FORM 2
THE PATENT ACT 1970 (39 of 1970)
The Patents Rules, 2003 COMPLETE SPECIFICATION
(See Section 10, and rule 13)
1. TITLE OF INVENTION
A DIAGNOSTIC KIT FOR QUANTITATIVELY TSH IN HUMAN SERUM AND/OR
PLASMA

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 diagnostic kit for quantitatively detecting TSH in human serum and/or plasma
FIELD OF THE INVENTION
The present invention relates to quantitative measurement of substances, such as hormones, in human serum and/or plasma based on specific binding assay techniques. More particularly, it relates to detection of antigens or hapten like small chemical compound, for example hormones, based on immunoassay techniques involving the use of labeled reagents, such as enzyme-labeled reagents. The invention provides a kit for quantitatively detecting thyroid stimulating hormone (TSH) in human serum and/ or plasma based on specific binding assay techniques.
BACKGROUND OF THE INVENTION
TSH is a glycoprotein, consisting of alpha and beta subunits with molecular weight approximately 28000 Daltons. The determination of serum and/or plasma level of TSH (thyrotropin) is recognized as important measurement in assessment of thyroid function. It is therefore, desirable to measure the amount of TSH in human serum and/ or plasma using a simple and reliable kit and method for measuring an amount of TSH in human serum and/or plasma. The appropriate technique for separating and measuring the amount of TSH molecules in human serum and/or plasma is solid-phase technique, which use antibodies covalently bound or physically adsorbed to an insoluble matrix. The antibody-antigen complex formed is then held by solid phase technique and bound fraction can be easily separated and measured using glowing reagents.

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, which is relatively a small and simple compound that may be determinant group of given antigen and that hapten is capable of binding with specific antibody but incapable of inducing an antibody production, unless it is bound to an antigenic carrier.
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The binding interaction between antigen or hapten and its antibody is specific and sensitive. Other materials that shows 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 technique known as specific binding assay technique. In one such assay method, the substance or group of substances to be determined (may referred as ligand) in a liquid sample is placed in competition with labeled form of the ligand or of binding analog thereof for binding to binding reagent. Where an enzyme label is used and the binding reagent is an antibody, the method is known as an enzyme-immunoassay method. Several alternative labeling materials are available for substituting the enzymes, such as radioisotopes, coenzymes, enzyme substrates, enzyme-modulators like inhibitors and allosteric effectors, fluorescent molecules, and luminescent molecules but these have inherent disadvantages of handling and test methods require sophisticated instruments and trained manpower for accurate results.
The afore mentioned system consists of antigen or hapten labeled with a enzyme marker, unlabeled native antigen in test sample and specific antibody, thereby there is competition between unlabeled antigen and labeled antigen for binding to limited amount of antibodies. Hence, greater the concentration of unlabeled antigen from the test sample less the labeled antigen will be bound to the antibodies. If the concentration of labeled antigen and antibody is fixed and the only variable is the level of unlabeled antigen, it becomes possible to establish an assay system for measuring unknown level of unlabeled antigen by physically separating the antigen-antibody complex from the remaining free antigen. The enzyme activity of the unknowns is compared with a standard curve plotting of values given by range of known amounts of the antigen treated in the same manner.


PRIOR ART OF THE INVENTION
Many methods are known for separating free unbound antigen or hapten from the complex antigen-antibody. One method is chromato-electrophoresis that combines paper chromatography and paper electrophoresis. Paper with a high affinity for free antigen like Whatman paper is used as carriers. This technique is discriminative and has been used in the assay of insulin, growth hormone, glucagons, parathyroid hormone, thyroid stimulating hormone and other peptide hormones. But it has number of prominent disadvantages, which limits its use. A Hmited amount of material may be applied to the absorbent and the separation is laborious and time-consuming.
Other known method is precipitation of antigen-antibody complex that 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 sulfate, ammonium sulfate, dioxane, trichloroacetic acid, polyethylene glycol, etc. The use of salts, solvents or organic materials has advantage that the separation is immediate, and a second incubation is not necessary. However, the chemical precipitation technique causes co-precipitation of other proteins, which causes incomplete separation of two fractions.
The double antibody technique is known and widely used for separation of bound and free antigen. Using this method, a second antibody that was raised against the first antibody is used to precipitate the primary antigen-antibody complex. More particularly, if the first antibody was raised in rabbit then the second antibody may be an antiserum to rabbit gammaglobulin 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 determination of small molecules such as thyroid hormones. One technique of this type used for separation of antigen-antibody complex from free antigen employs a column packed with material, which
V-S-

preferentially adsorbs either free antigen or antigen-antibody complex. The incubated aqueous reaction mixture is applied to the head of such a 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 method, free unbound antigens adsorbed onto adsorbent and then precipitated by centrifugation. Powdered talc like magnesium silicate, kaolin like aluminum silicate, QUSO like silica microgranules, cellulose powder, etc are some of the simple adsorbents used. Many separations are performed using adsorbent charcoal coated with dextran. The dextran behaves rather like a sieve, which allows the smaller molecules of free antigen to pass and these are then bound by the charcoal, leaving the bound antigen in solution, after the charcoal has been removed by centrifugation or filtration.
The solid-phase techniques are also known for separation of free and bound antigen, which use antibodies covalently bound or physically adsorbed to an 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 view of above mentioned prior art, inventors of the present invention, propose a kit for detecting amount of TSH in human serum and/or plasma that utilizes one monoclonal antibody covalently bonded with a solid support and second monoclonal antibody linked with horse redfish peroxidase (HRPO) as an enzyme conjugate for precipitating and measuring amount of TSH. An advantage of using the solid support like microwells containing microplate is that no centrifugation or filtration required for separation of solid and liquid phases.
OBJECT OF THE INVENTION
Accordingly, main object of this invention is to provide a diagnostic kit for quantitatively detecting the amount TSH present in the human serum and/or plasma. sZZ—-^'*^
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Another object of the present invention is to design a diagnostic kit for detecting amount of TSH in the human serum and/or plasma and assessing thyroid function.
Yet another object of the instant invention is to develop a simple, cost effective and reliable diagnostic kit for quantitatively detecting amount of TSH in the human serum and/or plasma samples obtained form patients whose thyroid function required to be assessed.
Still another object of the invention is to provide a diagnostic kit comprising one anti-TSH monoclonal antibody (MAb) as a coating material and second anti-TSH monoclonal antibody linked to enzyme conjugate for detecting amount of TSH in the samples of the patents suffering from problems related to thyroid functions.
Different object of the present invention is to develop method for preparing a diagnostic kit for detecting amount of TSH present in the human serum and/or plasma.
Still different object of the instant application is to provide a method for quantitatively detecting amount of TSH in human serum and/ or plasma.
SUMMARY OF THE INVENTION
According to main object of the present invention there is provided a diagnostic kit for quantitatively detecting amount of TSH in the human serum and/or plasma, which comprises a solid support containing covalently bound anti-TSH monoclonal antibodies; an conjugate containing anti-TSH monoclonal antibodies covalently linked with an enzyme; and immunoassay reagents of types such as sample diluent, thyrotropin standards, glow reagents and a washing solution.
Also provided is a method for preparing a diagnostic kit for quantitatively detecting amount of TSH in the human serum and/or plasma that comprises preparing a solid support and conjugate both containing covalently bound anti-TSH monoclonal antibodies by covalently binding anti-TSH monoclonal antibodies with the solid


support and enzyme; and preparing immunoassay reagents of types such as a sample diluent, thyrotropin standards, glow reagents and washing solution.
Further, a method for quantitatively detecting amount of TSH in the human serum and/or plasma is provided that comprises contacting human serum and/ or plasma comprising TSH molecules with solid support, which contains bound anti-TSH monoclonal antibodies and conjugated anti-TSH monoclonal antibodies, thereby forming antigen-antibody complex; and detecting the said antigen-antibody complex using glow reagents for quantitatively detecting amount of TSH in the samples.
DESCRIPTION OF THE INVENTION
In accordance with one object of the present invention, a diagnostic kit for quantitatively detecting amount of TSH in the human serum and/ or plasma comprises: (i) a microtitre plate consisting of plurality of microwells, which are coated with anti-TSH monoclonal antibodies by covalently bonding for iirimobilising TSH present in the human serum and/or plasma samples; (ii) a conjugate consisting of an enzyme, which is attached with anti-TSH monoclonal antibodies by covalently linkage for detecting TSH present in the human serum and/or plasma samples; and (iii) an immunoassay reagents consisting of thyrotropin standards, glow reagents and washing solution.

In accordance with another object of the instant invention, a method for preparing a diagnostic kit for quantitatively detecting amount of TSH in the human serum and/ or plasma comprises the steps of: (i) preparing a microtitre plate consisting of plurality of microwells coated with anti-TSH monoclonal antibodies, the process for preparing the microtitre plate comprises the steps of: (a) coating anti-TSH monoclonal antibodies dissolved in the bicarbonate buffer to microwells of microtitre plate by covalent bonding; (b) blocking the bound anti-TSH monoclonal antibodies using blocking solution comprising phosphate buffer, BSA and Trans-001; (c) stabilizing the blocked anti-TSH monoclonal antibodies using stabilising solution comprising PBS, Trans-002 and Tran-003 (bovine immunoglobulin); (ii) preparing
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conjugate consisting of an enzyme and anti-TSH monoclonal antibodies by covalently linkage between of enzyme and said monoclonal antibodies; and (iii) preparing solutions of thyrotropin standards, glow reagents and washing solution as immunoassay reagents.
In accordance with different object of the present invention, a method for quantitatively detecting amount of TSH in the human serum and/ or plasma comprises the steps of: (i) charging dispensed quantities of thyrotropin standards and TSH containing human serum and/or plasma samples into microwells of the microtitre plate containing coated anti-TSH monoclonal antibodies for immobilising TSH molecules; (ii) subsequently adding dispensed quantity of conjugate containing anti-TSH monoclonal antibodies linked with enzyme (HRPO) into the microwells, thereby forming stable antigen- antibody complex; (iii) washing the microwells containing said reaction mixture using washing solution to remove unbound fraction of conjugate; (iv) adding further dispensed quantity of working glow reagent into the said microwells, thereby generating the RLU; and (v) detecting the amount of TSH in the samples by reading RLU values on standard plot of RLU Vs. TSH.
DETAILED DESCRIPTION OF THE INVENTION
The present invention can be understood preferably with reference to following detailed and specific embodiments of the invention. Although, the invention has been disclosed with reference to particular and specific details of certain embodiments, it is not intended that such details should be regarded as limitations to the scope of the present invention. Further to that unless otherwise described, 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.
In preferred embodiment, the diagnostic kit of the present invention comprises a microtitre plate having plurality of microwells, wherein anti-TSH monoclonal

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antibodies are covalently bound to said microwells for immobilising TSH present in the human serum and/ or plasma samples.
In another preferred embodiment of the invention, the diagnostic kit comprises a conjugate, wherein anti-TSH monoclonal antibodies are covalently attached to an enzyme for quantitatively detecting the amount of TSH by detecting the antigen-antibody complex present the microwells of plate.
In more preferred embodiment of the instant invention, the enzyme used for preparing conjugate containing anti-TSH monoclonal antibodies is Horse Reddish Peroxidase (HRPO).
In still another preferred embodiment, immunoassay reagents for an immunoassay using the diagnostic kit of the present invention are the reagents of the types such as thyrotropin standards (3a to 3f), glow reagent-A and B and washing solution.
In different aspect of present invention, the anti-TSH monoclonal antibodies are bound to the microwell of microtitre plate by covalent bonding without adversely affecting structure and activity of the said monoclonal antibodies.
In still different aspect of the invention, the anti-TSH monoclonal antibodies are attached with HRPO of the conjugate by covalent linkage without adversely affecting structure and activity of the said monoclonal antibodies.
In preferred embodiment of the instant invention, the microtitre plate of the diagnostic kit comprises at least 24 microwells coated with said monoclonal antibodies for immobilising TSH present in the sample of human serum ands/or plasma.
In another preferred embodiment, the microtitre plate of the said diagnostic kit comprises not more than 96 microwells coated with said anti-TSH monoclonal antibodies.


In another separate embodiment, the kit comprises solutions of thyrotropin standards (3a to 3f) in concentrations of 0, 0.1, 0.5, 3, 6 and 10 uIU thyrotropin per millilitre with thimerosal and gentamycin.
In still different aspect of the invention, the kit comprises glow reagents, solution-A and solution-B, containing HRP substrates, component-A and component-B.
In still further aspect, at the time actual immunoassay, the kit comprises working glow reagents solution, which comprises equal volume of solution-A and solution-B.
In yet another embodiment, the washing solution (20X) comprises a TRIS buffer, NaCl, Tween-20 in deionized water.,
In an important embodiment the diagnostic kit of the present invention for quantitatively detecting the amount of TSH in human serum and/or plasma is stored at temperature between 2°C and 8 °C.
In another main embodiment of the present invention, the method for preparing the diagnostic kit comprises preparing a microtitre plate, wherein anti-TSH monoclonal antibodies dissolved in bicarbonate buffer are coated onto the microwells of the plate by covalently bonding. The method of preparing the microtitre plate further comprises blocking the coated anti-TSH monoclonal antibodies using blocking solution containing phosphate buffer, BSA and Trans-001; and stabilizing the blocked anti-TSH monoclonal antibodies using stabilising solution containing PBS, Trans-002 and Tran-003 (bovine immunoglobulin).
In another embodiment of method for preparing diagnostic kit of the present invention comprises preparing a conjugate, wherein the method comprises linking the anti-TSH monoclonal antibodies to HRPO by covalent bonding without affecting the activities of enzyme and monoclonal antibodies.
In still another preferred embodiment, the method comprises preparing solutions of thyrotropin standards by dissolving thyrotropin standards (3a to 3f) of

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concentrations 0, 0.1, 0.5, 3, 6 and 10 uIU thyrotropin in per millilitre of deionized water.
In different embodiment, the method comprises preparing solutions of glow reagents A and B by mixing independently HRP substrates component A and B in deionized water. The method further comprises preparing working glow reagent solution at the time use by mixing equal volume of glow reagent A and B.
In still different embodiment, the method comprises preparing washing solution by mixing a TRIS buffer, NaCl, Tween-20 in deionized water.
In yet another embodiment of the invention, the method for quantitatively detecting the amount of TSH in human serum and/or plasma samples comprises the steps of: adding thyrotropin standard solutions (3a to 3f) and human serum and/or plasma samples containing TSH into the microwells containing coated anti-TSH monoclonal antibodies for bringing said thyrotropin standards and TSH present in the human serum and/ or plasma samples in contact with said monoclonal antibodies; subsequently adding the conjugate containing anti-TSH monoclonal antibodies attached to HRPO, thereby forming stable antigen-antibody complex of said TSH molecules and monoclonal antibodies; and quantitatively detecting the amount of TSH present in the samples by detecting antigen-antibody complex using glow reagents.
In more preferred embodiment of the invention, the method for quantitatively detecting the amount of TSH comprises contacting thyrotropin molecules present in standard solutions and TSH molecules present in human serum and/or plasma samples with anti-TSH monoclonal antibodies of the microtitre plate and enzyme conjugate, thereby forming stable antigen-antibody complex.

In another preferred embodiment, the method comprises detecting antigen-antibody complex by adding glow reagents to the microwells containing said complex, thereby generating RLU, which are proportional to amount of thyrotropin standards or TSH present in the test samples.
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EXAMPLES

The following examples serve to illustrate the present invention by way of best method of performing the invention and not to be regarded as limitations to the scope of the invention.
Example: 1 - Quantitative detection of amount of TSH present in test samples
Adding thyrotropin standards or TSH containing human serum and/or plasma to the microwells of the microtitre plate; subsequently adding conjugate comprising anti-TSH monoclonal antibodies linked to HRPO, incubating the plate containing the reaction mixture for 60 minutes at room temperature between 20°C and 40°C, thereby forming the stable antigen-antibody complex with monoclonal antibodies coated onto the microwells and linked to HRPO of the conjugate; washing the microplate using washing solution, thereby removing unbound fraction of the conjugate; adding the glow reagents, which contain the HRP substrates, thereby generating RLU in the thyrotropin standards and human serum and/or plasma containing wells; and finally detecting the amount of TSH by reading RLU using standard plot of RLU Vs. TSH concentrations in ul U per milliliter.
Example: 2 - Test procedure for detecting amount of TSH in the test samples
(i) Bring all the reagents and test specimens at room temperature before use; (ii) add 50 ul of standards and test specimens to the respective wells; (iii) add 50 ul of conjugate to each well; (iv) incubate 60 minutes at room temperature (20 - 40°C), preferably 37 °C; (v) wash the microplate as per known washing procedure; (vi) add 50 ul of working glow reagents solution to each well; and (vii) read RLU vales after 1 minutes and before 20 minutes of glow reagents addition.
Example: 3- Interpretation of results

The results of typical standard run of the assay are shown in Table 1 and Figure 1 of accompanying drawing.
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Table 1: Profile of RLU generations as function of TSH concentration

Sr. No. TSH concentration (\xIU/ml) RLU
1 0 1234
2 0.1 3743
3 0.5 17643
4 3 75675
5 6 147696
6 10 312542
The results depicted in Table 1 and Figure 1 of the accompanying drawing are for just illustration purpose only and should not be used to calculate the concentrations of test specimens for testing.
Any further modifications in and/or improvements in any aspect of the
embodiments of this invention will also fall under the scope of this invention. In
view of the foregoing description and example, 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 this invention. Various features of the
invention hereinbefore described are set forth in the following claims.
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WE CLAIMS:

1. A diagnostic kit for quantitatively detecting an amount of TSH in human
serum and/or plasma samples comprises:
(i) a microtitre plate consisting of plurality of microwells, which are coated with anti-TSH monoclonal antibodies by covalently bonding for immobilising TSH present in the human serum and/or plasma samples;
(ii) a conjugate consisting of an enzyme, which is attached with anti-TSH monoclonal antibodies by covalently linkage for detecting TSH present in the human serum and/or plasma samples; and
(iii) an immunoassay reagents consisting of sample diluent, thyrotropin standards, glow reagents and washing solution.

2. The diagnostic kit of claim 1, wherein the microwells are coated with the anti-TSH monoclonal antibodies by covalent bonding for immobilising TSH without adversely affecting structure and activity of said antibodies.
3. The diagnostic kit of claim 1, wherein the enzyme linked with anti-TSH monoclonal antibodies of conjugate is HRPO and wherein anti-TSH monoclonal antibodies are linked with HRPO by covalent boning without adversely affecting structure and activity of said enzyme and antibodies.
4. The diagnostic kit of any of preceding claims, in which the microtitre plate comprises at least 24 and not more than 96 microwells coated with anti-TSH monoclonal antibodies for detecting TSH present in the human serum ands/or plasma samples.
5. The diagnostic kit of claim 1, wherein the immunoassay reagents for detecting antigen-antibody complex comprises sample diluent, thyrotropin standard solutions (3a to 3f), glow reagent-A and B and washing solution.
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6. The diagnostic kit of claim 5, wherein the solutions of thyrotropin standards (3a to 3f) comprise thyrotropin in amounts of 0, 0.1, 0.5, 3, 6 and 10 uIU per millilitres.
7. The diagnostic kit of claim 5, wherein the solutions of glow reagents A and B comprise HRP substrates component A and B, respectively.
8. The diagnostic kit of claim 5, wherein the washing solution comprises TRIS buffer, NaCl and Tween-20 in deionized water.
9. A method for preparing a diagnostic kit for quantitatively detecting an amount of TSH in the human serum and/or plasma samples comprises the steps of:
(i) preparing a microtitre plate consisting of plurality of microwells coated with anti-TSH monoclonal antibodies, the process for preparing the microtitre plate comprises the steps of:
(a) coating anti-TSH monoclonal antibodies dissolved in the bicarbonate buffer to microwells of microtitre plate by covalent bonding;
(b) blocking the bound anti-TSH monoclonal antibodies using blocking solution comprising phosphate buffer, BSA and Trans-001;
(c) stabilizing the blocked anti-TSH monoclonal antibodies using stabilising solution comprising PBS, Trans-002 and Tran-003 (bovine immunoglobulin);

(ii) preparing a conjugate consisting of an enzyme and anti-TSH monoclonal antibodies by covalently linkage between of enzyme (HRPO) and said monoclonal antibodies; and
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(iii) preparing solutions of thyrotropin standards, glow reagents and washing solution as immunoassay reagents.
A method for quantitatively detecting an amount of TSH in the human serum and/or plasma samples comprises the steps of:
(i) charging dispensed quantities of thyrotropin standards and TSH containing human serum and/or plasma samples into micro wells of the microtitre plate containing coated anti-TSH monoclonal antibodies for immobilising TSH molecules;
(ii) subsequently adding dispensed quantity of conjugate containing anti-TSH monoclonal antibodies linked with enzyme (HRPO) into the microwells, thereby forming stable antigen- antibody complex;
(iii) washing the microwells containing said reaction mixture using washing solution to remove unbound fraction of conjugate;
(iv) adding further dispensed quantity of working glow reagent into the said microwells, thereby generating the RLU; and
(v) detecting the amount of TSH in the samples by reading RLU values on standard plot of RLU Vs. TSH.
A diagnostic kit for quantitatively detecting an amount of TSH in the human serum and/or plasma samples such as hereinbefore described with reference accompanying examples and drawing.

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12. A method for preparing a diagnostic kit for quantitatively detecting an mount of TSH in the human serum and/or plasma samples such as hereinbefore described with reference accompanying examples and drawing.

Dated this 23rd day of May, 2007.

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