FORM 2
THE PATENT ACT 197 0 (39 of'1970)
The Patents Rules, 2003 COMPLETE SPECIFICATION See Section 10, and rule 13)
TITLE OF INVENTION
AN IMMUNOASSAY KIT FOR SCREENING AND DETECTION OF TB IgG ELISA KIT FOR DETECTION OF ANTIBODIES TO M. Tuberculosis IN HUMAN SERUM AND PLASMA AND A METHOD OF MANUFACTURING THE SAME

2. APPLICANT(S)
a) Name
b) Nationality
c) Address

TRANSASIA BIO-MEDICALS LTD.
INDIAN Company
TRANSASIA HOUSE,
8, CHANDIVALI STUDIO ROAD,
MUMBAI - 400 072
MAHARASHTRA

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

A SOLID PHASE IMMUNOASSAY KIT FOR DETECTION OF ANTIBODIES AGAINST MICROBACTERIUM TUBERCULOSIS
FIELD OF THE INVENTION
The present invention relates to a solid phase immunoassays test kit and method for detection of presence of antibodies in a patient to disease-related to antigens of Mycobacterium tuberculosis.
DESCRIPTION OF THE RELATED ART
Antibodies are naturally produced biomolecules that specifically react with foreign biomolecules called antigens. Disease-related microbial infections, e.g., Mycobacterium tuberculosis, which causes tuberculosis (TB), are usually characterized by the production of antibodies to the specific disease-related antigens. Antibodies are also produced with other diseases and afflictions, e.g., autoimmune diseases, wherein there is often a destructive antibody response to the host. In the case of autoimmune diseases, the host supplies the disease-related antigen, a host tissue. In this case, the corresponding host antibody synthesis may have been initiated by either a foreign substance or by a host tissue not normally encountered by the host's immune system. Subsequent antibody production may proceed in the absence of the foreign substance, due to similar structural nature of the host tissue. Herein the term "disease-related antigen" includes microbial antigens and antigens associated with the host antibody response in autoimmune diseases.
Infectious diseases are widespread in the world. Military personnel, including Navy and Marine personnel, are at particular risk because of their global deployment. According to the World Health Organization, the infectious disease tuberculosis caused by Mycobacterium tuberculosis (Mycobacterium) kills about three million people every year.

There are several conventional tests for mycobacterium, however these conventional tests all have disadvantages.
One conventional test is a purified protein derivative (PPD) skin test. In this lest, a PPD is injected subcutaneously into a patient, and after 2 to 3 days, the site of injection is observed to determine whether there is hardening and/or discoloration of the patient's skin. This test is believed to be only about 50-75% accurate. In persons that have been immunized with Mycobacterium bovis BCG, there are a high percentage of false positives. Another disadvantage of the PPD skin test is that it requires a needle injection, and patient compliance is low in coming back for observation by trained medical personnel for hardening and/or discoloration. This test is typically used as a screening test for people who do not have symptoms of tuberculosis. The specificity of purified protein derivative (PPD) skin test for the detection of tuberculosis infection is compromised by exposure to environmental mycobacteria.
Another conventional test is a culture test. However, this test takes up to 6 to 8 weeks since that is how long it takes for the mycobacterium organism to grow to an amount that is visibly detectable. Another disadvantage of the culture test is that typically only about half of all samples wherein the Mycobacterium tuberculosis is present actually grow to the point where it is detectable. Thus, this method has about 50% false negatives.
Another conventional test is a chest x-ray. However, this method does not work if the person undergoing the chest x-ray has been recently infected with Mycobacterium tuberculosis. Another disadvantage is that the chest x-ray can be mis-read. Other disadvantages are the cost of the x-ray and need to subject a person to x-rays.
Another conventional test is an acid fast staining test. This method detects the most infectious patients but not those with extrapulmonary disease. In this method,

patient sputum is smeared onto a glass slide, and a microscopic chemical stain for acid fast bacilli (AFB) is performed using the Ziehl Nielson method. The sample is then viewed under a microscope for the presence of the red staining acid-fast mycobacterium organisms. This test requires hours to complete and a high level of expertise of the microbiologist running the test. Only after sputum tests on three separate occasions have come up negative is the patient considered negative for AFB. Examination of three sputum samples detects up to 87% of pulmonary tuberculosis patients, but a considerably lower percentage of patients (43%) with extrapulmonary disease.
Another test is a MycoDot test. The MycoDot test detects anti-lipoarabinomannan IgG antibodies in serum/whole blood to diagnose cases of active TB. The MycoDot test employs the lipoarabinomannan (LAM) antigen bound to plastic combs. During incubation of the comb in the diluted serum sample for 6 minutes, the free anti-LAM antibodies, if present in the serum/blood of the individual, will bind to the antigen on the comb. Next, the excess antibodies that might be loosely attached to the comb are washed off. During incubation of the comb in a colloidal gold signal reagent for 10 minutes, the bound anti-LAM antibodies on the comb, if present, then bind to the protein ligand attached to the gold particles, providing a pink coloured aggregate at the antigen spot on the comb. Next, any excess signal reagent is washed off the comb. The presence of a pink coloured spot on the comb, generated as a result of the steps described above, is indicative of a positive reaction due to active TB, However, the test requires either serum or blood and takes at least 20 minutes to perform. It is also not a portable test and requires a laboratory facility to perform.
There are several screening tests for tuberculosis. The Mantoux test uses tuberculin purified protein derivative (PPD), which is injected intracutaneously. A delayed hypersensitivity reaction develops in individuals having previous exposure to Mycobacterium tuberculosis. The injection site is normally read within 48 to 72 hours after intracutaneous injection of the antigen; a palpable induration measuring

10 mm in diameter or more is considered a positive reaction. This procedure is accepted as an aid in the diagnosis of tuberculosis infection.
The Heaftest uses a multiple puncture disk, which introduces needles through concentrated Old Tuberculin applied to the skin. The tine test uses tuberculin adhering to metal tines, which is then inoculated by simple pressure into the skin. The Heat and tine tests are acceptable for screening but should be confirmed by the Mantoux test, Antigenic material can also be applied by scratch, i.e., Pirquet's test. Similar to the Mantoux test, these invasive tests generally require 48 to 72 hours after inoculation before results can be determined. The Bacillus of Calmette and Guerin (BCG) is a live, attenuated strain of Mycobacterium bovis, which has been used with varying success as a vaccine against tuberculosis in countries where the prevalence of tuberculosis is high. BCG causes tuberculin conversion to positive; it has also been used to stimulate the immune system against a variety of medical conditions. Both PPD and BCG can serve as suitable antigens to detect general mycobacterial infections with this method.
Other antigens have been described. Maes has described A60-antigen from mycobacteria and use thereof as tuberculin vaccine in U.S. Pat. No. 4,965,192. This patent describes the A60-antigen as being effective for detecting prior exposure of an individual to mycobacterial infections through the use of a cutaneous test. This patent is similar to other invasive inoculation tests mentioned earlier except that a new antigen is used and 24 to 48 hours are required to observe the responses at the test site.
To summarize, the conventional serological methods do not provide a non-invasive, rapid diagnostic test that measures exposure to mycobacteria, A simple, rapid and non-invasive screening method would be especially useful in evaluating mycobacterium exposure.

BRIEF SUMMARY OF THE INVENTION
The solid phase immunoassay kit and method of the present invention provides a rapid test to determine the presence of different anti-M. Tb. antibodies in the serum, CSF and in sputum. A set of M. tuberculosis specific antigens are first time, which absent in BCG vaccine strain and are proprietary to applicant of the present invention, have been used in the kit of the present invention. Anti-M. Tb. antibodies are absent in healthy individuals. Patients, suffering form tuberculosis infection, show presence of IgG antibodies and these antibodies can be detected by the kit of the present invention.
Subsequent to M. tuberculosis infection, serologicai analysis ensures the presence of antibodies to M. tuberculosis proteins. The kit of the present invention is a solid phase immunoassay, utilizing a mixture of M tuberculosis specific recombinant protein antigens for detection of M. tuberculosis antibodies present in human serum and/or plasma.
More specifically, the present invention comprises the kit and method for detecting the presence of at least one predesignated, target IgG antibody to a mycobacterium in a sample selected from one or more patient bodily fluids. The kit comprises a mixture of recombinant proteins of M. tuberculosis, that are absent in BCG vaccine, immobilized in bicarbonate buffer on to a solid support, blocked with phosphate buffer and stabilized using stabilizing solution, comprising PBS. The kit of the present invention further comprises a conjugate, an anti-TB positive control, a TB negative control, a colour reagent, a sample diluent, a stopping solution and a washing solution. The present invention also relates to a method for preparing a solid phase immunoassay kit for detection of anti-M. Tb. IgG antibodies in the human serum and/or plasma. The method comprises immobilizing on to a solid support a plurality of layers of mixture of recombinant proteins ofM tuberculosis in bicarbonate buffer; blocking said antigens using blocking solution, comprising phosphate buffer and stabilising using stabilizing solution, comprising PBS. The

present invention still further relates to an in vitro serological method for solid phase immunoassay for detection of anti-M. Tb. IgG antibodies in the human serum and/or plasma. The method comprises contacting the sample of one or more patient bodily fluids with at least one mycobacterium specific recombinant antigens that are absent in BCG vaccine strain and first time identified by the applicant of the present invention, on a solid support to bind to the target IgG antibody in the sample; previously, simultaneously or subsequently to step of contacting, binding the at least one said mycobacterial specific antigen with a conjugated label, thereby reducing amount of substrate of the enzyme linked to the conjugate; and detecting the enzyme activity, whereby the presence of the target IgG antibody is determined in the sample.
In one or more preferred embodiment, said bodily fluids may be selected from the group consisting of saliva, oral rinse expectorant, oral fluid including oral mucosal transudate and gingival crevicular fluid, urine, sweat, tears, blood, serum, stool, gastric fluid, synovial fluid, phlegm, culture media and other clinical and laboratory specimens and samples. In the most preferred embodiment, the one or more bodily fluids used in the method are serum and/or plasma.
In another preferred embodiment, the sample is added in to the wells of the microtitre plate as a solid support. The anti-M. Tb. IgG antibodies present in the sample bind to a mycobacterium specific recombinant protein antigen immobilized on to the solid support. The conjugate label binds to IgG antibodies in the sample to form complexes. The complexes of mycobacterium specific IgG antibodies, if present, bind to the at least one mycobacterium antigen that is immobilized on to the plate. Formation of this labelled conjugate antibody complex with its mycobacterial antigen results in a detectable colour change, indicating a positive result that mycobacterium specific antibodies are present in the sample.
The results of the present invention can be used to evaluate immunization status of a patient. For example, if the sample tests positive, and the sample came from a person

who has received a TB vaccine, then the positive result will indicate that the appropriate immune response was elicited from that person, particularly if there are no other indications or symptoms of TB in the person. If the sample tests negative, and the sample came from a person who has received a TB vaccine, then the negative result will indicate that the person has not been properly immunized.
Thus, the immunoassays of the present invention provide markers of vaccine status by detecting specific mycobacterium antibodies in patient bodily fluids. Possible long-term applications of this diagnostic kit include continuing field surveillance of vaccinated personnel to verify protective immunization and to assist in the determination of the optimum dose schedule for vaccination. This immunoassay method may also be employed to monitor personnel and to follow the progression and treatment of TB.
DETAILED DESCRIPTION OF THE INVENTION
The present invention preferably relates to the solid phase immunoassay kit, utilizing a mixture of protein antigens ofM tuberculosis for the detection anti-M. Tb. IgG antibodies present in the human serum and/or plasma.
The present invention more preferably relates to the solid phase diagnostic kit comprising a mixture of mycobacterium specific recombinant protein antigens of M. tuberculosis for the detection anti-M. Tb. IgG antibodies present in the human serum and/or plasma.
The present invention most preferably relates to the diagnostic kit for the detection of anti-M. Tb. IgG antibodies in the in the human serum and/or plasma, which comprises plurality of coating on the solid support of mixture of recombinant protein antigens specific to M. tuberculosis that are absent in BCG vaccine strain and first time identified by the applicant of the present.

More specifically, the present invention relates to the diagnostic kit for the detection of anti-M. TB. IgG antibodies in the human serum and/or plasma comprising a mixture of mycobacterium specific recombinant protein of M. tuberculosis immobilized on to the solid support, such as microtitre plate; an enzyme linked conjugate; an anti-TB positive control; a TB negative control; a colour reagent; a sample diluent; a stopping solution and a washing solution, wherein said solid support having at least three coatings of a homogenous mixture of said mycobacterium specific recombinant protein antigens.
Most specifically, the present invention relates to the diagnostic kit comprising a microtitre plate immobilized with plurality of coating of mixture of mycobacterium specific recombinant M. tuberculosis protein antigens, such as, those which are absent in BCG vaccine and first time identified by the applicant; an enzyme linked conjugate, such as, goat anti-human IgG-HRPO. The said recombinant mycobacterium specific protein antigens are immobilized in bicarbonate buffer, blocked in blocking solution, comprising phosphate buffer and Bovine Serum Albumin and stabilized with stabilizing solution, comprising PBS.
In a preferred aspect of the invention, the buffer solution used in the present invention is selected form the phosphate group mixed with surfactant, stabilizer, sodium chloride and preservative.
Another buffer used in the present invention is selected from the group consisting ofdisodium hydrogen phosphate and sodium dihydrogen phosphate having molar ratio of 8-12 irtillimolar each.
The stabilizer used in the said buffer solution is a protein stabilizer preferably bovine serum albumin.

The surfactant may be selected from the non-ionic, anionic and zwitterionic surfactant; preferably the surfactant used is non-ionic surfactant, The preservative used in the said buffer solution is selected from thimerosal and gentamycin.
In a preferred aspect of the invention, the enzyme linked conjugate comprises goat anti-human IgG-HRPO in conjugate diluent with thimerosal and gentamycin.
In another preferred aspect of the invention, the anti-TB positive control is an inactivated anti-TB containing human serum along with thimerosal and gentamycin.
In still preferred aspect of the invention, the TB negative control is an inactivated normal human serum along with thimerosal and gentamycin.
In further aspect of the invention, the colour reagent is 3,3', 5,5'-tetra methyl benzidine dimethyl sulfoxide along with H2O2, thimerosal and gentamycin.
In another aspect of the invention, the sample diluent is a phosphate buffer along with BSA, Tween-20, Triton X 100, MgC12, thimerosal, gentamycin and bovine immunoglobulin.
In still another aspect of the invention, the stopping solution is a concentrated phosphoric acid and deionized water.
In a preferred aspect of the invention, the washing solution concentrate is a TRIS buffer along with NaCI and Tween-20 in deionized water,
According to another object of the present invention, a method for preparing a solid phase immunoassay kit for the detection of anti-M. Tb. IgG antibodies of M. tuberculosis comprises: immobilizing on to a solid support a mixture of mycobacterium specific recombinant protein antigens of M. tuberculosis, such as, those which are absent in BCG vaccine strain and first time identified by the

applicant of the present invention, in bicarbonate buffer, blocking using blocking solution, containing phosphate buffer and stabilizing using stabilizing solution; providing a coupling product as an enzyme linked conjugate obtained by covalently binding an goat anti-human IgG-HRPO and providing solutions of an anti-TB positive control, a TB negative control, a colouring reagent, a sample diluent, a stopping solution and a washing solution.
In preferred embodiment of the said process, the immobilized antigens are mycobacterium specific recombinant protein of M. tuberculosis, which are absent in BCG vaccine strain and first time identified by the applicant of the present invention
In another preferred embodiment of the said process, the enzyme linked conjugate is goat anti-human IgG-HRPO.
In still preferred embodiment of the invention, the method comprises preparing a solution of an inactivated anti-TB containing human serum in thimerosal and gentamycin as an anti-TB positive control.
In still another preferred embodiment of the invention, the method comprises preparing a solution of an inactivated normal human serum in thimerosal and gentamycin as a TB negative control.
In preferred embodiment of the method, preparing the colouring reagent comprises preparing a solution of 3,3', 5,5'-tetra methyl benzidine dimethyl sulfoxide in H202, thimerosal and gentamycin.
In one more embodiment of the invention, the method comprises preparing a solution of a sample diluent containing a phosphate buffer, BSA, Tween-20, Triton X 100, MgC12, thimerosal, gentamycin and bovine immuno globulin.

In a separate embodiment of the invention, the method comprises preparing a solution of a stopping solution containing a concentrated phosphoric acid and deionized water.
In a different embodiment of the invention, the method comprises preparing a solution of a washing solution concentrate containing a TR1S buffer, NaCI, Tween-20 and deionized water.
Pursuant to distinct object of the invention, the present invention also provides an in vitro serological immunoassay method for detection of anti-M. Tb. IgG antibodies in the human serum and/or plasma using the solid phase immunoassay diagnostic kit of the present invention. The method comprises contacting a liquid sample, for example, serum and/or plasma, containing antibodies against M. tuberculosis to at least one of the antigen from a mixture of mycobacterium specific recombinant protein antigen bound to the surface of a microtitre plate used as a solid support. The liquid sample is then incubated with this immobilized solid support. After washing, the foregoing solid phase is contacted with covalently linked enzyme of the conjugate in the liquid phase. After removing unbound fraction, determining the enzyme activity for the presence of anti-M. Tb. IgG antibodies in the sample.
In a preferred embodiment of the invention, the method for the detection of anti-M. Tb. IgG antibodies comprises: providing a mixture of mycobacterium specific recombinant protein antigens of M. tuberculosis, which are absent in BGC vaccine strain immobilized on to the solid support as a solid phase and an enzyme linked conjugate obtained by covalently binding a goat anti-human IgG-HRPO to an enzyme; contacting a given quantity of liquid sample containing anti-M. Tb. IgG antibodies with said solid phase antigens, thereby forming a stable complex and deterrnining the enzyme activity of the conjugate added for the presence of anti-M. Tb. IgG antibodies.

The method for the detection of anti-M. Tb.IgG antibodies using the diagnostic kit of the present invention most preferably, comprises: providing a mixture of mycobacterium specific recombinant protein antigens of M tuberculosis, such as, those which are not present in BCG vaccine and first time identified by the applicant of the present invention, immobilized on to the microtitre plate; providing a coupling product as an enzyme linked conjugate, such as, goat anti-human IgG-HRPO; contacting a liquid sample under testing with said immobilized antigens, thereby forming a stable complex with anti-M. Tb, IgG antibodies present in the sample or anti-TB positive control and determining the enzyme activity for the presence of anti-M. Tb. IgG antibodies.
The solid support to which antigen is bound may be any water-insoluble, water-in suspensible, solid support. An example of suitable solid support includes microtitre plate. The immunological component may be bound to the solid support by covalent bonds or by adsorption. The advantage of the use of a solid support is that no centrifugation is required for the separation of solid and liquid phases.
The enzyme linked conjugate consists of the immunological component covalently linked to one or more enzyme molecules. Such linking can be achieved either by direct condensation or by using external bridging molecules, in accordance with methods those that are known to skilled in the art.
The choice of the enzyme that is to form a part of the coupling product is specific binding activity. For example, a peroxidase is an enzyme that can be suitably used in the method of the present invention. Preferably, horseradish peroxidase is used.
The assay of the present invention is a rapid, sensitive and specific screening test. It can be suitable for field use by ancillary medical personnel. No electrical equipment is required to perform the method of the present invention.

Obviously, many modifications and variations of the present invention are possible in light of the above teaching. It is therefore to be understood that, within the scope of the appended claims, the invention may be practiced otherwise than as specifically described. The principles described above can be readily modified or adapted for various applications without departing from the generic concept, and therefore such adaptations and modifications are intended to be comprehended within the meaning and range of equivalents of the enclosed embodiments. It is to be understood that the terminology and phraseology herein is for the purpose of description and not of limitation.
EXAMPLE
The following examples serve to illustrate the use of the invention, but are not to be regarded as limitations for the scope of the invention:
Example: 1 - Detection of anti-M. Tb. IgG
When human plasma and/or serum is added to sample diluent containing wells, the bound mycobacterium specific recombinant antigens will form a stable complex, if anti-M. Tb. IgG antibodies are present. Followed by a wash step, goat anti-human-IgG-HRPO is added to the wells. A second wash step will remove free goat-anti-human-IgG-HRPO. Colour reagent containing the substrate of HRPO is then added to the wells. Wells containing negative control sample will remain colourless and blue colour will develop in wells containing positive control and test specimens containing anti-M. Tb. IgG. Upon addition of stop solution, blue colour changes to yellow. The intensity of yellow colour is directly proportional to the amount of the bound anti-M. Tb. IgG antibodies to the well.

Example: 2 - Test procedure for detection of anti-M. Tb. IgG
(i) Bring all the reagents and test specimens at room temperature before use; (ii) except the blank, add lOOul of sample diluent to each well, in each run, there will be one blank, one negative control and three positive controls, add l0 ml of control and test specimens to the respective wells, mix properly with pipettor and cover the plate with black cover and incubate 1 hour at 20-30°C; (iii) wash the plate as per microplate washing procedure known to person skilled in the art; (iv) add 50ul conjugate to each well (except blank well), cover the plate with black cover and incubate for 15 minutes at 20-30°C; (v) repeat step (iii); (vi) add 50^1 of colour reagent to each well and cover the plate with black cover and incubate for 15 minutes in dark at 20-30°C ,• (vii) add 100ml of stopping buffer to each well; and (viii) read absorbance at 450 nm and deduct the blank absorbance from the control and test wells.
Example: 3 - Calculation for cut-off value determination
Blank value: Absorbance of blank values should be less than 0.1- Positive Control; Absorbance of individual positive control should be grater than 0.2. Negative Control: Absorbance of individual negative control should be less than 0.1. PCx:
Average value of positive controls.
Calculation of PCx: For example:
PC Absorbance
1 0.270
2 0.275
3 0.280 PCx: (0.27+0.275+0.28)/3-0.275

Cut-off value formula; 1.0 x PCx Cut-off vale: 1.0 x 0.275=0.275
Interpretation of results:
Non-reactive: If the absorbance of the test serum and/or plasma is less than the 0.8 times of cut-off value, then it is considered as non-reactive.
Doubtful: If the absorbance of the test serum is in between 0.8 time to 1.0 times of cut-off value, then the sample is considered as doubtful,
Reactive: If the absorbance of the test serum and/or plasma is greater than the cutoff value, then it is considered as initial reactive.

CLAIM:
1. A solid phase immunoassay kit for detection of target antibodies against M. tuberculosis antigens in a sample selected from one or more patient bodily fluids comprises: a solid support containing immobilized on to it a mixture of mycobacterium specific recombinant protein antigens; an enzyme conjugate; and immunochemically acceptable regents required for the detection of the antibodies against M. tuberculosis.
2. The immunoassay kit as claimed in claim 1, wherein the target antibodies against M. tuberculosis antigens are anti-M.Tb. IgG antibodies.
3. The immunoassay kit as claimed in claim 1, wherein the immobilized antigens selected are mycobacterium specific recombinant protein antigens that are absent in BCG vaccine strain,
4. The immunoassay kit as claimed in claim 1, wherein the mycobacterium specific recombinant antigens that are absent in BCG vaccine strain are first time identified by the applicant of the present invention.
5. The immunoassay kit as claimed in claim 1, wherein the immunochemically acceptable reagents are selected form the group consisting of a conjugate, an anti-TB positive control, a TB negative control, a colour reagent, a sample diluent, a stopping solution and a washing solution.
6. The immunoassay kit as claimed in claim 1, wherein the bodily fluids selected from the group consisting of saliva, oral rinse expectorant, oral fluid including oral mucosal transudate and gingival crevicular fluid, urine, sweat, tears, blood, serum, stool, gastric fluid, synovial fluid, phlegm, culture media and other clinical and laboratory specimens and samples.

7. The immunoassay kit as claimed in claim 1, wherein the bodily fluid selected is human serum and/ or plasma.
8. The immunoassay kit as claimed in claim 1, wherein the solid support used for immobilizing the recombinant antigens is microtitre plate.
9. The immunoassay kit as claimed in claim 1, wherein the microtitre plate comprises plurality of coating of mixture of recombinant protein antigens specific to M. tuberculosis.
10. The immunoassay kit as claimed in claim 1, wherein the microtitre plate comprises at least three coatings of a homogenous mixture of said recombinant antigens.
11. The immunoassay kit as claimed in claim 1, wherein the enzyme linked conjugate comprises goat anti-human IgG-HRPO in conjugate diluent along with thimerosal and gentamycin.
12. The immunoassay kit as claimed in claim 1, wherein the anti-TB positive control is an inactivated anti-TB containing human serum with thimerosal and gentamycin.
13. The immunoassay kit as claimed in claim 1, wherein the TB negative control is an inactivated normal human serum with thimerosal and gentamycin.
14. The immunoassay kit as claimed in claim 1, wherein the colour reagent is 3,3'/ 5,5'-tetra methyl benzidine dimethyl sulfoxide in H2O2 with thimerosal and gentamycin.

15. The immunoassay kit as claimed in claim 1, wherein the sample diluent is phosphate buffer with BSA, Tween-20, Triton X 100, MgC12, thimerosal, gentamycin and bovine immunoglobulin.
16. The immunoassay kit as claimed in claim 1, wherein the stopping solution is concentrated phosphoric acid in deionized water.
17. The immunoassay kit as claimed in claim 1, wherein the washing solution concentrate is TRIS buffer with NaCI and Tween-20 in deionized water.
18. A method for preparing a solid phase immunoassay kit for detection of anti-M. Tb. IgG antibodies of M tuberculosis as claimed in claims 1 to 16 comprises: immobilizing on to a solid support a mixture of mycobacterium specific recombinant protein antigens ofM tuberculosis, such as, those which are absent in BCG vaccine strain and first time identified by the applicant of the present invention in bicarbonate buffer; blocking and stabilizing said antigens using blocking and stabilizing solution, respectively; providing a coupling product as an enzyme linked conjugate obtained by covalently binding an goat anti-human IgG-HRPO and providing solutions of anti-TB positive control, TB negative control, colouring reagent, sample diluent, stopping solution and washing solution.

19. An in vitro serological immunoassay method for detection of anti-M. Tb. IgG antibodies in human serum and/or plasma using the solid phase immunoassay kit as claimed in claims 1 to 16 comprises contacting a liquid sample containing anti-M. Tb. IgG antibodies against M. tuberculosis to at least one of the antigen from a mixture of mycobacterium specific recombinant protein antigens bound to the surface of a microtitre plate; followed by washing, contacting said antigens with covalently linked enzyme of the conjugate; followed by second washing, determining the enzyme activity for the presence of anti-M. Tb. IgG antibodies in the sample.
Dated this 3rd day of August, 2006.