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 DIAGNOSTIC KIT FOR MEASURING AN AMOUNT OF PRL IN 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 0 72
MAHARASHTRA

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

An diagnostic kit for measuring an amount of PRL in human serum and/or plasma
Field Of The Invention
The present invention relates to quantitative measurement of substances, preferably hormones, in human serum and/or plasma based on specific binding immunoassay techniques. Particularly, it relates to detection of antigens or hapten like small chemical compound, more particularly prolactin (PRL), based on solid phase immunoassay techniques employing labeled reagents, such as enzyme-labeled reagents for detection of said antigens or hapten. The present invention provides a diagnostic kit for measuring an amount of PRL in human serum and/or plasma based on specific binding assay techniques.
Background Of The Invention
The human prolactin is secreted from the anterior pituitary gland as a single polypeptide hormone with molecular weight of approximately 23000 daltons. During pregnancy, prolactin levels increase progressively to between 10 and 20 times of normal values and declining to non-pregnant levels by 3-4 weeks post partum. The determination of PRL concentration is helpful in diagnosis of hypothalamic-pituitary disorders. It is therefore beneficial to measure an amount of PRL in human serum and/or plasma samples using a simple and reliable diagnostic kit and a process for measuring its concentration. The most suitable technique for separating and estimating the amount of PRL molecules in human serum and/ or plasma is solid-phase immunoassay method, which use one antibody covalently bound or physically adsorbed to an insoluble matrix and second antibody covalent linked to an enzyme. The antigen-antibody complex so formed is then held by solid phase immunoassay method and bound fraction can easily be separated and estimated using glowing reagent.
A living system promptly responds to the presence of foreign antigen like protein, virus, bacteria, etc. by generating a specific antibody against that particular antigen. Then, there is a reaction between antibody and antigen to form a complex. An

antibody once generated is also capable of binding a hapten, which is relatively a small and simple molecule that can 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 rapidly emerging analytical methods known as specific binding immunoassay methods. In one such assay method, the substance or group of substances to be determined, which 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 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, trained manpower and special care to handle them.
The above said system consists of antigen or hapten labeled with an 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 estimating unknown level of unlabeled antigen by physically separating the antigen-antibody complex from the remaining free antigen. The enzyme activity of the


unknown 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
There are several known methods in the prior art 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 method 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 disadvantages that limits its use. A limited amount of material may be applied to the absorbent and the separation process is laborious and time-consuming.
The precipitation of antigen-antibody complex that involves use of salts, organic material or solvents under the conditions, which do not affect free antigens is another known method. 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. The chemical precipitation technique, however, causes co-precipitation of other proteins molecules too, which causes incomplete separation of two fractions.
Further, double antibody method 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 gamma-globulin raised in goats. But the disadvantage of this method is that use of second antibody requires an additional incubation.

The 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.
There is still another method by which free unbound antigens are adsorbed onto adsorbent and then precipitated by the centrifugation. Powdered talc like magnesium silicate, kaolin like aluminum silicate, QUSO like silica micro-granules, 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 molecules 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 methods are also known for separation of free and bound antigen, which use antibodies covalently bound or physically adsorbed onto 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.
The inventors of the present invention therefore proposed a solid phase diagnostic kit for measuring the amount of PRL in human serum and/or plasma, which comprises one monoclonal antibody absorbed on a solid support as a coating material and second monoclonal antibody conjugated with an enzyme (horse radish peroxidase, HRPO) as a conjugated material. An advantage of using the solid support like microwells containing microtitre plate is that no centrifugation or filtration step is required for separation of solid and liquid phases.

Object Of The Invention
Accordingly, an object of the present invention is to offer a diagnostic kit for measuring an amount of PRL in human serum and/or plasma,
Further object of the present invention is to provide a diagnostic kit for measuring an amount of PRL in human serum and/or plasma, which comprises one monoclonal antibody covalently bound with a solid support and second monoclonal antibody linked to an enzyme.
Still further object of the invention is to design a solid phase diagnostic kit for measuring an amount of PRL for the diagnosis of hypothalamic-pituitary disorders.
Another object of the present invention is to develop a simple, cost effective and reliable diagnostic kit for measuring an amount of PRL in human serum and/or plasma samples obtained from women those are suffering form the hypothalamic-pituitary disorders.
Yet another object of the invention is to provide a diagnostic kit that uses anti-PRL monoclonal antibodies as coating material for solid support and conjugate material for conjugated enzyme.
Still another object of the present invention is to offer a process for preparation of a diagnostic kit for measuring an amount of PRL in the human serum and/or plasma.
Yet another object of the invention is to provide a process for measuring an amount PRL in human serum and/or plasma for the diagnosis of hypothalamic-piruitary disorders in women.
Statement Of The Invention
According to an object of the present invention, a solid phase diagnostic kit for measuring an amount of PRL in human serum and/or plasma is provided, which comprises a solid support onto which anti-PRL monoclonal antibodies are coated as



coating material; and an enzyme conjugate in which second group of anti-PRL monoclonal antibodies are linked as conjugate material. The solid phase diagnostic kit further comprises immunochemical reagents required for measuring an amount of PRL in the human serum and/or plasma samples.
According to another object of the present invention, a process for the preparation of diagnostic kit for measuring the amount of PRL in human serum and/or plasma is provided, which comprises preparing a solid support containing anti-PRL monoclonal antibodies coated; further preparing an enzyme conjugate containing anti-PRL monoclonal antibodies linked; and preparing different immunochemical reagents required for measuring the amount of PRL in the human serum and/or plasma.
According to yet another object of the present invention, a process for measuring the amount of PRL in human serum and/or plasma samples for the diagnosis of hypothalamic-pituitary disorders is provided, which comprises contacting the human serum and/or plasma sample from the patient with a solid support containing anti-PRL monoclonal antibodies; adding an enzyme conjugate containing a second group of anti-PRL monoclonal antibodies into said reaction, thereby producing stable antigen-antibody complex for separating PRL molecules; and finally measuring an amount of PRL using glowing reagent in the samples.
Summary Of The Invention
The solid phase diagnostic kit for measuring the amount of PRL in the human serum and/or plasma samples comprises (i) a microtitre plate as a solid support having plurality of microwells onto which first anti-PRL monoclonal antibodies are coated using for separating PRL molecules in the human serum and/or plasma; (ii) an enzyme conjugate in which second anti-PRL monoclonal antibodies are couped with enzyme using covalent bonding for separating PRL molecules in said human serum and/ or plasma; and (iii) an immunochemical reagents required for measuring the amount of PRL in said samples.

The process for the preparation a solid phase diagnostic kit for measuring the amount of PRL in the human serum and/or plasma comprises the steps of: (i) preparing a microtitre plate having plurality of microwells and containing anti-PRL monoclonal antibodies, which comprises: (a) coating the microwells with anti-PRL monoclonal antibodies in bicarbonate buffer; (b) blocking said coated anti-PRL monoclonal antibodies in blocking solution containing mixture of phosphate buffer, BSA and Trans-001; (c) stabilizing said blocked anti-PRL monoclonal antibodies in stabilising solution containing mixture of PBS, Trans-002 and Tran-003 (bovine immunoglobulin); (d) preparing an enzyme conjugate that comprises linking anti-PRL monoclonal antibodies to an enzyme, such as HRPO using covalent bonding; and (e) preparing immunochemical reagents that comprises preparing solutions of said immunochemical reagents.
The process for measuring the amount of PRL in human serum and/or plasma for the diagnosis of hypothalamic-pituitary disorders comprises the steps of: (i) charging expended volume of PRL standards and PRL containing human serum and/or plasma samples into the microwells of plate containing anti-PRL monoclonal antibodies coated for separating PRL molecules present in the standards and PRL containing test samples; (ii) subsequently charging expended volume of enzyme conjugate containing anti-PRL monoclonal antibodies linked, thereby producing antigen-antibody complex; (hi) discarding unbound fraction of enzyme conjugate by washing using washing solution; (iv) charging expended volume of working glow reagent to the microwells, thereby generating RLU; and (v) finally measuring the amount of PRL in the samples by reading RLU on standard plot of RLU Vs PRL concentration.
Detailed Description Of The Invention
The subject matter of the present invention can be understood preferably with reference to following detailed and specific embodiments of the invention. Although, the features of the invention have been disclosed with reference to particular and specific details of certain embodiments that should not 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 particular embodiment of the present invention, the solid phase diagnostic kit for measuring the amount of PRL in the human serum and/or plasma samples obtained form the patients suffering form the hypothalamic-pituitary disorders comprises the microtitre plate as a solid support having plurality of micro wells, wherein anti-PRL monoclonal antibodies are coated onto the surface of microwells for separating PRL molecules in the human serum and/ or plasma samples.
In another particular embodiment, the solid phase diagnostic kit of the present invention comprises the enzyme conjugate, wherein anti-PRL monoclonal antibodies are linked with an enzyme by covalent bonding for separating PRL molecules in said human serum and/ or plasma samples.
In more particular embodiment of present invention, the enzyme used for the preparation of conjugated enzyme material is Horse Reddish Peroxidase (HRPO).
In still another particular embodiment of the invention, the solid phase diagnostic kit comprises the immunochemical reagents necessary for the diagnostic assay, which includes PRL standards (a - f), glow reagents (A and B) and washing solution.
In yet another particular embodiment, the microwell of the plate are coated with anti-PRL monoclonal antibodies by absorption without adversely affecting the structure and activity of said monoclonal antibodies for separating PRL molecules in the human serum and/or plasma samples.
In different embodiment of the present invention, the HRPO molecules of the conjugate are coupled with second group of anti-PRL monoclonal antibodies by covalent bonding without adversely affecting the structure and activity of said antibodies and enzyme for separating PRL molecules in test samples.

In more specific embodiment of the invention, the microtitre plate prepared as a solid support of said diagnostic kit comprises minimum 48 and maximum 96 microwells that are coated with anti-PRL monoclonal antibodies for separating PRL molecules in the human serum and/or plasma samples.
In one specific embodiment, the PRL standards provided in the kit comprise six standard solutions (a - f) of PRL in the concentrations of 0, 1, 5, 25, 100 and 200 ng per millilitre with thimerosal and gentamycin.
In another specific embodiment of the invention, the diagnostic kit provided comprises two glow reagents (A and B) each comprising HRP substrate A and B.
In preferred embodiment for immediate use during an assay method, the working glow reagent comprises mixture of glow reagent A and B in equal volumes.
In yet another specific embodiment of the invention, the washing solution provided in the kit comprises a admixture of TRIS buffer, NaCl, Tween-20 in deionized water.
In more preferred embodiment, the diagnostic kit of the present invention for measuring the amount of PRL in human serum and /or plasma samples is stored at temperature between 2°C and 8 °C.
In one specific embodiment of the present invention, the process for the preparation of the solid phase diagnostic kit for measuring the amount of PRL in human serum and/or plasma comprises preparing the microtitre plate having plurality of microwells, wherein the process comprises coating the microwells with anti-PRL monoclonal antibodies in bicarbonate buffer using covalent bonding; and subsequently blocking said anti-PRL monoclonal antibodies in blocking solution containing admixture of phosphate buffer, BSA and Trans-001 and stabilizing said anti-PRL monoclonal antibodies in stabilizing solution containing admixture of PBS, Trans-002 and Tran-003 (bovine immunoglobulin).

In another specific embodiment of the process, it comprises preparing the enzyme conjugate, wherein anti-PRL monoclonal antibodies are coupled with HRPO of the conjugate material by absorption without affecting structure and activity of said enzyme and monoclonal antibodies.
In yet another specific embodiment of the invention, the process comprises preparing six PRL standard solutions (a - f) by dissolving six PRL standards (a - f) in the concentrations 0,1, 5, 25,100 and 200 ng per millilitre in deionized water.
In one particular embodiment, the process for the preparation of diagnostic kit comprises preparing solutions of glow reagents A and B by admixing HRP substrates A and B in deionized water.
In more preferred embodiment of the said process, for immediate use at the time assay the working glow reagent is prepared by admixing the glow reagent A and B in equal volumes.
In another particular embodiment, the process for the preparation of diagnostic kit comprises preparing the washing solution by admixing TRIS buffer, NaCl and Tween-20 in deionized water.
In different embodiment of the present invention, the process for measuring the amount of PRL in human serum and/or plasma samples for the diagnosis the hypothalamic-pituitary disorders comprises the steps of pouring expended volume of PRL standards and PRL containing human serum and/or plasma samples into the microwells containing coated anti-PRL monoclonal antibodies for separating PRL molecules present in the test samples, thereby forming stable antigen-antibody complex; subsequently pouring expended volume of enzyme conjugate containing linked anti-PRL monoclonal antibodies, thereby generating antigen-antibody complex; discarding unbound fraction of enzyme conjugate by washing using washing solution; pouring expended volume of working glow reagent to the microwells, thereby producing RLU; and finally measuring the amount of PRL in the samples by reading RLU on standard plot of RLU Vs PRL concentration.

In more preferred embodiment of the process, it comprises contacting the PRL standard solutions and PRL containing human serum and/or plasma samples with anti-PRL monoclonal antibodies coated onto the surfaces of microwells and linked to conjugate, thereby forming stable antigen-antibody complex that separates PRL molecule in the standard solution and test sample for measuring the amount of PRL using standard plot of RLU Vs PRL concentration.
In still more preferred embodiment, it comprises separating the antigen-antibody complex formed as a function of binding of PRL molecules and anti-PRL monoclonal antibodies. The detection of separated PRL molecules is completed by adding working glow reagent, which produces RLU due to catalytic reaction between HRPO and HRP substrate of the glow reagent and are proportional to the amount of PRL standards or PRL content of test samples.
Examples
The following examples serve to illustrate the instant 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 PRL in test samples
Pouring expended volume of PRL standards or PRL containing human serum and/or plasma samples to the microwells of plate; subsequently pouring expended volume of enzyme conjugate 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 discarding unbound fraction of enzyme conjugate; pouring working glow reagent, containing HRP substrate to the microwells, containing said antigen-antibody complex, thereby generating RLU in PRL standards and human serum and/or plasma samples containing microwells; and finally estimating the amount of PRL by reading RLU values on standard plot of RLU Vs PRL (ng/ml).

Example: 2 - Test procedure for measuring the amount PRL in the test samples
(i) Bring all reagents and test specimens at room temperature before use; (ii) six standards in each run; (iii) add 50 ml of standards and test specimens to the respective wells; (iv) add 50 ul of conjugate to each well; (v) incubate 60 minutes at room temperature (20-400(1), preferably 37°C; (vi) wash the microplate as per known washing procedure; (vii) add 50 ul of working glow reagent to each well; and (viii) read RLU vales after 1 minutes and before 20 minutes of glow reagent addition.
Example: 3- Interpretation of results
The results of typical standard assay run performed 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 a function of PRL concentration

Sr. No. PRL concentration (ng/ml) RLU
1 0 1234
2 1 3167
3 5 12865
4 25 51639
5 100 196752
6 200 376432
The results presented 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.

WE CLAIM:
1. A solid phase diagnostic kit for measuring an amount of PRL in human serum
and/or plasma samples comprises:
(i) a microtitre plate having plurality of microwells onto which first group of anti-PRL monoclonal antibodies are coated for separating PRL molecules in the human serum and/or plasma;
(ii) an enzyme conjugate in which second group of anti-PRL monoclonal antibodies are coupled with enzyme using covalent bonding for separating PRL molecules in said human serum and/or plasma; and
(iii) an immunochemical reagents required for measuring the amount of PRL in said samples.
2. The diagnostic kit according to claim 1, wherein the first group of anti-PRL monoclonal antibodies are coated onto the said microwells by absorption for separating PRL molecules in the human serum and/or plasma samples of the patients.
3. The diagnostic kit according to claim 1, wherein the second group of anti-PRL monoclonal antibodies are coupled with enzyme, preferably HRPO, by covalent bonding for separating PRL molecules in the human serum and/or plasma samples of the patients.
4. The diagnostic kit according to any one of the claims 1 to 3, wherein the microtitre plate of the kit comprise minimum 48 and maximum 96 microwells that are coated with anti-PRL monoclonal antibodies.
5. The diagnostic kit according to claim 1, wherein the immunochemical reagents of the said kit are PRL standard solutions (a - f), glow reagent-A and B and washing solution.

6. The diagnostic kit according to claim 1, wherein the PRL standard solutions (a - f) comprise PRL standards in the concentration of 0, 1, 5, 25, 100 and 200 ng per millilitres.
7. The diagnostic kit according to claim 1, wherein the glow reagents A and B comprise HRP substrates A and B respectively.
8. The diagnostic kit according to claim 8, wherein the working glow reagent comprises the glow reagent A and B in equal volumes for immediate use.
9. The diagnostic kit according to claim 1, wherein the washing solution is composed of an admixture of TRIS buffer, NaCl and Tween-20 in deionized water.
10. A process for the preparation of the solid phase diagnostic kit for measuring the amount of PRL in the human serum and/or plasma samples according to claims 1-10 comprises the steps of:
(i) preparing a microtitre plate having plurality of microwells and containing anti-PRL monoclonal antibodies, which comprises:
(a) coating the microwells with anti-PRL monoclonal antibodies in bicarbonate buffer using absorption;
(b) blocking said coated anti-PRL monoclonal antibodies in blocking solution containing mixture of phosphate buffer, BSA and Trans-001;
(c) stabilizing said blocked anti-PRL monoclonal antibodies in stabilising solution containing mixture of PBS, Trans-002 and Tran-003 (bovine immunoglobulin);

(ii) preparing an enzyme conjugate that comprises linking anti-PRL monoclonal antibodies to an enzyme, such as HRPO using covalent bonding; and
(iii) preparing immunochemical reagents that comprises preparing solutions of said immunochemical reagents.
11. A process for measuring the amount of PRL in human serum and/or plasma samples for the diagnosis of hypothalamic-pituitary disorders using the solid phase diagnostic kit according to claims 1-10 comprises the step of:
(i) charging expended volume of PRL standards and PRL containing human serum and/or plasma samples into the microwells of plate containing anti-PRL monoclonal antibodies coated for separating PRL molecules present in the standards and PRL containing test samples;
(ii) subsequently charging expended volume of enzyme conjugate containing anti-PRL monoclonal antibodies linked, thereby producing antigen-antibody complex;
(iii) discarding unbound fraction of enzyme conjugate by washing using washing solution; removing unbound fraction of enzyme conjugate by washing using washing solution;
(iv) charging expended volume of working glow reagent to the microwells, thereby generating RLU; and
(v) finally measuring the amount of PRL in the samples by reading RLU on standard plot of RLU Vs PRL concentration.

A solid phase diagnostic kit and a process for the preparation the same for measuring the amount of PRL in human serum and /or plasma samples such as herein described with reference to examples and drawing.
31st day of August, 2007.