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
A kit for quantitative measurement of 3,5,3,' tri- iodothyronine in human serum and/or plasma;
APPLICANT(S)
a) Name
b) Nationality
c) Address
TRANSASIA BIO-MEDICALS LTD. INDIAN Company TRANSASIA HOUSE, 8, CHANDIVALI STUDIO ROAD, MUMBAI - 4 00 0 72 MAHARASHTRA
3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which it is to be performed : -

A kit for quantitative measurement of 3,5,3' tri-iodothyronine in human serum and/or plasma
Field of the Invention
The present invention relates to quantitative measurement of substances 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 based on immunoassay techniques involving the use of labeled reagents, such as enzyme-labeled reagents. The invention provides a kit for quantitative measurement of 3,5,3' tri-iodothyronine in human serum and/or plasma based on specific binding assay techniques.
Background of the Invention
3,5,3' tri-iodothyronine (T3) molecules circulates in bloodstream mostly bound to plasma protein, Thyroxin Binding Globulin (TBG). The concentration of T3 is much less than that of L-Thyroxin (T4), but its metabolic potency is comparatively much greater. The measurement of T3 has uncovered a variant of hyperthyroidism in thyrotoxic patients with elevated T3 values and normal T4 values. Likewise, an increase without increase in T4 is frequently a forerunner of recurrent thyrotoxicosis in previously treated patients. It is therefore, desirable to measure the amount of T3 in human serum and/or plasma using a simple and reliable detection kit and method for measuring the quantity of T3 in human serum and/or plasma. The appropriate technique for separating and detecting the T3 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 detected 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.
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, 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 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 limited amount of material may be applied to the absorbent and the separation is laborious and time-consuming. y


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 (T3 and T4). One technique of this type used for separation of antigen-antibody complex from free antigen employs a column packed with material, which 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 afore-said prior art and problems associated with it, inventors of the present invention, propose a kit for measuring amount of T3 in human serum and/or plasma that utilizes one monoclonal antibody covalently bonded with a solid support and second monoclonal antibody linked with horse raddish peroxidase (HRPO) as an enzyme conjugate for immobilizing and measuring amount of T3. 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
Therefore, an object of present invention is to provide a kit based on the solid phase immunoassay technique for quantitative measurement of amount 3,5,3' tri-iodothyronine (T3) present in the human serum and/or plasma.
Another object of the invention is to develop a kit for measuring amount of T3 in the human serum and/or plasma and diagnosing recurrent thyrotoxicosis in previously treated patients.
Yet another object of the instant invention is to provide a simple, cost effective and reliable kit for quantitative estimation of T3 amount in the human serum and/or plasma samples obtained form the recurrent thyrotoxicosis patients.
Still another object of the invention is to designs a kit comprising one monoclonal antibody to T3 as coating material and T3-HRPO enzyme conjugate for measuring amount of T3 in the samples obtained from the patents suffering from hypo- or hyperthyroidism.
Different object of the present invention is to provide a process for preparing a kit for measuring the amount of T3 present in the human serum and/or plasma.
Still different object of the instant application is to provide a process for measuring the amount of T3 in human serum and/or plasma.

Summary of the Invention
According to object of the present invention there is provided the kit for measuring the amount of 3,5,3' tri-iodothyronine (T3) in the human serum and/or plasma that comprises a solid support comprising covalently bound anti-T3 monoclonal antibodies to T3; an enzyme conjugate comprising covalently linked T3 to HRPO; and immunoassay reagents of the types such as T3 standards, glow reagents and a washing solution. A process for preparing the kit for measuring the amount of T3 in the human serum and/or plasma is also provided that comprises preparing a solid support, anti-T3 monoclonal antibody bound to solid support and enzyme conjugate by covalently binding T3 ; and preparing immunoassay reagents of the types such as a sample diluent, T3 standards, glow reagents and washing solution. Further, a process for quantitative measurement of T3 in the human serum and/or plasma is provided that comprises putting the human serum and/or plasma in to the solid support comprising anti-T3 monoclonal antibodies to T3 for said monoclonal antibodies with T3 molecules and detecting antigen- antibody complex using immunoassay reagent such as glow reagents for quantitative estimation of T3 amount in the samples.
Description of the invention
As per one aspect, the kit for quantitatively estimating the amount of T3 in the human serum and/or plasma comprises: (a) a microplate with plurality of microwells, which comprises covalently attached to said microwells an anti-T3 monoclonal antibodies for precipitating the molecules of T3 present in the human serum and/or plasma samples; (b) an enzyme conjugate, which comprises covalently linked with HRPO to T3; and (c) immunoassay reagents, such as T3 standards, glow reagents and a washing solution.
As per another aspect, the process for preparing a kit for quantitatively measuring the amount of T3 in the human serum and/or plasma comprises the steps of: (a) preparing a microplate, the process of which comprises: (al) covalently binding anti-T3 monoclonal antibodies dissolved in bicarbonate buffer to the microwells of the plate; (a2) blocking said covalently bound anti- T3 monoclonal antibodies using a blocking solution, comprising phosphate buffer, BSA and Trans-001; (a3) stabilizing the blocked anti-T3 monoclonal antibodies using a stabilising solution, comprising PBS, Trans-002 and Tran-003 (bovine immunoglobulin); (b) preparing an enzyme conjugate by covalently linking to T3 with HRPO; and (c) preparing solutions of immunoassay reagent of the types such as T3 standards, glow reagents and a washing solution.


As per different aspect, the process for quantitatively measuring the amount of T3 in the human serum and/or plasma comprises the steps of: (a) adding T3 standards and the human serum and/or plasma containing T3 molecules into the microwells of the plate, which comprise coated anti-T3 monoclonal antibodies; (b) adding further an enzyme conjugate, which comprises T3 linked to HRPO, thereby forming the stable antigen-antibody complex; adding a glow reagent to the wells comprising the said stable antigen-antibody complex, thereby generating the RLU; and measuring the amount of T3 in the samples by reading RLU using standard plot of RLU Vs. T3 concentrations
Detailed description of the invention
The instant invention can be understood more preferably with reference to following detailed and specific embodiments of the present invention. Although, the present 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 invention. Further to that unless otherwise described, all the technical and scientific terms used herein have the same meaning as commonly understood by the person skilled in the art to which this invention belongs.
In one embodiment of the present invention, the kit comprises a microplate plate comprising plurality of microwells, wherein said microwells are coated with the anti-T3 monoclonal antibodies by covalent bonding for precipitating the molecules of T3 present in the human serum and/or plasma samples.
In another embodiment of instant invention, the kit comprises an enzyme conjugate comprising HRPO, T3 are covalently linked with HRPO for quantitatively measuring the amount of T3 molecules by detecting the antigen-antibody complexes present the microwells of plate.
In still another embodiment of the invention, the kit comprises an immunoassay reagents, required for performing the estimation test, and separating and/or detecting the antigen- antibody complexes, which are the reagents of types selected form the group of sample diluent, T3 standards (3a to 3f), glow reagent-A and B and washing solution.
In one preferred embodiment of present application, the anti-T3 monoclonal antibodies are coated onto the microwell by covalent bonding without any adverse effect on their structure and activity.


In another preferred embodiment, the T3 are linked to HRPO by covalent linking without any adverse effect on their structure and activity.
In different embodiment, the microplate of kit of instant invention comprises at least 24 microwells coated with said antibodies for precipitating T3 molecules present in the sample of human serum ands/or plasma.
In another preferred embodiment, the microplate of the kit comprises not more than 96 microwells coated with said anti-T3 monoclonal antibodies.
In yet another preferred aspect of the invention, the enzyme conjugate comprises horse raddish peroxidase (HRPO) as an enzyme label.
In still separate preferred embodiment, the kit comprises solutions of T3 standards (3a to 3f) in concentrations of 0, 0.5, 1, 2.5, 5, 7.5 and 10 ng of T3 per ml.
In yet different aspect of the invention, the kit comprises glow reagents, solution-A and solution-B, containing HRP substrates, component-A and component-B.
In one embodiment, the washing solution (20X) comprises a TRIS buffer, NaCl, Tween-20 in deionized water.
In an important embodiment the solid phase immunoassay kit of the present invention is stored at temperature between 2°C and 8 °C.
According to another object, the process for preparing the kit comprises the steps of preparing a microplate in which anti-T3 monoclonal antibodies dissolved in bicarbonate buffer are covalently bound to the microwells of the plate; blocking said covalently bound anti-T3 monoclonal antibodies in which said antibodies are blocked using a blocking solution, which comprises phosphate buffer, BSA and Trans-001; and stabilizing the said blocked anti-T3 monoclonal antibodies in which said antibodies are stabilized using stabilising solution, which comprises PBS, Trans-002 and Tran-003 (bovine immunoglobulin).
In other preferred aspect of the process for preparing the kit comprises step of preparing an enzyme conjugate in which T3 are covalently linked with HRPO.


In still another preferred embodiment of process, it comprises preparing the solution of sample diluent in which phosphate buffer, BSA, Tween-20 are dissolved with thimerosal and gentamycin in deionized water.
In yet another preferred embodiment, the process comprises preparing the solutions of T3 standards in which T3 standards (3a to 3f) of concentrations 0, 0.5, 1, 2.5, 5, 7.5 and 10 ng of T3 are mixed in deionized water.
In different aspect, the process comprises preparing solutions of glow reagents in which glow reagents A and B are prepared by independently mixing HRP substrates component A and B in deionized water.
In still different aspect, the process comprises preparing washing solution in which a TRIS buffer, NaCl, Tween-20 dissolved in deionized water.
According to different object, the method for quantitatively measuring the amount of T3 in the human serum and/or plasma comprises the steps of, adding T3 standard solutions (3a to 3f) and the human serum and/or plasma containing T3 molecules into the microwells comprising coated anti-T3 monoclonal antibodies for bringing said T3 standards and T3 molecules of the human serum and/or plasma in contact with said monoclonal antibodies; adding further the enzyme conjugate comprising linked T3 to HRPO, thereby forming antigen-antibody complex of said T3 molecules and monoclonal antibodies; and quantitatively measuring the T3 molecules in samples by detecting antigen-antibody complex using glow reagents.
In preferred embodiment of the process for quantitatively measuring the amount of T3, the method comprises contacting T3 molecules present in the standard solutions and human serum and/or plasma with anti-T3 monoclonal antibodies present in the microwells and enzyme conjugate, thereby forming stable antigen-antibody complex.
In another preferred embodiment of said process, it comprises detecting antigen-antibody complex by adding glow reagents to the microwells containing said complex, thereby generating Relative Light Units (RLU), which are inversely proportional to the concentrations of standard solutions or amount quantity of T3 present in the test samples.
In still another preferred embodiment, the enzyme conjugate consists of immunological components that covalently linked to the enzyme molecule, which is achieved either by direct

condensation or by using external bridging molecules. Thus, the enzyme coupling products is produced by employing the covalent bond, which is effected by using the reagents, for example, carbodiimides, diisocyanates, glutaric aldehyde and bis-diazobenzidine.
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 measurement of amount of T3 present in test samples
Adding T3 standard solutions or T3 containing human serum and/or plasma to the microwells of the plate; adding further the enzyme conjugate comprising the T3 linked with 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 enzyme 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 T3 standard and human serum and/or plasma containing wells; and finally measuring the amount of T3 by reading RLU using standard plot of RLU Vs. T3 concentrations in ng per ml.
Example: 2 - Test procedure for measuring amount of T3 in the test samples
(i) Bring all the reagents and test specimens at room temperature before use; (ii) add 50 ul of standard solutions and test specimens to the respective wells; (iii) add 50 fxl 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 p.1 of working glow reagents solution to each well; and (vii) read RLU 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 below and Figure 1 of accompanying drawing.


Sr. No. T3 concentration (ng/ml) RLU
1 0 312764
2 0.5 2218843
3 1 156612
4 2.5 116721
5 5 72341
6 10 46439
These results depicted in Table 1 and figure 1 of accompanying drawing are for just illustration only and should not be used to calculate the concentrations of test specimens.

WE CLAIM :
1. A kit for quantitatively measuring an amount of T3 in human serum and/or plasma samples comprises:
(a) a microplate having plurality of microwells, which comprises covalently attached an anti-T3 monoclonal antibodies to the microwells for precipitating T3 molecules present in the human serum and/or plasma samples;
(b) an enzyme conjugate, which comprises covalently linked T3 with HRPO for detecting T3 molecules present in the human serum and/or plasma samples; and
(c) immunoassay reagents, which comprise the reagent of the types such as T3 standards, glow reagents and washing solution.
2. The kit according to claim 1, wherein the microwells are coated with the anti-T3 monoclonal antibodies by covalent bonding for precipitating the molecules of T3 without adversely affecting structure and activity.
3. The kit according to claim 1, wherein HRPO is linked with T3 by covalently linking for quantitatively measuring the amount of T3 molecules without adversely affecting structure and activity.
4. The kit according to any one of preceding claims, in which the microplate comprises at least 24 and not more than 96 microwells coated with said monoclonal antibodies for precipitating the T3 molecules present in the human serum ands/or plasma samples.
5. The kit according to claim 1, wherein the immunoassay reagents, required for performing the test, and separating and/or detecting the antigen-antibody complex, are the reagents of types such as sample diluent, T3 standard solutions (3a to 3f), glow reagent-A and B and washing solution.
6. The kit according to claim 5, wherein the solutions of T3 standards (3a to 3f) comprise the concentrations of 0, 0.5, 1, 2.5, 5, 7.5 and 10 ng of T3 standard per ml of solution.
7. The kit according to claim 5, wherein the solutions of glow reagents, A and B, comprise HRP substrates component A and B.


The kit according to claim 5, wherein the washing solution comprises a solution of TRIS buffer, NaCl and Tween-20 in deionized water.
A process for preparing a kit for quantitatively measuring the amount of T3 in the
human serum and/or plasma samples comprises the steps of:
(a) preparing a microplate having plurality of microwells in which it comprises the steps of:
(al) covalently binding the anti-T3 monoclonal antibodies dissolved in the bicarbonate buffer to the microwells of the plate;
(a2) blocking the said attached anti-T3 monoclonal antibodies using the blocking solution, which comprises phosphate buffer, BSA and Trans-001;
(a3) stabilizing the said blocked anti-T3 monoclonal antibodies using the stabilising solution, which comprises PBS, Trans-002 and Tran-003 (bovine immunoglobulin);
(b) preparing an enzyme conjugate by covalently linking the T3 with HRPO; and
(c) preparing solutions of immunoassay reagent of the types such as T3 standards, glow reagents and washing solution.
A process for quantitatively measuring amount of T3 in the human serum and/or
plasma samples comprises the steps of:
(a) adding T3 standard solutions and human serum and/or plasma samples containing T3 into the microwells of the plate, which comprises the coated anti-T3 monoclonal antibodies for precipitating T3;
(b) further adding an enzyme conjugate, which comprises the linked T3 to the microwells, thereby forming stable antigen- antibody complex; and
(c) adding a glow reagent to the wells comprising the said stable antigen-antibody complex, thereby generating the RLU; and

(d) measuring the amount of T3 in the samples by reading RLU using standard plot of RLU Vs. T3 concentrations
11. A kit for quantitatively measuring the amount of T3 in the human serum and/or plasma samples such as herein disclosed with reference to the description and accompanying examples.
12. A process for preparing the kit for quantitatively measuring the amount of T3 in the human serum and/or plasma samples such as herein disclosed with reference to the description and accompanying examples.
Dated this 24th day of March, 2007.

CHAIRMAN AND MANAGING DIRECTOR TRANSASIA BIO-MEDICALS LTD.