacterium Tuberculosis in human serum or plasma as claimed in claim 1, where in the enzyme conjugate is prepared by linking TB antigen with HRPO by linking method as given below: a. dissolving the 15mg HRP in 10mM PBS pH 7.0- 7.5 at a concentration of 20mg/ml; b. adding 5mg of Sulfo- SMCC [ Sulfo Succinimidyl 4-(N-Maleimidomethyl) Cyclohexane - 1- Carbooxylate] to the HRP solution and mixing and reacting for 30 minutes at room temperature; c. Immediately purifying the Sulfo-SMCC activated HRP from dialysis or desalting column adjust the HRP concentration to 10mg/ml for the conjugation; d. dissolving above TB antigen and ambination ( same antigen which is coated on the plate) in 100Mm PBS pH 7.2+ 10mM EDTA in 1 - 5 mg /ml; e. adding 10 molar excess of Traut Reagent to each of antigen and mix; f. incubating for 37°C for 1-2 hours; g. purifying the reduced TB antigens using dialysis against lOOmM PBS pH 7.2 for 12-18 hours; h. mixing the reduced TB antigen with activated HRP and incubate for 1 hour at 37°C and further purified by dialysis and store at 2- 8°C. 5. A kit for analyzing the presence of IgM, IgA, IgG antibodies of Mycobacterium Tuberculosis in human serum or plasma as claimed in claim 1, where in the TMB concentrate comprising 1- 3 gm of TMB ( Tetra methyl benzidene) powder dissolved in 100 ml of Dimethyl Sulfoxide. 6. A kit for analyzing the presence of IgM, IgA, IgG antibodies of Mycobacterium Tuberculosis in human serum or plasma as claimed in claim 1, where in the substrate solution is TMB concentrate diluted in 1:100 in TMB diluent. 7. A kit for analyzing the presence of IgM, IgA, IgG antibodies of Mycobacterium Tuberculosis in human serum or plasma as claimed in claim 1, where in TMB diluent comprises 100mM acetate buffer and 0,.005% Hydrogen peroxide. 8. A kit for analyzing the presence of IgM, IgA, IgG antibodies of Mycobacterium Tuberculosis in human serum or plasma as claimed in claim 1, where in the negative control is dilute normal human sera (1:100-1:1000) in PBS. 9. A kit for analyzing the presence of IgM, IgA, IgG antibodies of Mycobacterium Tuberculosis in human serum or plasma as claimed in claim 1, where in the positive control is dilute TB positive human sera ( 1: 100 - 1: 1000) in PBS. 6. A kit for analyzing the presence of IgM, IgA, IgG antibodies of Mycobacterium Tuberculosis in human serum or plasma as claimed in claim 1, where in substantially described with the help of accompanying drawing.

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449-del-2006-abstract.pdf

449-DEL-2006-Claims-(10-10-2008).pdf

449-del-2006-claims.pdf

449-DEL-2006-Correspondence-Others-(10-10-2008).pdf

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

449-del-2006-correspondence-others.pdf

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

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

449-del-2006-drawings.pdf

449-DEL-2006-Form-1-(10-10-2008).pdf

449-del-2006-form-1.pdf

449-del-2006-form-18.pdf

449-DEL-2006-Form-2-(10-10-2008).pdf

449-del-2006-form-2.pdf

449-del-2006-form-26.pdf

449-del-2006-form-3.pdf

449-del-2006-form-5.pdf

449-del-2006-form-9.pdf