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 SOLID PHASE IMMUNOASSAY KIT FOR ESTIMATING FSH AMOUNT 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 - 400 072
MAHARASHTRA

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

A solid phase immunoassay kit for estimating FSH amount in human serum and/or plasma
FIELD OF THE INVENTION
The present invention relates to quantitative estimation 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 Follicle Stimulating Hormone, 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 solid phase immunoassay kit for quantitatively estimation of FSH in human serum and/or plasma based on specific binding assay techniques.
BACKGROUND OF THE INVENTION
Follicle stimulating Hormone (FSH) is a glycoprotein hormone, having alpha and beta subunits, which is secreted by basophilic cells of the anterior pituitary gland. FSH levels are elevated after menopause, castration and in premature ovarian failure. Although there are significant exceptions, ovarian failure is indicated when random FSH concentrations exceed 40 mlU/ml. It is therefore beneficial to measure the concentration of FSH in human serum and/or plasma samples using a simple and reliable immunoassay kit and method for estimation of its concentration. The most suitable technique for separating and estimating the concentrations of FSH molecules in human serum and/or plasma is solid-phase immunoassay technique, which use one antibodies covalently bound or physically adsorbed to an insoluble matrix and second antibody covalent linked to an enzyme of conjugate material. The antigen-antibody complex so formed is then held by solid phase immunoassay technique and bound fraction can be easily separated and estimated using glow reagent.
A living system promptly 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 immunoassay technique. 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 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 estimating 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
Several methods are known in the 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 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 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.
Another known method is precipitation of antigen-antibody complex that involves use of salts, organic material or solvents under the conditions, which 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 molecules too, which causes incomplete separation of two fractions.
Also, 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.
Further, ion exchange and other resins are 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 still 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.
Therefore, the inventors of the present invention have proposed a solid phase immunoassay kit for quantitatively estimating FSH amount in human serum and/or plasma, which utilizes one monoclonal antibody covalently bonded with a solid support as coating material and second enzyme (horse redfish peroxidase, HRPO) conjugated monoclonal antibody as conjugate 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
Therefore, an object of the present invention is to provide a solid phase assay kit for quantitatively estimating FSH amount in human serum and/or plasma,
Further object of the present invention is to provide a diagnostic kit for quantitatively estimating FSH amount in human serum and/or plasma, which utilizes one monoclonal antibody covalently bound with a solid support and second monoclonal antibody linked to an enzyme.
Another object of the instant invention is to design an immunoassay kit for estimating an amount of FSH for diagnosis premature ovarian failure in women.
Yet another object of the invention is to develop a simple, cost effective and reliable an diagnostic kit for quantitatively estimating FSH amount in human serum and/or plasma samples from women those are suffering form premature ovarian failure.
Still another object of this invention is to provide a quantitative estimation assay kit that comprises anti-FSH monoclonal antibody as a coating material for solid support and conjugate material for conjugated enzyme.
Further object of the invention is to tender a method for producing a solid phase immunoassay kit for estimating an amount of FSH present in the human serum and/or plasma.
Still further object of instant invention is to offer a method for estimating an amount FSH in human serum and/or plasma for diagnosis a premature ovarian failure in women.
SUMMARY OF THE INVENTION
According to an object of the present invention, a solid phase immunoassay kit for estimating an amount of FSH in human serum and/or plasma comprises solid support in which monoclonal antibodies to FSH (anti-FSH monoclonal antibodies)


are coated as coating material and an enzyme conjugate in which second group of monoclonal antibody to FSH are linked as conjugated material, wherein said monoclonal antibodies are coated and linked by covalently bonding to said solid support and enzyme conjugate. The solid phase immunoassay kit also comprises immunochemical reagents required for estimating amount of FSH in the human serum and/or plasma samples.
According to further object of the invention, a method for producing the assay kit for estimating the amount of FSH in human serum and/ or plasma comprises preparing a solid support having anti-FSH monoclonal antibodies coated and enzyme conjugate having anti-FSH monoclonal antibodies linked; and preparing different immunochemical reagents required for estimating amount of FSH in the human serum and/or plasma.
According to still further object of this invention, a method for estimating the amount of FSH in human serum and/ or plasma samples for diagnosis of premature ovarian failure comprises contacting human serum and/ or plasma samples from the patients with solid support having anti-FSH monoclonal antibodies and enzyme conjugate having second group of anti-FSH monoclonal antibodies, thereby forming stable antigen-antibody complex for separating FSH molecules; and finally detecting FSH molecules using glow reagent for estimating its amount in the samples.
DESCRIPTION OF THE INVENTION
The solid phase immunoassay kit for estimating the amount of FSH in the human serum and/or plasma samples comprises: (a) a microtitre plate as a solid support with number of microwells in which anti-FSH monoclonal antibodies are being coated using covalently bond for confining FSH molecules in the human serum and/or plasma samples; (ii) a conjugate material with enzyme to which anti-FSH monoclonal antibodies are being couped using covalent bond for separating FSH molecules in said human serum and/or plasma samples; and (iii) an


immunochemical reagents, which include FSH standards, glow reagents and washing solution.
The method for producing a solid phase immunoassay kit for estimating amount of FSH in human serum and/or plasma sample comprises the steps of: (a) preparing a microtitre plate as a solid support with number of microwells, containing anti-FSH monoclonal antibodies in which the method comprises: (al) coating the microwells with anti-FSH monoclonal antibodies in bicarbonate buffer using covalent bond; (a2) blocking said coated anti-FSH monoclonal antibodies using blocking solution, containing mixture of phosphate buffer, BSA and Trans-001; (a3) stabilizing said blocked anti-FSH monoclonal antibodies using stabilising solution, containing mixture of PBS, Trans-002 and Tran-003 (bovine immunoglobulin); (b) preparing an enzyme conjugate with enzyme in which the method comprises joining anti-FSH monoclonal antibodies to enzyme, such as HRPO using covalent bond; and (c) preparing immunochemical reagents, in which the method comprises preparing solutions FSH standards, (a - f) glow reagents A and B and washing solution.
The method of estimation of FSH amount in human serum and/or plasma samples for diagnosis of premature ovarian failure comprises the steps of: (a) adding given volume of FSH standard solutions and FSH containing human serum and/or plasma samples into the microwells of plate having anti-FSH monoclonal antibodies coated for confining FSH molecules present in the standards and FSH containing test samples; (b) adding thereafter given volume of enzyme conjugate having anti-FSH monoclonal antibodies affixed, thereby generating antigen-antibody complex; (c) removing unbound fraction of enzyme conjugate by washing using washing solution; (d) adding given volume of working glow reagent to the microwells, thereby producing RLU; and (e) finally estimating the amount of FSH in the samples by reading RLU on standard plot of RLU Vs FSH.

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DETAILED DESCRIPTION OF THE INVENTION
The present invention can be understood preferably with reference to following detailed and specific embodiments of the invention. Although, the invention has been disclosed with reference to particular and specific details of certain embodiments, it is not intended that such details should be regarded as limitations to the scope of the 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 detailed aspect of the present invention, the solid phase immunoassay kit for estimating the amount of FSH in the human serum and/or plasma samples obtained form the patients supposed to be suffering form premature ovarian failure comprises the microtitre plate as a solid support with multiplicity of microwells in which anti-FSH monoclonal antibodies are being coated onto the surface of microwells by covalently bonding technique for confining FSH molecules in the human serum and/or plasma samples.
In another detailed aspect, the solid phase immunoassay kit of present invention further comprises the enzyme conjugate in which anti-FSH monoclonal antibodies are being labelled with an enzyme by covalent bonding for detecting FSH molecules in said human serum and/or plasma samples.
In more detailed aspect of present invention, the enzyme label used for preparing the conjugated enzyme is Horse Reddish Peroxidase (HRPO).
In still another detailed aspect, the solid phase immunoassay kit of the present invention also comprises the immunochemical reagents necessary for the immunoassay, which include FSH standards (a - f), glow reagents (A and B) and washing solution.


In yet another detailed aspect, the microwell of the plate are coupled with anti-FSH monoclonal antibodies by covalent bonding without adversely affecting the structure and activity of said monoclonal antibodies as per as confining FSH molecules in the human serum and/or plasma samples is concerned.
In alternate detailed aspect of the kit of the present invention, the HRPO molecules of the conjugate are adjoined with another group of anti-FSH monoclonal antibodies by covalent linkage without adversely affecting the structure and activity of said antibodies and enzyme as per as confining FSH molecules in test samples and catalytic function of HRPO are concerned.
In separate aspect of the invention, the microtitre plate provided as a solid support in said immunoassay kit comprises at least 48 microwells affixed with anti-FSH monoclonal antibodies.
In another separate embodiment, the microtitre plate provided as a solid support in said kit comprises at least 96 microwells affixed with anti-FSH monoclonal antibodies.
In another further embodiment, the FSH standards included in the kit comprise six solutions (a - f) of FSH standards of the concentrations 0,1, 5, 30, 60 and 120 mlU per millilitre with thimerosal and gentamycin.
In still another embodiment of the invention, the immunoassay kit comprises two glow reagents (A and B) each comprising HRP substrate A and B.
In more preferred embodiment, for immediate use during an assay the working glow reagent comprises mixture of glow reagent A and B in equal volumes.
In yet another aspect, the washing solution included in the kit comprises a mixture of TRIS buffer, NaCl, Tween-20 in deionized water.

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In an important embodiment, the immunoassay kit of the present invention for estimating an amount of FSH in human serum and/ or plasma samples is stored at temperature between 2°C and 8 °C.
In another detailed aspect of the invention, the method for producing the solid phase immunoassay kit for estimating the amount of FSH in human serum and/or plasma comprises producing the microtitre plate as a solid support with number of microwells that contain anti-FSH monoclonal antibodies, wherein the method comprises coating the microwells with anti-FSH monoclonal antibodies dissolved in bicarbonate buffer using covalent bond; further blocking said coated anti-FSH monoclonal antibodies using blocking solution, containing a mixture of phosphate buffer, BSA and Trans-001; and stabilizing thereafter said blocked anti-FSH monoclonal antibodies using stabilising solution, containing a mixture of PBS, Trans-002 and Tran-003 (bovine immunoglobulin);
In further detailed embodiment, the method for producing immunoassay kit comprises producing the enzyme conjugate, containing anti-FSH monoclonal antibodies, wherein anti-FSH monoclonal antibodies are couples with HRPO of the conjugate material by covalent bond without affecting structures and activities of said enzyme and monoclonal antibodies.
In still another detailed aspect, the method comprises preparing standard solutions of FSH by dissolving FSH standards (a - f) in concentrations of 0,1, 5, 30, 60 and 120 mlU per millilitre of deionized water.
In different detailed embodiment, the method for producing the immunoassay kit comprises preparing solutions of glow reagents A and B by mixing HRP substrates A and B in deionized water. For immediate use at the time assay, the working glow reagent is prepared by mixing the glow reagent A and B in equal volumes.
In separate detailed aspect, the method for producing the kit comprises preparing the washing solution by mixing TRIS buffer, NaCl, Tween-20 and deionized water.


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.In yet another detailed embodiment, the method of estimation of FSH amount in human serum and/or plasma samples for diagnosis of premature ovarian failure comprises the steps of adding given volume of FSH standard solutions and FSH containing human serum and/or plasma samples into the microwells of plate having anti-FSH monoclonal antibodies coated for confining FSH molecules present in the standards and FSH containing test samples, thereby forming stable antigen-antibody; adding thereafter given volume of enzyme conjugate having anti-FSH monoclonal antibodies affixed, thereby generating antigen-antibody complex; removing unbound fraction of enzyme conjugate by washing using washing solution; adding given volume of working glow reagent to the microwells, thereby producing RLU; and finally estimating the amount of FSH in the samples by reading RLU on standard plot of RLU Vs FSH.
In more detailed embodiment, the method comprises contacting the FSH standard solutions and FSH containing human serum and/or plasma samples with anti-FSH monoclonal antibodies affixed and onto the surfaces of microwells of microtitre plate and joined to HRPO of conjugate material, thereby forming stable antigen-antibody complex that confine and separate FSH molecules in the standard solutions and test samples for estimating amount of FSH using standard plot of RLU Vs FSH.
In still more detailed aspect of the said method, it comprises separating the antigen-antibody complex formed as a result of interaction between said antigens, such as FSH and said monoclonal antibodies, such as anti-FSH monoclonal antibodies. The separation is as result of addition of working glow reagent to said microwells, thereby generating RLU as a function of catalytic reaction of HRPO on its substrate present in the glow reagent. The amount of RLU is proportional to the amount of FSH standards or FSH 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
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Example: 1 - Quantitative estimation of amount of FSH in test samples
Adding given volume of FSH standards or FSH containing human serum and/or plasma samples to the microwells of plate; adding thereafter measured 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 removing unbound fraction of enzyme conjugate; adding working glow reagent, containing HRP substrate to the microwells, containing said antigen-antibody complex, thereby generating RLU in FSH standards and human serum and/or plasma samples containing microwells; and finally estimating the amount of FSH by reading RLU values on standard plot of RLU Vs FSH (mlU/ml).
Example: 2 - Test procedure for estimating FSH amount in the test samples
(i) Bring all reagents and test specimens at room temperature before use; (ii)
add 50 (il of standards and test specimens to the respective wells; (iii) add 50 JLXI 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 |j.l of working glow reagent to each well; and (vii) 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 function of FSH concentration

Sr. No. FSH concentration (mlU/ml) RLU
1 0 1234
2 1 5127
13

3 5 17964
4 30 102549
5 60 199765
6 120 396541
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.
|g(WUMeA72',HJ 14

WE CLAIMS :
1. A solid phase immunoassay kit for estimating an amount of FSH in human
serum and/or plasma samples comprises:
(a) a microtitre plate as a solid support with number of microwells therein in which anti-FSH monoclonal antibodies are being coated using covalently coupling for confining FSH molecules in the human serum and/ or plasma samples;
(b) a conjugate material with enzyme therein to which anti-FSH monoclonal antibodies are being joined using covalent bonding for separating FSH molecules in said human serum and/or plasma samples; and
(c) an immunochemical reagents, which include FSH standards, glow
reagents and washing solution.

2. The solid phase immunoassay kit as claimed in claim 1, wherein one group of anti-FSH monoclonal antibodies are covalently coupled with the microwells of the plate for specifically confining circulating FSH molecules in the human serum and/or plasma samples derived from the patients.
3. The solid phase immunoassay kit as claimed in claim 1, wherein second group of anti-FSH monoclonal antibodies are covalently affixed with the enzyme, preferably HRPO, for separating FSH molecules in the human serum and/or plasma samples derived from the patients.
4. The solid phase immunoassay kit as claimed in claim 1, wherein the microtitre plate at least contains 48 microwells coupled with anti-FSH monoclonal antibodies.
5. The solid phase immunoassay kit as claimed in claims 1, wherein the microtitre plate at least contains 96 microwells coupled with anti-FSH monoclonal antibodies.
15

6. The solid phase immunoassay kit as claimed in claim 1, wherein the immunochemical reagents comprise FSH standard solutions (a - f), glow reagent-A and B and washing solution.
7. The solid phase immunoassay kit as claimed in claim 1, wherein the FSH standards (a - f) comprise FSH standards in the concentration of 0, 1, 5, 30, 60 and 120 mlU per millilitres.
8. The solid phase immunoassay kit as claimed in claim 1, wherein the solutions A and B of glow reagents comprise HRP substrates A and B and the working solution of glow reagent comprises the glow reagent A and B in equal volumes.
9. The solid phase immunoassay kit as claimed in claim 1, wherein the washing solution comprises a mixture of TRIS buffer, NaCl, Tween-20 and deionized water.
10. A method for producing a solid phase immunoassay kit for estimating amount of FSH in the human serum and/or plasma samples comprises:
(i) preparing a microtitre plate as a solid support with number of microwells, containing anti-FSH monoclonal antibodies in which the method comprises:
(a) coating the microwells with anti-FSH monoclonal antibodies in bicarbonate buffer using covalent coupling;
(b) blocking said coated anti-FSH monoclonal antibodies using blocking solution, comprising a mixture of phosphate buffer, BSA and Trans-001;
(c) stabilizing said blocked anti-FSH monoclonal antibodies using stabilising solution, comprising a mixture of PBS, Trans-002 and Tran-003 (bovine immunoglobulin);

(ii) preparing an enzyme conjugate with enzyme in which the method comprises joining anti-FSH monoclonal antibodies to enzyme, such as HRPO using covalent bonding; and
(iii) preparing immunochemical reagents, in which the method comprises preparing solutions of FSH standards, (a - f) glow reagents A and B and washing solution.
\\, tf- A method of estimating an amount of FSH in human serum and/or plasma samples for diagnosis of premature ovarian failure using solid phase immunoassay kit comprises:
(a) adding given volume of FSH standard and FSH containing human serum and/or plasma samples into the microwells having anti-FSH monoclonal antibodies coated for confining FSH molecules present in the standards and FSH containing test samples;
(b) immediately adding given volume of enzyme conjugate having anti-FSH monoclonal antibodies affixed, thereby generating antigen-antibody complex;
(c) removing unbound fraction of enzyme conjugate by washing using washing solution;
(d) adding given volume of working glow reagent to the microwells, thereby producing RLU; and
(e) finally estimating the amount of FSH in the samples by reading RLU on standard plot of RLU Vs FSH.

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\Z- $t- A solid phase immunoassay kit and a method of producing the same for estimating an amount of FSH in human serum and/or plasma samples such as herein described with reference to examples and drawing.
Dated this 2nd day of June, 2007.

MiDICALS LIMITED
FOR TRANSASI

tty
RAVIN DR^B- YF"' r VICE PRESIDENT-LEGAL AND COMPANY SECRETARY
SUR&H VAZ^RANI
CHAIRMAN AND^MANAGING DIRECTOR
TRNASASIA BKD-MEDlfcALS LTD.
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