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 SOLID PHASE IMMUNOASSAY KIT FOR ESTIMATING L-THYROXINE 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 - 4 00 0 72
MAHARASHTRA

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

A solid phase immunoassay kit for estimating L-Thyroxine 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 L-Thyroxine in human serum and/or plasma based on specific binding assay techniques.
BACKGROUND OF THE INVENTION
L-Thyroxine (T4) is a hormone that is synthesized and stored in thyroid gland. More than 99% of it is bound to plasma protein, Thyroxine Binding Globulin, Thyroxine binding pre-albumin and albumin. Normally around 0.03% is circulated in fee form. Measurement of serum thyroxine concentration is generally regarded as an important in vitro diagnostic test for assessing thyroid function. It is therefore, desirable to measure the amount of T4 in human serum and/ or plasma using a simple and reliable kit and method for measuring an amount of T4 in human serum and/or plasma. The appropriate technique for separating and measuring the amount of T4 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.
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.
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 adsorbeti 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 measuring amount of T4 in human serum and/or plasma that utilizes one monoclonal antibody covalently bonded with a solid support and T4- linked with horse redfish peroxidase (HRPO) as an enzyme conjugate for precipitating and measuring amount of T4. 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, main object of this invention is to provide a solid phase immunoassay kit for quantitatively measuring the amount L-Thyroxine (T4) present in the human serum and/or plasma.
Another object of the present invention is to develop a solid phase immunoassay kit for measuring the amount of T4 in the human serum and/or plasma and assessing thyroid function.
Yet another object of the instant invention is to provide a simple, cost effective and reliable solid phase immunoassay kit for quantitatively estimating the amount of T4 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 designs a solid phase immunoassay kit comprising one anti-T4 monoclonal antibody (MAb) as a coating material and T4 linked to enzyme conjugate for measuring the amount of T4 in the samples of the patents suffering from problems of either hypo- or hyperthyroidism.
Different object of the present invention is to provide a process for preparing a solid phase immunoassay kit for measuring the amount of T4 present in the human serum and/or plasma.
Still different object of the instant application is to provide a process for quantitatively measuring the amount of T4 in human serum and/or plasma.

SUMMARY OF THE INVENTION
In accordance with main object of the present invention there is provided the solid phase immunoassay kit for quantitatively measuring the amount of L-Thyroxine (T4) in the human serum and/or plasma comprising of a solid support, which comprises covalently bound anti-T4 monoclonal antibodies; an enzyme conjugate, which comprises T4 linked with HRPO; and reagents required for immunoassay of the types such as sample diluent, T4 standards, glow reagents and a washing solution. A process for preparing the solid phase immunoassay kit for quantitatively measuring the amount of T4 in the human serum and/or plasma is also provided, which comprises preparing a solid support and enzyme conjugate by covalently binding T4 with HRPOwith; and preparing reagents required for immunoassay of the types such as T4 standards, glow reagents and washing solution. Further, a process for quantitatively measuring the amount of T4 in the human serum and/or plasma is provided, which comprises contacting the human serum and/or plasma containing T4 molecules into the solid support that comprising bound anti-T4 monoclonal antibodies and conjugated T4-HRPO, thereby forming antigen-antibody complex; and detecting the said antigen-antibody complex using the reagent required for immunoassay, such as, glow reagents for quantitatively estimating the amount of T4 in the samples.
DESCRIPTION OF THE INVENTION
According to one object, the kit for quantitatively measuring the amount of T4 in the human serum and/or plasma comprises: (a) a microplate having plurality of microwells and comprising covalently bound to said microwells an anti-T4 monoclonal antibodies for precipitating the T4 present in the human serum and/or plasma samples; (b) an enzyme conjugate comprising T4 covalently linked with HRPO; and (c) reagents required for the immunoassay comprising T4 standards, glow reagents and a washing solution.

According to another object, the process for preparing a solid phase immunoassay kit for quantitatively measuring the amount of T4 in the human serum and/or plasma comprises the steps of: (a) preparing a microplate that further comprises the steps of: (al) covalently binding anti-T4 monoclonal antibodies dissolved in bicarbonate buffer to the microwells of the plate; (a2) blocking the covalently bound anti-T4 monoclonal antibodies using the blocking solution, containing phosphate buffer, BSA and Trans-001; (a3) stabilizing the blocked anti-T4 monoclonal antibodies using the stabilising solution, containing PBS, Trans-002 and Tran-003 (bovine immunoglobulin); (b) preparing an enzyme conjugate by covalently linking T4 to HRPO; and (c) preparing solutions of the reagent required for immunoassay of the types such as T4 standards, glow reagents and washing solution.
According to different object of the invention, the process for quantitatively measuring the amount of T4 in the human serum and/or plasma comprises the steps of: (a) adding T4 standards and the human serum and/or plasma containing T4 into the microwells of the plate comprising coated anti-T4 monoclonal antibodies; (b) subsequently adding an enzyme conjugate comprising T4 linked to HRPO, thereby forming the stable antigen-antibody complex; (c) washing the microwells after completion of incubation using the washing solution; (d) further adding glow reagents to "the microwells containing the stable antigen-antibody complex, thereby generating the RLU; and (e) measuring the amount of T4 in the samples by reading RLU using standard plot of RLU Vs. T4 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 before and after have the same meanings as commonly understood by the person skilled in the art to which this invention belongs.
In one embodiment, the solid phase immunoassay kit of the present invention comprises a microplate plate having plurality of microwells, wherein said microwells are coated with the anti-T4 monoclonal antibodies by covalent bonding for precipitating the T4 present in the human serum and/or plasma samples.
In another embodiment, the solid phase immunoassay kit of instant invention comprises an enzyme conjugate, wherein T4 covalently linked to HRPO for quantitatively measuring the amount of T4 by detecting the antigen-antibody complex present the microwells of plate.
In still another embodiment, the solid phase immunoassay kit of the invention comprises an immunoassay reagents for performing the assay and detecting the antigen-antibody complex, which are the reagents of types such as sample diluent, T4 standards (3a to 3f), glow reagent-A and B and washing solution.
In one preferred embodiment of present application, the anti-T4 monoclonal antibodies are coated onto the microwell by covalent bonding without any adverse effect on their structure and activity.
In another preferred embodiment, T4 are linked to HRPO by covalent linking without any adverse effect on their structure and activity.
In different embodiment, the microplate of the solid phase immunoassay kit of instant invention comprises at least 24 microwells coated with said antibodies for precipitating T4 present in the sample of human serum ands/or plasma.
In another preferred embodiment, the microplate of the said kit comprises not more than 96 microwells coated with said anti-T4 monoclonal antibodies.

In yet another preferred aspect of the invention, the enzyme conjugate comprises horse reddish peroxidase (HRPO) as an enzyme label.
In still separate preferred embodiment, the kit comprises solutions of T4 standards (3a to 3f) in concentrations of 0, 5, 10, 25, 50, 100 and 200 nmol of T4 per litre with thimerosal and gentamycin.
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.
Accordingly, the process for preparing the solid phase immunoassay kit comprises: preparing a microplate in which anti-T4 monoclonal antibodies dissolved in the bicarbonate buffer are covalently bound to the microwells of the plate; blocking the covalently bound anti-T4 monoclonal antibodies in which said antibodies are blocked using the blocking solution containing phosphate buffer, BSA and Trans-001; and stabilizing the blocked anti-T4 monoclonal antibodies in which said antibodies are stabilized using the stabilising solution containing PBS, Trans-002 and Tran-003 (bovine immunoglobulin).
In other preferred aspect of the process for preparing the solid phase immunoassay kit of the present invention comprises preparing an enzyme conjugate in which T4 are covalently linked to HRPO.
In yet another preferred embodiment, the process comprises preparing the solutions of
T4 standards in which T4 standards (3a to 3f) of concentrations 0, 5, 10, 25, 50, 100 and
200 nmol T4 per litre 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.
Accordingly, the method for quantitatively measuring the amount of T4 in the human serum and/or plasma comprises the steps of: adding T4 standard solutions (3a to 3f) and the human serum and/ or plasma containing T4 into the microwells comprising coated anti-T4 monoclonal antibodies for bringing said T4 standards and T4 present in the human serum and/or plasma samples in contact with said monoclonal antibodies; subsequently adding the enzyme conjugate T4 linked to HRPO, thereby forming antigen-antibody complex of said T4 molecules and monoclonal antibodies; and quantitatively measuring the T4 molecules in samples by detecting antigen-antibody complex using glow reagents.
In preferred embodiment of the process for quantitatively measuring the amount of T4, the method comprises contacting T4 molecules present in the standard solutions and human serum and/or plasma with anti-T4 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 T4 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 T4 present in test samples
Adding T4 standard solutions or T4 containing human serum and/or plasma to the microwells of the plate; subsequently adding the enzyme conjugate comprising the T4 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 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 T4 standard and human serum and/or plasma containing wells; and finally measuring the amount of T4 by reading RLU using standard plot of RLU Vs. T4 concentrations in nmol per liter.
Example: 2 - Test procedure for measuring amount of T4 in the test samples
(i) Bring all the reagents and test specimens at room temperature before use; (ii)keep
one blank (50 ul sample diluent + 50 ul enzyme conjugate) and six standards in each
run; (iii)add 50 ul of standards and test specimens to the respective wells; (iv0add 50 ml
of conjugate to each well; (v)incubate 60 minutes at room temperature (20 - 40°C),
preferably 37 °C; (vii) wash the microplate as per known washing procedure; (vii) add

50 (j.1 of working glow reagents solution to each well; and (viii) 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. .
Table 1: Profile of RLU generations as function of T4 concentration

Sr. No. T4 concentration (nmol/L) RLU
1 0 363542
2 10 257432
3 25 206743
4 50 154972
5 100 124321
6 200 96432
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.

WE CLAIM :
1. A solid phase immunoassay kit for quantitatively measuring an amount of T4 in
human serum and/or plasma samples comprises:
(a) a microplate having plurality of microwells, wherein anti-T4 monoclonal antibodies are covalently bound to the microwells of the plate for precipitating T4 present in the human serum and/or plasma samples;
(b) an enzyme conjugate, T4 are covalently linked to HRPO for detecting T4 present in the human serum and/or plasma samples; and
(c) reagents required for immunoassay, comprising the reagent of the types such as, T4 standards, glow reagents and washing solution.

2. The solid phase immunoassay kit of claim 1, wherein the microwells are coated with the anti-T4 monoclonal antibodies by covalent bonding for precipitating the T4 without adversely affecting structure and activity.
3. The solid phase immunoassay kit of claim 1, wherein the HRPO is linked with T4 molecules by covalent linkage for quantitatively measuring the T4 without adversely affecting structure and activity.
4. The solid phase immunoassay kit of any one of preceding claims, in which the microplate comprises at least 24 and not more than 96 microwells coated with anti-T4 monoclonal antibodies for precipitating the T4 molecules present in the human serum ands/or plasma samples.
5. The solid phase immunoassay kit of claim 1, wherein the reagents required for performing the immunoassay and detecting the antigen-antibody complex are the reagents of types such as T4 standard solutions (3a to 3f), glow reagent-A and B and washing solution.

.6. The solid phase immunoassay kit of claim 5, wherein the solutions of T4 standards (3a to 3f) contain the concentrations of 0, 5,10, 25, 50,100 and 200 nmol of T4 standard per litre of the solution.
7. The solid phase immunoassay kit of claim 5, wherein the solutions of glow reagents, A and B, contain HRP substrates component A and B.
8. The solid phase immunoassay kit of claim 5, wherein the washing solution contains a solution of TRIS buffer, NaCl and Tween-20 in deionized water.
9. A process for preparing a solid phase immunoassay kit for quantitatively measuring the amount of T4 in the human serum and/or plasma samples comprises the steps of:
(a) preparing a microplate having plurality of microwells, which further
comprises the steps of:
(al) covalently binding anti-T4 monoclonal antibodies dissolved in the bicarbonate buffer to the microwells of the plate;
(a2) blocking the bound anti-T4 monoclonal antibodies using the blocking solution comprising phosphate buffer, BSA and Trans-001;
(a3) stabilizing the blocked anti-T4 monoclonal antibodies using the stabilising solution comprising PBS, Trans-002 and Tran-003 (bovine immunoglobulin);
(b) preparing an enzyme conjugate by covalently linking T4 molecules to HRPO; and
(c) preparing solutions of sample diluent, T4 standards, glow reagents and washing solution as immunochemical reagents required for immunoassay.

10. A process for quantitatively measuring amount of T4 in the human serum
and/ or plasma samples comprises the steps of:
(a) adding T4 standard solutions and T4 containing human serum and/or plasma samples into the microwells of the plate comprising coated anti-T4 monoclonal antibodies for precipitating T4 molecules;
(b) subsequently adding an enzyme conjugate comprising linked anti-T4 monoclonal antibodies to the microwells, thereby forming stable antigen-antibody complex;
(c) washing the microwells containing said reaction mixture using a washing solution to remove unbound fraction of conjugate molecule;
(d) adding a glow reagent to the wells containing stable antigen-antibody complex, thereby generating the RLU; and
(e) measuring the amount of T4 in the samples by reading RLU using standard plot of RLU Vs. T4 concentrations

11. A solid phase immunoassay kit for quantitatively measuring the amount of T4 in the human serum and/or plasma samples such as herein disclosed with reference to description, and accompanying examples and drawing.
12. A process for preparing a solid phase immunoassay kit for quantitatively measuring the amount of T4 in the human serum and/ or plasma samples such as herein disclosed with reference to description and accompanying examples drawing.