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
AN ASSAY KIT FOR MEASURING CONCENTRATION LH AND/OR PLASMA

IN HUMAN SERUM

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

TRANSASIA BIO-MEDICALS LTD.
INDIAN Company
TRANSASIA HOUSE,
8, CHANDIVALI STUDIO ROAD,
MUMBAI - 4 00 072
MAHARASHTRA

3.PREAMBLE TO THE DESCRIPTION

The following specification particularly invention and the manner in which it is to

describes be

An assay kit for measuring concentration LH 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 Luteinizing Hormones (LH), based on immunoassay techniques involving the use of labeled reagents, such as enzyme-labeled reagents. The invention provides a kit for quantitatively measuring LH in human serum and/or plasma based on specific binding assay techniques.
BACKGROUND OF THE INVENTION
LH is a glycoprotein hormone, having alpha and beta subunits with molecular weight approximately 30000 Daltons. The variation in LH concentrations in women is subject to the complex ovulatory cycle of healthy menstruating women, and depends upon a sequence of hormonal events along with the gonado-hypothalamic-pituitary axis. Patients suffering from hypogonadism show increased concentration of serum LH. In differential diagnosis of hypothalamic, pituitary or gonadal dysfunction, assays of LH concentration are routinely performed in conjunction with FSH assay as their roles are closed interrelated. It is therefore, desirable to measure the concentration of LH in human serum and/or plasma using a simple and reliable kit and method for measurement of its concentration. The most appropriate technique for separating and measuring the concentration of LH molecules in human serum and/or plasma is solid-phase technique that 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,
2

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, 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 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

4

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 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 measuring the concentration of LH in human serum and/or plasma that utilizes one monoclonal antibody covalently bonded with a solid support and other monoclonal antibody linked with horse redfish peroxidase (HRPO) as a conjugate.


An advantage of using the solid support like microwells containing microtitre plate is that no centrifugation or filtration required for separation of solid and liquid phases.
OBJECT OF THE INVENTION
Therefore, the object of present invention is to provide an assay kit for measuring the concentration of LH in the human serum and/or plasma.
Therefore, another object of the instant invention is to design an assay kit for detecting the concentration of LH for diagnosis of hypothalamic, pituitary or gonadal dysfunction.
Therefore, yet another object of this invention is to develop a simple, cost effective and reliable an assay kit for estimating an amount of LH in the human serum and/or plasma samples obtained from patients those are suffering form hypothalamic, pituitary or gonadal dysfunction.
Therefore, still another object of the invention is to provide an assay kit, which comprises one anti-LH monoclonal antibody as a coating material and another anti-LH monoclonal antibody linked to conjugated enzyme for measuring LH in the test samples obtained form the patients suffering from hypothalamic, pituitary or gonadal dysfunction.
Therefore, a separate object of the present invention is to provide process for preparing an assay kit for measuring the amount of LH present in the human serum and/or plasma.

Therefore, still separate object of this application is to provide a process for measuring concentration of LH in human serum and/or plasma for diagnosis of hypothalamic, pituitary or gonadal dysfunction.
6

SUMMARY OF THE INVENTION

Pursuant to an object of present invention, an assay kit is provided for measuring the concentration of LH in human serum and/or plasma, which comprises solid support coated with anti-LH monoclonal antibodies by covalently bonding; a conjugated enzyme linked with anti-LH monoclonal antibodies by covalently linkage; and immunochemical reagents required for measuring concentration of LH in the human serum and/or plasma.
Further, a process for preparing an assay kit for measuring concentration of LH in human serum and/or plasma is provided, which comprises preparing a solid support and conjugated enzyme that are covalently bound with anti-LH monoclonal antibodies; and preparing immunochemical reagents required for measuring concentration of LH in the human serum and/or plasma.
Still further, a process for measuring concentration of LH in human serum and/or plasma samples is provided, which comprises bringing human serum and/or plasma samples form the patients suffering form hypothalamic, pituitary or gonadal dysfunction in contact with solid support coated with anti-LH monoclonal antibodies and conjugated enzyme, thereby forming stable antigen-antibody complex; and finally detecting said antigen-antibody complex using working glow reagent for measuring the concentration of LH in said samples.
DESCRIPTION OF THE INVENTION
The assay kit for measuring concentrations of LH in the human serum and/ or plasma samples provided by this invention comprises: (a) a microtitre plate, containing plurality of microwells in which anti-LH monoclonal antibodies are coated onto said wells using covalently bonding for binding with LH molecules present in the human serum and/or plasma samples; (ii) a conjugated enzyme, containing enzyme in which anti-LH monoclonal antibodies are affixed to said

7

enzyme using covalent linkage for detecting LH molecules present in said human serum and/or plasma samples; and (iii) an immunochemical reagents, such as, LH standards, glow reagents and washing solution for performing the assay.
The process for preparing an assay kit for measuring concentrations of LH in human serum and/or plasma sample of present invention comprises the steps of: (a) preparing a microtitre plate, containing plurality of microwells coated with anti-LH monoclonal antibodies, wherein the process for coating the microwells comprises the steps of: (al) coating anti-LH monoclonal antibodies mixed with bicarbonate buffer onto the microwells by covalent bond coating; (a2) fastening the coated anti-LH monoclonal antibodies using blocking solution of phosphate buffer, BSA and Trans-001; (a3) further stabilizing the fastened anti-LH monoclonal antibodies using stabilising solution of PBS, Trans-002 and Tran-003 (bovine immunoglobulin); (b) preparing a conjugated enzyme, which comprises affixing anti-LH monoclonal antibodies with enzyme (HRPO) by covalently bonding; and (c) preparing immunochemical reagent, which includes preparing the solutions of LH standards, (a - f) glow reagents A and B and washing solution.
The process for measuring concentrations of LH in human serum and/or plasma samples of this invention comprises the steps of: (a) loading dispensed amount of LH standard solutions and LH containing human serum and/or plasma samples form the patients suffering form hypothalamic, pituitary or gonadal dysfunction into the microwells of microtitre plate coated with anti-LH monoclonal antibodies for binding to LH molecules; (b) subsequently loading dispensed amount of conjugated enzyme affixed with anti-LH monoclonal antibodies, thereby forming stable antigen-antibody complex; (c) washing the microwells using washing solution so as to remove unbound fraction of conjugated enzyme; (d) adding dispensed amount of working glow reagent to said microwells, thereby generating RLU; and (e) measuring the concentration of LH in the samples by reading RLU on standard plot ofRLUVsLH.

8
DETAILED DESCRIPTION OF THE INVENTION

The instant 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 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 specific embodiment of present invention, the assay kit comprises the microtitre plate that contains plurality of microwells, wherein anti-LH monoclonal antibodies are coated onto said microwells by covalently bonding so that said antibodies will specifically bind LH molecules present in human serum and/or plasma samples obtained form the patients suffering form hypothalamic, pituitary or gonadal dysfunction.
In another specific embodiment, the assay kit of instant invention comprises the conjugated enzyme, wherein anti-LH monoclonal antibodies are covalently attached to the enzyme so that said antibodies will specifically bind LH molecules present in human serum and/or plasma samples obtained form said patients, thereby detecting the antigen-antibody complex present the microwells.
In more specific embodiment of the kit of present invention, the enzyme label used for preparing the conjugated enzyme is Horse Reddish Peroxidase (HRPO).
In still another specific embodiment, the immunochemical reagents provided in the
LH assay kit of present invention are selected form the group of I standards (3a to 3f),

glow reagent-A and B and washing solution.
9

In yet another specific embodiment, the anti-LH monoclonal antibodies are coated onto the microwell by covalent bonding without adversely affecting the structure and activity of said antibodies.
In alternate specific embodiment of the invention, the anti-LH monoclonal antibodies are linked to conjugated HRPO by covalent linkage without adversely affecting the structure and activity of said antibodies.
In separate specific embodiment, the microtitre plate provided in assay kit of present invention at least comprises 24 microwells coated with monoclonal antibodies directed to LH molecule in of human serum ands/or plasma samples.
In another embodiment of the invention, the microtitre plate comprises not more than 96 microwells coated with said monoclonal antibodies.
In another aspect of the invention, the LH standards provided in the assay kit comprises six solutions (a - f) of LH standards of the concentrations 0, 1, 5, 25, 100 and 200 mlU per millilitre along with thimerosal and gentamycin.
In still another aspect, the assay kit of this invention comprises two solutions of glow reagents (A and B), which contain HRP substrate component-A and B, respectively.
In more preferred aspect of the invention, at the time assay, fresh working glow reagent is being prepared by mixing equal volumes of glow reagent A and B.
In yet another aspect, the washing solution provided in the kit comprises a mixture of TRIS buffer, NaCl, Tween-20 in deionized water.
In detailed embodiment, the assay kit of the invention used for measuring the concentration of LH in human serum and/or plasma samples is stored at temperature between 2°C and 8
10
In another specific embodiment, the process for preparing the assay kit for measuring the concentration of LH in human serum and/or plasma samples of the present invention comprises preparing the microtitre plate, which contains plurality of microwells, wherein said process for preparing microtitre plate comprises coating a mixture of anti-LH monoclonal antibodies in bicarbonate buffer onto said microwells by covalent binding; fastening the coated anti-LH monoclonal antibodies onto said microwells using blocking solution, comprising a mixture of phosphate buffer, BSA and Trans-001; stabilizing further the fastened anti-LH monoclonal antibodies onto the microwells using stabilising solution, comprising mixture of PBS, Trans-002 and Tran-003 (bovine immunoglobulin).
In further specific embodiment, the process for preparing assay kit comprises preparing the conjugated enzyme, wherein the anti-LH monoclonal antibodies are liked to HRPO of the conjugate by covalent bonding without affecting structures and activities of said enzyme and monoclonal antibodies.
In still another specific embodiment, the said process comprises preparing standard solutions of LH by dissolving LH standards (a - f) in concentrations of 0,1, 5, 25,100 and 200 mlU per millilitre of deionized water.
In different specific embodiment, the process for preparing the kit comprises preparing solutions of glow reagents A and B by mixing HRP substrates component A and B, respectively in deionized water. Further to that, at the time use working glow reagent is prepared by mixing equal volume of said glow reagent A and B.
In separate specific embodiment, the process of preparation also comprises preparing washing solution by mixing TRIS buffer, NaCl, Tween-20 and deionized water.

11

In yet another specific embodiment of the present invention, the process for measuring the concentration of LH in human serum and/or plasma samples form the patients suffering form hypothalamic, pituitary or gonadal dysfunction comprises loading dispensed amount of LH standards and LH containing human serum and/or plasma samples into the micro wells of plate coated with anti-LH monoclonal antibodies for separating LH molecules, thereby forming stable antigen-antibody; subsequently loading dispensed amount of conjugated enzyme having affixed to it anti-LH monoclonal antibodies, thereby forming enzyme labelled antigen-antibody complex; removing unbound conjugated enzyme by washing the microwells using washing solution; further loading dispensed amount of freshly prepared working glow reagent to said microwells, thereby generating RLU in the wells containing LH standards and LH containing human serum and/or plasma samples; and measuring at the end the concentration of LH in the samples by reading RLU on standard plot of RLU Vs LH.
In more specific embodiment, the method comprises bringing the LH standard solutions and LH containing human serum and/or plasma samples in contact with coated anti-LH monoclonal antibodies of the microtitre plate and conjugated enzyme, thereby forming stable antigen-antibody complex, which separate LH molecules for measurement of their amount in the samples using said standard plot.
In still more specific embodiment, the method comprises separating the antigen-antibody complex formed as a result of immunochemical bonding between said antigens, such as LH and antibodies, such as anti-LH monoclonal antibodies. The step of separation is performed by adding working glow reagent to the microwells, thereby generating RLU as a result of catalytic reaction of enzyme, such as HRPO, on the substrate present in the glow reagent. Therefore, an amount of RLU is proportional to the amount of LH standards or LH content of test samples.

12

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 LH in test samples
Adding measured amount LH standards or LH containing human serum and/or plasma samples to the microwells of plate; subsequently adding measured amount of conjugated enzyme to said microwells and incubating the said plate for 60 minutes at room temperature between 20°C and 40°C, thereby forming stable antigen-antibody complex; washing the microtitre plate using washing solution, thereby removing unbound fraction of conjugated enzyme; adding working glow reagent, containing HRP substrate to the microwells, containing said antigen-antibody complex, thereby generating RLU in LH standards and human serum and/or plasma samples containing microwells; and finally measuring the concentration of LH by reading RLU values on standard plot of RLU Vs LH (mlU/ml).
Example: 2 - Test procedure for measuring of LH in the test samples
(i) Bring all reagents and test specimens at room temperature before use; (ii) add 50 (j.1 of standards and test specimens to the respective wells; (iii) add 50 ju.1 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 reagent to each well; and (vii) read RLU vales after 1 minutes and before 20 minutes of glow reagent addition.

13

Example: 3- Interpretation of results
The results of typical standard assay run carried out using the kit of the present invention are shown in Table 1 and Figure 1 of accompanying drawing..
Table 1: Profile of RLU generations as function ofLH concentration

Sr. No. LH concentration (mlU/ml) RLU
1 0 1234
2 1 3245
3 5 12543
4 25 51325
5 100 189626
6 200 362743
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.
14

WE CLAIMS:
1. An assay kit for measuring a concentration of LH in human serum and/or
plasma samples comprises:
(a) a microtitre plate, containing plurality of microwells in which anti-LH monoclonal antibodies are coated onto said wells using covalently bonding for binding with LH molecules present in the human serum and/or plasma samples;
(b) a conjugated enzyme, containing enzyme in which anti-LH monoclonal antibodies are affixed to said enzyme using covalent linkage for detecting LH molecules present in said human serum and/or plasma samples; and
(c) an immunochemical reagents, such as, sample diluent, LH standards, glow reagents and washing solution for performing the assay.

2. The assay kit as according to claim 1, in which the microwells comprises one coated anti-LH monoclonal antibodies onto surfaces of said microwells for separating LH molecules from pool of molecules present in the human serum and/or plasma samples obtained from the patients.
3. The assay kit according to claim 1, in which the conjugate comprises second linked anti-LH monoclonal antibodies to an enzyme, such as HRPO for detection of LH molecules in the human serum and/ or plasma samples obtained from the patients.
4. The assay kit according to claims 1 and 2, in which the microtitre plate comprises at least 24 and 96 microwells coated with anti-LH monoclonal antibodies.
15

5. The assay kit according to claim 1, in which the immunochemical reagents provided in the kit include sample'diluent, LH standard solutions (a - f), glow reagent-A and B and washing solution.
6. The assay kit according to claims 1 and 5, in which the LH standard solutions (a - f) comprise LH standards in the concentration of 0, 1, 5, 25, 100 and 200 mlU per millilitres.
7. The assay kit according to claims 1 and 5, wherein the solutions of glow reagents A and B comprise HRP substrates A and B and wherein the working glow reagent comprises equal volumes of glow reagent A and B.
8. The assay kit according to claims 1 and 5, in which the washing solution comprises a mixture of TRIS buffer, NaCl, Tween-20 and deionized water.
9. A process for preparing an assay kit according to claims 1 to 9 for measuring the concentration of LH in the human serum and/or plasma samples comprises:
(a) preparing a microtitre plate, containing plurality of microwells coated with anti-LH monoclonal antibodies, wherein the process for coating the microwells comprises the steps of:
(al) coating anti-LH monoclonal antibodies mixed with bicarbonate buffer onto the microwells by covalent bond coating;
(a2) fastening the coated anti-LH monoclonal antibodies using blocking solution of phosphate buffer, BSA and Trans-001;

16

(a3) further stabilizing the fastened anti-LH monoclonal antibodies using stabilising solution of PBS, Trans-002 and Tran-003 (bovine immunoglobulin);
(b) preparing a conjugated enzyme, which comprises affixing anti-LH
monoclonal antibodies with enzyme (HRPO) by covalently bonding;
and
(c) preparing immunochemical reagent, which includes preparing the
solutions of LH standards, (a - f) glow reagents A and B and washing
solution.
10. A process for measuring the concentration of LH in human serum and/or plasma samples using the kit according to claims 1-9 comprises the steps of:
(a) loading dispensed amount of LH standard solutions and LH corttaining human serum and/or plasma samples form the patients suffering form hypothalamic, pituitary or gonadal dysfunction into the microwells of microtitre plate coated with anti-LH monoclonal antibodies for binding to LH molecules;
(b) subsequently loading dispensed amount of conjugated enzyme affixed complex; 3
(c) washing the microwells using washing solution so as to remove unbound fraction of conjugated enzyme;
(d) adding dispensed amount of working glow reagent to said microwells,


(e) measuring the concentration of LH in the samples by reading RLU on standard plot of RLU Vs LH.
11. An assay kit for measuring the concentration of LH in human serum and/or
plasma samples such as hereinbefore described with reference to examples
t and drawing.
12. A process for preparing an assay kit for measuring the concentration of LH in
human serum and/or plasma samples such as hereinbefore described with
reference to examples and drawing.
Dated this 24th day of May, 2007.
18