FORM 2
THE PATENT ACT 1970 (39 of 1970)
The Patents Rules, 2003 COMPLETE SPECIFICATION
See Section 10, and rule 13


TITLE OF INVENTION
A SOLID PHASE IMMUNOASSAY KIT FOR THE DETECTION OF ANTIBODIES
TO M. TUBERCULOSIS

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 : -



2009

A solid phase immunoassay kit for the detection of antibodies to M. tuberculosis

Field of the invention :
The instant invention in principle relates to a serological analysis of human serum and plasma for the detection of IgG and IgM antibodies to Mycobacterium tuberculosis proteins. Preferably, the invention relates to an in vitro diagnostic kit for the detection of IgG and IgM antibodies directed against the proteins of M. tuberculosis in the human serum and plasma. The invention also relates to a method for the preparation of solid phase immunoassay kit for the detection of IgG and IgM antibodies to M. tuberculosis proteins in the human serum and plasma. Further, the invention relates to an in vitro solid phase immunoassay method for the detection of IgG and IgM antibodies to M. tuberculosis proteins in the human serum and plasma samples.
Background and prior art of the invention :
Significant number of population in the world is being infected regularly with_M.
tuberculosis. Patients suffering from tuberculosis infection show presence of different
anti-M tuberculosis antibodies (anti-MTB) in serum, CSF & in Sputum. A set of M.
tuberculosis specific synthetic peptides has been synthesized and used in the kits for
detecting anti-MTB in the serum and plasma. Anti-MTB antibodies are absent in healthy
individuals. Patients suffering from tuberculosis infection show the presence of
IgG+IgM antibodies and these antibodies can be detected. Subsequent to M. tuberculosis
infection, serological analysis of human serum and plasma ensures the presence of said
antibodies to M. tuberculosis proteins. Therefore, it is highly desirable to detect said
antibodies to M. tuberculosis in the human serum and plasma. An appropriate technique
for separating and detecting said antibodies to M. tuberculosis in the human serum and
plasma is solid phase immunoassay, which utilizes synthetic peptides cr antigens
covalently bound or physically adsorbed to an insoluble matrix. The antibody-antigen
complex so formed is then held by solid phase technique and bound fraction can be
easily detected and estimated using colour reagent.
2
21 APR 2009

A living system responds to the presence of foreign antigen like protein, virus, bacteria, etc by producing specific antibody against that particular antigen. Then, there is a specific reaction between antibody and antigen to form a complex. An antibody once produced is also capable of binding a hapten that is relatively a small and simple molecule, which may be determinant group of given antigen and is capable of binding with specific antibody, but incapable of inducing an antibody production, unless it is bound to an antigen carrier. The binding interaction between antigen or hapten and its antibody is specific and sensitive. Other materials that show similar specific and sensitive binding interactions are enzymes and their substrates, hormones, vitamins, metabolites and pharmacological agents and their receptors or binding substances and such other substances known in the art. These specific and sensitive binding reactions have given rise to a rapidly emerging analytical teclmique known as specific binding assay technique. In one such assay, the substances or group of substances to be determined, which may be referred as ligand, in a liquid sample is placed in competition with labelled form of the ligand or of binding analogue thereof for binding to binding reagent. Where an enzyme label is used and the binding reagent is antibody, the technique is known as an enzyme-immunoassay technique. Several alternative labelling materials are available for substituting the enzymes, such as radioisotopes, co¬enzymes, enzyme substrates, enzyme-modulators like inhibitors and allosteric effectors, fluorescent molecules, and luminescent molecules, but these have inherent disadvantages of handling and test requires sophisticated instruments and trained manpower for accurate results.
The above system consists of antigen or hapten labelled with an enzyme marker, unlabelled native antigen in test sample and specific antibody, thereby there is competition between unlabelled antigen and labelled antigens for binding to limited amount of antibodies. Hence, greater the concentration of unlabelled antigen from the test sample less the labelled antigen will be bound to the antibodies. If the concentration of labelled antigen and antibody is fixed and the only variable is the level of unlabelled
3
21 APR 2009

antigen, it becomes possible to establish an assay system for detecting unknown level of unlabelled antigens by physically separating the antigen-antibody complex from the remaining free antigens. The enzyme activity of the unknown is compared with standard values given by the range of known amount of antigens treated in the same manner.
In the prior art, many techniques are known for separating free unbound antigens or haptens from the complexes of antigen-antibody. One such technique is chromato-ekctrophoresis, which combines paper chromatography and paper electrophoresis. The paper with a high affinity for the free antigens, like Whatman paper, is used as carriers. This technique is discriminative and has been used in tlie assay of insulin, growth hormone, glucagons, parathyroid hormone, thyroid stimulating hormone and other peptide hormones, but it has number of disadvantages, which limit its use. One such disadvantage is limited amount of material can be applied to the absorbent and the separation and detection is laborious and time-consuming.
Other known technique is precipitation of antigen-antibody complexes, which involves use of salts, organic material or solvents under the conditions that do not affect free antigens. Among these, salts, materials and solvents used are ethanol, acetone, sodium sulphate, ammonium sulphate, dioxane, trichloroacetic acid, polyethylene glycol, etc. The use of salts, solvents or organic materials has advantage that the separation is immediate, and second incubation is not necessary, but the chemical precipitation technique causes co-precipitation of other proteins, which causes incomplete separation of two fractions.
The double antibody technique is also known and widely used for the separation of bound and free antigens. Using this technique, a second antibody that was raised against the first antibody is used to precipitate the primaiy antigen-antibody complex. Particularly, if the first antibody was raised in rabbit then the second antibody may be
4
21 APR 2009

an antiserum to rabbit gamma globulin raised in goats, but the disadvantage of this technique is that use of second antibody requires an additional incubation. Ion exchange and other resins are also known to use for binding free antigens by electrostatic forces and mainly used for the detection of small molecules, such as thyroid hormones. One technique of this type used for the separation of antigen-antibody complex from either free antigen employs a column packed with material that preferably adsorbs either free antigen or antigen-antibody complex. The incubated aqueous mixture is applied on to the head of such column and the column is then eluted. The radioactivity of either the column or the eluate is then determined and the content of the antigen in the starting solution is calculated from the count.
By yet another technique, free unbound antigens adsorbed onto adsorbent and then precipitated by cenrrifugation. Powdered talc (magnesium silicate), kaolin (aluminium silicate), QUSO (silica micro-granules), cellulose powder, etc are some of the simple adsorbents used for precipitation. Many separations are performed using adsorbent charcoal coated with dextran, which behaves rather like a sieve that allows the smaller molecules of free antigen to pass and these are bound by the charcoal, leaving the bound antigen in the solution, after the charcoal has been removed by cenrrifugation or filtration.
Also, the solid phase techniques are known for the separation of free and bound antigens that use antibodies covalently bound or physically adsorbed onto insoluble matrix. The formed antibody-antigen complex is held by the solid phase and the bound fraction can be easily separated from the free fraction by filtration.
In order to overcome aforesaid drawbacks, an in vitro serological method comprising detecting anti-MTB, such as IgG, IgM or both, in the human plasma and/or serum using ELISA technique may be used. Therefore, the inventors of the present invention have invented a solid phase immunoassay kit that utilizes a mixture of M. tuberculosis
5
21 APR 2009

synthetic peptides for the detection M. tuberculosis antibodies present in human serum and plasma, wherein said synthetic peptides of M. tuberculosis are noncovalently bonded with a solid support for separating and detecting the antibodies to M. tuberculosis proteins. An advantage of using the solid support like microwells containing microplate is that no centrifugation or filtration required for the separation of solid and liquid phases.
Object of the invention :
In view of above, an object of the present invention is to provide a solid phase immunoassay kit for the detection of IgG and IgM antibodies to M. tuberculosis in the human serum and plasma.
Further object of the invention is to provide a solid phase immunoassay kit for the qualitative detection of IgG and IgM antibodies to M. tuberculosis in the human serum and/or plasma, which utilizes a mixture of synthetic peptide antigens of M. tuberculosis that are noncovalently bound to a solid support and an antibody conjugated enzyme conjugates G and M.
Still further object of the invention is to provide a solid phase immunoassay kit for the quantitative detection of IgG and IgM antibodies to M. tuberculosis in the human serum and/or plasma for the diagnosis of M. tuberculosis infection in the human beings.
Yet further object of the invention is to develop a simple, cost effective and reliable a solid phase immunoassay kit for the quantitative detection of IgG and IgM antibodies to M. tuberculosis in the human serum and/or plasma samples from the patients suffering from tuberculosis.

Another object of the present invention is to provide a method for the preparation of solid phase immunoassay kit for the detection of IgG and IgM to M.
6
21 APR 2009

proteins in the human serum and plasma that is based on the solid phase immunoassay technique.
Still another object of this invention is to provide an in vitro solid phase immunoassay method for the detection of IgG and IgM antibodies to M. tuberculosis proteins in the human serum and plasma samples.
Statement of the invention :
In accordance with main object of the invention, the solid phase immunoassay kit for the detection of IgG and IgM antibodies to M. tuberculosis in the human serum and plasma is provided, which comprises a solid support coated with a mixture of synthetic peptide antigens of M. tuberculosis, IgG and IgM conjugated enzyme conjugates G and M and immunochemical reagents required for performing the detection of IgG and IgM antibodies to M. tuberculosis proteins in the human serum and plasma.
In accordance with another object of the invention, a method for the preparation of solid phase immunoassay kit for the detection of IgG and IgM antibodies to M. tuberculosis proteins in the human serum is provided, which comprises providing a solid support coated with a mixture of synthetic peptide M. tuberculosis antigens and IgG and IgM conjugated enzyme conjugates G and M and preparing immunochemical reagents for required for performing the detection of IgG and IgM antibodies to M. tuberculosis proteins in the human serum and plasma.
In accordance with still another object of the invention, an in vitro solid phase immunoassay method for the detection of IgG and IgM antibodies to M. tuberculosis proteins in the human serum and plasma samples is provided, which comprises interacting human serum and/or plasma samples with a solid support coated with a mixture of synthetic peptide antigens of M. tuberculosis and IgG and IgM conjugated enzyme, conjugate-G and conjugate-M and finally performing the detection of said antibodies using immunochemical reagent provided in the kit of the present invention.
21 APR 2009

Description of the invention :

The solid phase immunoassay kit for the detection of IgG and IgM antibodies to M. tuberculosis in the human serum and plasma comprises (i) a solid support containing number of microwells coated with a mixture of antigens containing twelve synthetic peptide antigens of M. tuberculosis; (ii) IgG conjugated enzyme conjugates-G coupled with one or more goat anti-human IgG[Fc] polyclonal antibodies; (iii) IgM conjugated enzyme conjugates-M coupled with one or more goat anti-human IgM[Mu] polyclonal antibodies; and (iv) irrvmunochemical reagents, such as TB positive control, TB negative control, colour reagent, sample diluent, stopping solution and washing solution required for performing the detection of IgG+IgM antibodies to M. tuberculosis proteins in the human serum and/or plasma.
The method for the preparation of solid phase immunoassay kit for the detection of IgG and IgM antibodies to M. tuberculosis proteins in the human serum and plasma, wliich is based on the solid phase immunoassay technique comprises (i) providing a solid support containing number of microwells, which are coated with a mixture of antigens containing twelve synthetic peptide of M. tuberculosis and IgG and IgM conjugated enzyme conjugates G and M and containing one or more goat anti-human IgG[Fc] and IgM[Mu] polyclonal antibodies, respectively; and (ii) providing solutions of immunochemical reagents, such as TB positive control, TB negative control, colour reagent, sample diluent, stopping solution and washing solution for performing the detection of IgG and IgM antibodies to M. tuberculosis proteins in the human serum and plasma.
The in vitro solid phase immunoassay method for the detection of IgG and IgM antibodies to M. tuberculosis proteins in the human serum and plasma samples comprises (i) interacting human serum and/or plasma samples with a solid support containing microwells coated with a mixture of antigens containing twelve synthetic peptide of M. tuberculosis, thereby forming stable antigen-antibody complexes in the
21 APR 2009

said microwells; (ii) interacting said antigen-antibody complexes so formed with IgG and IgM conjugated enzyme conjugates G and M coupled with one or more goat anti-human IgG[Fc] and IgM[Mu] polyclonal antibodies, respectively; and (iii) detecting said antibodies using colour reagent for detecting the amount of antibodies in the samples.
Detailed description of the invention :
What is claimed in the present invention can be understood with reference to the foregoing preferred and particular embodiments. Although, the subject matter has been disclosed with reference to particular and specific embodiments, it is not intended that sudi embodiments should be regarded as limitations to the scope of the invention. Also, unless otherwise specified, all the technical and scientific terms used herein before and after have the same meanings as commonly understood by the person skilled in the art to which this invention belongs or assigned to them.
In preferred aspect of the present invention, the solid phase immunoassay kit for the detection of IgG and IgM antibodies to M. tuberculosis proteins in the human serum and plasma comprises a solid support, which is a microtitre plate containing number of microwells coated with a mixture of synthetic peptide antigens of tuberculosis by using covalent linkage of said antigens with the microwells.
In more preferred aspect of the invention, the said microwells of the plate are coated with a mixture of tuberculosis antigens containing twelve synthetic peptides of M. tuberculosis designated as TBPEP1, TBPEP2, TBPEP3, TBPEP4, TBPEP5, TBPEP6, TB-PEP7, TBPEP8, TBPEP9, TBPEP10, TBPEP11 and TBPEP12. (These are Immunogenic peptides of different proteins of M. Tuberculosis).
In most preferred aspect of the invention, the solid phase immunoassay kit for the detection of IgG and IgM antibodies to M. tuberculosis proteins in the human serum and plasma comprises a microtitre plate, which contains number of micro-wells coated with number of homogenous layers of tuberculosis antigen, which contain twelve syntlietic
9
21 APR 2009

peptide antigens of M. tuberculosis selected from TBPEP1, TBPEP2, TBPEP3, TBPEP4, TBPEP5, TBPEP6, TBPEP7, TBPEP8, TBPEP9, TBPEP10, TBPEP11 and TBPEP12.
In another preferred aspect of tlie present invention, the solid phase immunoassay kit for the detection of IgG and IgM antibodies to M. tuberculosis proteins in the human serum and plasma comprises IgG and IgM conjugated enzyme conjugates G and M, which are respectively enzyme labelled conjugates coupled with one or more goat anti-human Ig[Fc] and IgM[Mu] polyclonal antibodies by covalent bonding for detecting IgG and IgM antibodies M. tuberculosis proteins.
In another more preferred aspect of tlie invention, the enzyme label of conjugates G and M preferably used for preparing tlie conjugated enzyme material is Horse Reddish Peroxidase (HRPO).
In another most preferred aspect of the invention, the said IgG and IgM conjugated enzyme conjugates G and M are goat anti-human-IgG[Fc]-HRPO and goat anti-human-IgM[Mu]-HRPO for the separation of antigen-antibody complexes of said tuberculosis antigens coated onto the microwells and IgG and IgM antibodies to M. tuberculosis proteins.
In still another preferred aspect of the invention, the said IgG and IgM conjugated enzyme conjugates G and M are goat anti-human-IgG[Fc]-HRPO and goat anti-human-IgM[Mu]-HRPO for separating antigen-antibody complexes is provided in the solution of conjugate diluent containing thimerosal and gentamycin as preservatives.
In further preferred aspect of the present invention, the solid phase inimunoassay kit for the detection of IgG and IgM antibodies to M. tuberculosis proteins in the human serum and plasma comprises TB positive control, TB negative control, colour reagent, sample diluent, stopping solution and washing solution as immunochemical reagents required for performing detection of IgG and IgM antibodies to M. tuberculosis proteins in the human serum and plasma.
10
21 APR 2009

In still further aspect of the present invention, the said microwells of the microtitre plate are coated with a mixture of twelve synthetic peptides of M. tuberculosis antigens using non-covalent linkage without affecting structural and functional integrity of said antigens. Similarly, said IgG and IgM conjugated enzyme label, HRPO, is coupled with said goat anti-human IgG[Fc] and goat anti-human IgM[Mu] polyclonal antibodies using covalent bonds without affecting structural and functional integrity cl said antibodies.
In yet further aspect of the present invention, the microtitre plate used as a solid support for the solid phase immunoassay comprises minimum 48 and maximum 96 microwells that are immunochemically coated with a mixture of twelve synthetic peptides antigens of M. tuberculosis.
In one differed aspect of the present invention, the said TB positive control.is an inactivated anti-TB containing human serum preserved by addition of thimerosal and gentamycin.
In still different aspect of the invention/ the said TB negative control is an inactivated normal human serum containing thimerosal and gentamycin.
In yet different aspect of the invention, the said colour reagent comprises 3,3', 5,5'-tetra methyl benzidine dimethyl sulfoxide and H2O2 containing thimerosal and gentamvcin.
In separate aspect of this invention, the said sample diluent comprises a solutions of TRIS buffer, animal serum, BSA, Tween-20, TritonXlOO MgCI2, and thimerosal and gentamycin.
In still separate aspect of the invention/ the said stopping solution comprises a solution of concentrated phosphoric acid and deionized water.
solution comprises a solution
In yet separate aspect of the invention/ the said washing
of TRIS buffer, NaCl and Tween-20 in deionized water.
11
21 APR 2009

In an essential aspect of the present invention, the solid phase immunoassay kit for the detection of IgG and IgM antibodies to M. tuberculosis proteins in the human serum and plasma samples is stored at temperature between 2°C and 8 °C.
In accordance with different object of the present invention, there is provided a method for the preparation of solid phase immunoassay kit for the detection of IgG and IgM antibodies to M. tuberculosis proteins in the human serum and plasma, which is based on the solid phase immunoassay technique more preferably comprises coating over a solid support, which is a microtitre plate containing number of microwells, a mixture of synthetic peptides of M. tuberculosis antigens in bicarbonate buffer using non-covalent linkage; blocking and stabilizing said synthetic peptide antigens using blocking and stabilizing solutions.
In most preferred aspect of the invention, the method for coating the solid support comprises coating onto the microwells number of homogenous layers of a mixture containing twelve synthetic peptides antigens of M. tuberculosis designated as TBPEP1, TBPEP2, TBPEP3, TBPEP4, TBPEP5, TBPEP6, TBPEP7, TBPEP8, TBPEP9, TBPEP10, TB-PEP11 and TBPEP12 ( These are Immunogenic peptides of different proteins of M. Tuberculosis) in bicarbonate buffer using covalent linkage; blocking said tuberculosis antigens using blocking solution containing phosphate buffer, BSA and Trans-001; and stabilising said blocked tuberculosis antigens using stabilizing solution containing phosphate buffer saline of pH 7.4, Trans-002 and Tran-003 (bovine immunoglobulin).

In another preferred aspect of the invention, the method for the preparation of solid phase immunoassay kit for the detection of IgG and IgM antibodies to M. tuberculosis proteins in the human serum and plasma comprises preparing enzyme conjugates G and M containing one or more goat anti-human IgG[Fc] and IgM[Mu] polyclonal antibodies using covalent coupling of said goat anti-human IgG[Fc] and IgM[Mu] monoclonal antibodies with HRPO as a suitable enzyme label.
12
21 APR 2009

In further preferred aspect of the invention, the method for the preparation of solid phase immunoassay kit for the detection of IgG and IgM antibodies to M. tuberculosis proteins in the human serum and plasma comprises preparing solutions of immunochemical reagents, such as TB positive control/ TB negative control, colour reagent, sample diluent, stopping solution and washing solution required for performing detection of IgG and IgM antibodies to M- tuberculosis proteins in the human serum and plasma.
In still further aspect, the method of present invention comprises preparing a solution of TB positive control, which comprises an inactivated anti-TB containing human serum and thimerosal and gentamycin as preservatives.
In yet further aspect, the method of present invention comprises preparing a solution of TB negative control, which contains an inactivated normal human serum and thimerosal and gentamycin.
In next aspect, the method of present invention comprises preparing a solution of colouring reagent, which comprises 3,3', 5,5'-tetra methyl benzidine dimethyl sulfoxide and H2O2 along with thimerosal and gentamycin.
In still next aspect, the method of present invention comprises preparing a solution of sample diluent, which comprises TRIS buffer, animal serum, BSA, Tween-20, Triton X 100, MgCh, thimerosal, gentamycin and bovine immunoglobulin.
In yet next aspect, the method of present invention comprises preparing a stopping solution, which comprises concentrated phosphoric acid and deionized water.
In further aspect, the method of present invention comprises preparing a washing solution concentrate, which comprises TRIS buffer, NaCl, Tween-20 in deionized water.
In accordance with still preferred object of the invention, there is provided an in vitro
solid phase immunoassay method for the detection of IgG and IgM antibodies to M.
13
21 APR 2009

tuberculosis proteins in the human serum and plasma samples comprises interacting human serum and/or plasma samples with a mixture of twelve synthetic peptide antigens of M. tuberculosis coated onto the microwells of microtitre plate by adding the human serum and plasma samples into the microwells containing sample diluents/ thereby forming a stable antigen-antibody complexes in the said microwells, if IgG and IgM antibodies to M. tuberculosis are present in the samples; interacting further said antigen-antibody complexes so formed with IgG and IgM conjugated enzyme conjugates G and M containing one or more goat anti-human IgG[Fc] and IgM[Mu] polyclonal antibodies by adding said enzyme into the microwells; detecting IgG and IgM antibodies to M. tuberculosis proteins by adding colour reagent containing substrate of HRPO into said microwells, thereby making catalytic reaction on the substrate of HRPO in the microwells that contain positive control and test samples; and detecting the enzyme activity for the presence of target IgG and IgM antibodies to M. tuberculosis proteins in the sample.
In yet another preferred aspect, the in vitro solid phase immunoassay method for the detection of IgG and IgM antibodies to M. tuberculosis proteins in the human serum and/or plasma samples comprises contacting human serum and/or plasma samples with the microwells of microtitre plate containing mixture of twelve synthetic peptide antigens of M. tuberculosis designated as TBPEP1, TBPEP2, TBPEP3, TBPEP4, TBPEP5, TBPEP6, TBPEP7, TBPEP8, TBPEP9, TBPEP10, TBPEPll and TBPEP12 ( These are Immunogenic peptides of different proteins of M. Tuberculosis) in the sample diluents, thereby producing stable antigen-antibody complexes, if said IgG and IgM antibodies are present in the positive control and test samples; contacting said antigen-antibody complexes with IgG and IgM conjugated enzyme conjugates G and M, which contain one or more goat anti-human IgG[Fc] and IgM[Mu] Polyclonal antibodies, thereby reducing the amount of HRPO substrate in the microwells that contain positive control and test samples; and measuring the enzyme activity spectrophotometrically for
14
21 APR 2009

performing detection IgG and IgM antibodies to M. tuberculosis proteins in the test samples.
In most preferred aspect of the invention, the in vitro solid phase immunoassay method for the detection of IgG and IgM antibodies to M. tuberculosis proteins in the human plasma and/or serum samples comprises adding measured quantity of human serum and/or plasma samples to sample diluent containing microwells, which are coated with mixture of twelve synthetic peptide antigens of M. tuberculosis, thereby forming stable antigen-antibody complexes, if said IgG and IgM antibodies to M. tuberculosis proteins are present in the positive control and test samples; washing the microwells by known microtitre plate washing procedure and adding measured quantity of goat anti-human IgG[Fc]-HRPO (conjugate-G) and goat anti-human IgM[Mu]-HRPO (conjugate-M) to said microwells; further washing the microwells, thereby removing free conjugate G and M; adding measured quantity of colour reagent containing substrate of HRPO, thereby producing a blue colour in the microwells containing positive control and test samples, which contain IgG and IgM antibodies to M. tuberculosis proteins and the microwells containing negative control remain colourless; further adding measured quantity of stopping solution to the microwells, thereby stopping catalytic reaction of enzyme conjugate-G and changing the blue colour to yellow; measuring an intensity of yellow colour using spectrophotometer, which is directly proportional to the amount of said IgG and IgM antibodies present therein.
In separate aspect of the invention, for simultaneous detection of IgG and IgM antibodies to M. tuberculosis proteins in the human serum and/ or plasma in a single assay and one attempt, the foregoing reagents and/or samples are employed in predetermined quantities.
In different aspect of the invention, the solid support to which said synthetic peptide antigens of M. tuberculosis are coated may be any water-insoluble, water-insuspensible, solid support. A suitable example of said solid support is microtitre plate containing
15
21 APR 2009

number of microwells. The immunological components may be coated to the microtitre plate by covalent bonds or adsorption. The advantage of such microtitre plate is that no centrifugation step is required for the separation of solid and liquid phases. Also, the antibodies of conjugated enzyme-G consist of the immunological components that are covalently linked to one or more enzyme molecules. The linking can be achieved either by direct condensation or using external bridging molecules known in the prior art. Thus, the reagents such as carbodiimides, diisocyanates, glutaric aldehyde and bis-diazobenzidine can be used for the production of enzyme-conjugated products employing the covalent bond.
The choice of an enzyme that is used with coupling product is determined by the properties such as specific binding activity i.e. a high conversion rate, as it increases the sensitivity of the test kit. The determination of an enzyme catalysing a conversion, in which coloured reaction components are involved, is simple. Such colorimetric determinations can be automatic in a simple manner. It is also possible to employ enzymes catalysing those conversions in which reaction components are involved that can be determined spectrophotometrically. These determinations are also suitable for automation, which is an additional advantage. As an enzyme suitably employed in the present invention is peroxidase, preferably Horse radish peroxidase.
Examples
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 IgG and IgM antibodies to M. tuberculosis proteins
A mixture of twelve synthetic peptide antigens of M. tuberculosis designated as TBPEP1, TBPEP2, TBPEP3, TBPEP4, TBPEP5, TBPEP6, TBPEP7, TBPEP8, TBPEP9, TBPEP10, TB-PEP11 and TBPEP12 are coated onto the microwells of microtitre plate. When the human plasma and/or serum samples added to sample diluent containing microwells
16
21 APR 2009

of said plate, the coated M. tuberculosis antigens will form a stable antigen-antibody complexes, if IgG and IgM antibodies to M, tuberculosis proteins (anti-MTB) are present in the said samples. Followed by a wash step, goat anti-human-IgG[Fc]-HRPO (conjugate-G) and goat anti-human-IgM[Mu]-HRPO (conjugate-M) are added cO the microwells. A second wash step will remove free goat anti-human-IgG[Fc]-HRPO (conjugate-G) and goat anti-human-IgM[Mu]-HRPO (conjugate-M). The colour reagent containing the substrate of HRPO is then added to the microwells. The microwells containing negative control will remain colourless and blue colour will develop in wells containing TB positive controls and test samples containing IgG and IgM antibodies to M. tuberculosis proteins. Upon addition of stopping solution, blue colour changes to yellow. The intensity of yellow colour is directly proportional to the amount of bound IgG and IgM antibodies to M. tuberculosis proteins in the respective microwells.
Example 2: Test procedure for detecting anti-MTB IgG and IgM antibodies
(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; (iii) add lOul of control and test samples to the respective microwells, mix properly with pipettor; (iv) cover the plate with black cover and incubate 45 minutes at 20-30°C; (v) wash the microtitre plate as per microplate washing procedure known to person skilled in the art; (vi) except blank well, add 50ul conjugate-G and conjugate-M to each well and cover the microtitre plate with black cover and incubate for 15 minutes at 20-30°C; (vi) repeat the step (v) for washing; (vii) add 50|LL1 of colour reagent to each well and cover the plate with black cover and incubate for 15 minutes in dark at 20-30°C; (viii) add 100pl of stopping buffer to each well; and (ix) read absorbance at 450 nm and deduct the blank absorbance from the control and test wells.
17
21 APR 2009

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 0.4.
Negative Control: Absorbance of individual negative control should be less than 0.2.
CoCx: Average value of positive controls.
Calculation of CoCx:
For example:
PC Absorbance
1 0.750
2 0.760
3 0.770
CoCx: (0.75+ 0.76+ 0.77)/3 = 0.76
Cut-off value formula: 1.0 x CoCx
Cut-off vale: 1.0 x 0.76 = 0.76
Interpretation of results :
If the absorbance of the test serum and/or plasma is less than cut-off value, then the sample is considered as non-reactive. If the absorbance of the test serum and/or plasma is greater than the cut-off value, then it is considered as initial reactive.

Any further modifications in and/or improvements in any aspect of the embodiments of the present invention will also fall under the scope of the present invention. In
18
21 APR 2009


of the foregoing description and examples, it will become apparent to those of ordinary skill in the art that equivalent modification thereof may be made without departing from the spirit and scope of the present invention. Various features of the invention hereinbefore described are set forth in the foregoing claims.
19
21 APR 2009

WE CLAIM:
1. A solid phase immunoassay kit for the detection of IgG and IgM antibodies to M.
tuberculosis proteins in human serum and/or plasma comprises:
(i) a solid support containing number of microwells coated with a mixture of antigens containing twelve synthetic peptide antigens of M. tuberculosis;
(ii) IgG conjugated enzyme conjugates-G coupled with one or more goat anti-human IgG[Fc] polyclonal antibodies;
(iii) IgM conjugated enzyme conjugates-M coupled with one or more goat anti-human IgM[Mu] polyclonal antibodies; and
(iv) immunochemical reagents, such as TB positive control, TB negative control, colour reagent, sample diluent, stopping solution and washing solution required for performing the detection of IgG+IgM antibodies to M. tuberculosis proteins in the human serum and/or plasma.
2. The solid phase immunoassay kit as claimed in claim 1, wherein said solid support is a microtitre plate containing number of microwells that are coated with a mixture of synthetic peptide antigens of M. tuberculosis using non-ccvalent linkage of said antigens with the microwells.
3. The solid phase immunoassay kit as claimed in claim 1, wherein said microwells are coated with a mixture twelve synthetic peptides antigens of M. tuberculosis designated as TBPEP1, TBPEP2, TBPEP3, TBPEP4, TBPEP5, TBPEP6, TBPEP7, TBPEP8, TBPEP9, TBPEP10, TBPEP11 and TBPEP12. ( These are Immunogenic peptides of different proteins of M. Tuberculosis).

4. The solid phase immunoassay kit as claimed in claim 1, wherein said IgG and IgM conjugated enzyme conjugate G and M are respectively coupled with one o
20
21 APR 2009

more goat anti-human IgG[Fc] and goat anti-human IgM[Mu] polyclonal antibodies by covalent bonding.

5. The solid phase immunoassay kit as claimed in claim 1, wherein said IgG and IgM conjugated enzyme label of conjugate G and M, which are coupled with said goat anti-human IgG[Fc] and IgM[Mu] polyclonal antibodies is preferably Horse Reddish Peroxidase (HRPO).
6. The solid phase immunoassay kit as claimed in claim 1, wherein said IgG and IgM conjugated enzyme conjugates G and M are goat anti-human-IgG[Fc]-HRPO and goat anti-human-IgM[Mu]-HRPO, respectively and are used for the separation of antigen-antibody complexes of said peptide antigens and anti-MTB IgG and IgM antibodies.
7. The solid phase immunoassay kit as claimed in claim 1, wherein said kit further comprises TB positive control, TB negative control, colour reagent, sample diluent, stopping solution and washing solution for performing the detection of anti-MTB IgG and IgM antibodies in the human serum and plasma.
8. The solid phase immunoassay kit as claimed in claim 1, wherein said the microtitre plate used as a solid support comprises minimum 48 and maximum 96 coated microwells.
9. The solid phase immunoassay kit as claimed in claim 1, wherein said TB positive control and TB negative control, respectively comprise an inactivated anti-TB containing human serum and an inactivated normal human serum with thimerosal and gentamycin as preservatives.
21
21 APR 2009

10. The solid phase immunoassay kit as claimed in claim 1, wherein said colour reagent comprises 3,3'/ 5,5'-tetra methyl benzidine dimethyl sulfoxide and H2O2 with thimerosal and gentamycin.
11. The solid phase immunoassay kit as claimed in claim 1, wherein said sample diluent comprises a solution of TRIS buffer, animal serum, BSA, Tween-20, TritonXlOO and MgCb, with thimerosal and gentamycin.
12. The solid phase immunoassay kit as claimed in claim 1, wherein said stopping solution and washing solution, respectively comprises a concentrated phosphoric acid and deionized water, and TRIS buffer, NaCl and Tween-20 in deionized water.
13. A method for the preparation of solid phase immunoassay kit for the detection of IgG and IgM antibodies to M. tuberculosis proteins in the human serum and plasma comprises:
(i) providing a solid support containing number of microwells coated with a mixture of twelve synthetic peptide antigens of M. tuberculosis and IgG and IgM conjugated enzyme conjugates G and M containing one or more goat anti-human IgG[Fc] and goat anti-human IgM[Mu] polyclonal antibodies, respectively; and

(ii) providing solutions of immunochemical reagents, such as TB positive control, TB negative control, colour reagent, sample diluent, stopping solution and washing solution for performing the detection of IgG+IgM antibodies to M. tuberculosis proteins in the human serum and plasma.
21 APR 2009

14. An in vitro solid phase immunoassay method for the detection of IgG and IgM antibodies to M. tuberculosis proteins in the human serum and plasma samples comprises:
(i) interacting human serum and/ or plasma samples with a solid support containing microwells coated with a mixture of twelve synthetic peptide antigens of M. tuberculosis, thereby forming stable antigen-antibody complexes in the said microwells;
(ii) interacting said antigen-antibody complexes so formed with IgG an i IgM conjugated enzyme conjugates G and M containing one or more goat anti-human IgG[Fc] and goat anti-human IgM[Mu] polyclonal antibodies, respectively; and
(iii) detecting said antibodies using colour reagent for detecting the amount of antibodies in the samples.
Dated this 25th day of April 2009.
NSASIA BIO-MEDICALS LTD.

RAVINDRA YEVLE VICE PRESIDENT - LEGAL, IP AND COMPANY SECRETARY
23
21 APR 2009