FIELD OF THE INVENTION:

The present invention generally relates to amethod of developing a graphene (oxide) based chemiluminescence and/or Enzyme Linked Immuno Sorbent Assay (ELISA) diagnostic technology and kits for determining the precise concentration of Anti-Mullerian hormone (AMH) in human serum.

BACKGROUND OF THE INVENTION:

Anti-Mullerian hormone (AMH) is a protein in humans, encoded by AMH gene. AMH is a substance produced by granulosa cells in ovarian follicles and it is one potential test of ovarian reserve. It can be measured in the blood at any time in the menstrual cycle and used for assessing the age-related decline of the ovarian pool in healthy women. It has a potential ability to predict future reproductive lifespan.
AMH assay has clinical importance in predicting the success of in vitro fertilization (IVF). There are other tests that are currently used for evaluation of the remaining egg supply. But, none of these tests are sufficient to determine the individual's capability to become pregnant.
In the present scenario, a number of immunodiagnostic kits were developed for the detection and quantitative determination of different biomolecules and biochemical substances. Immunodiagnostic technology uses antigen-antibody reaction as their primary means of detection. Well known immunodiagnostic tests include ELISA (Enzyme Linked Immuno Sorbent Assay), chemiluminescence, immunoblotting,detection tests andpregnancy testsetc.
ELISA procedure involves immobilizing antigen to a solid surface like Micro Well Plate (MWP) and adding antigen specific antibodies on the MWP so that it can bind to the antigen. This antibody is linked to an enzyme. The substrate suitable to the enzyme is added and this produces subsequent enzyme substrate reaction and results in indication of presence of the desired biomolecuie which is in general a color change.

Immunoassaysarebiochemical tests which are used to measure the concentration of macromolecule in a solution with the help of immunoglobulins, alternatively known as antibodies.Chemiluminescence is the reaction of enzymes with specific reagents resulting in the formation of light which can be used as an indication or detection signal for the presence of biomolecules. Hence, ELISA can also be characterised by chemiluminescence yielding light indication. Substrates yielding enhanced chemiluminescent or fluorescent signal can also be used in ELISA test as substrates in order to provide improved results.
Thus, the above said tests have made a rapid change in medical field leading to the detection of diseases. The sensitivity of these tests is considerably good.
PCT PublicationWO2014056896 to Vashistet al. entitled "One-step biomolecular immobilisation procedure and products thereof relates toa method for covalent immobilisation of a biomolecule to a solid .material and products arising there from.It provides a solid material with an activated surface, preferably by treatment with an oxidising agent, and subsequently contacting said material with a mixture of biomolecule to be immobilised and one or more silane components, preferably present in a pre-mixed binding solution.
PCT PublicationWO2013165318 to VashistSandeep Kumar et al. entitled "A mediator-less electrochemical glucose sensing procedure employing the leach-proof covalent binding of an enzyme(s) to electrodes and products thereof relates to devices and procedures for the development of glucose oxidase-bound electrodes by covalent binding of glucose oxidase on amine- functionalizcd electrodes. A secondary substrate such as graphene or other secondary substrate on the electrode provides electrochemical signal enhancement due to its large surface area, which increases the sensitivity of glucose detection.
Chinese document CN103728459 to Xia Xi andGuan Yachuentitled "Preparation method and application of kit for double-sandwich immunofluorescence quantitative detection of human anti-Mullerian hormone (AMH) on basis of quantum dots" relates to a double sandwich immunofluorescence quantitative detection of quantum dot-based anti-human Mullerian hormone AMH kit in which the detection antibody used is a CdTe quantum dot label for

monoclonal antibodies and the kit has the advantages of high fluorescence intensity and long stabilization time.
Chinese document CN103913573 to QianXiaohong et al. entitled "Double signal amplification ELISA detection method based on nanometer gold and graphene oxide" discloses the use of nanometer gold and graphene oxide in ELISA detection signal amplification.
PCT Publication WO2014175635 to KimJungbae and Kim Jae-Hyun entitled "Enzyme-graphene oxide complex used for electrochemical applications and method for preparing same" relates to an enzyme-graphene oxide complex used for electrochemical applications. The enzyme-graphene oxide complex can significantly improve stability and output, and thus is applicable to a biofuel cell requiring enzyme activity stable for a long time as well as a biosensor, an enzymatic column, enzyme-linked immunosorbent assay (ELISA), a bio-purification device, an anti-fouling road, and the like.
Though the above prior art overcome the disadvantages posed by the traditional ELISA method, there is a need to develop a kit to determine the precise concentration of Anti-Mullerian hormone (AMH) present in human serum with the help of graphene oxide- based chemiluminescencc (and/or) Enzyme Linked Immuno Sorbent Assay (ELISA). This is because grapheme oxide retains enormous amount for capturing antibodies and hence can enhance the sensitivity, accuracy and stability of the diagnostic technology. The conventional systems do not use such diagnostic technology to enhance the sensitivity
of the diagnostic kit. Hence, a significant need remains for enhanced diagnostic technology to determine the concentration of AMH present in human serum.

SUMMARY OF THE INVENTION:
The present invention provides a kit and method for determining precise concentration of Anti-MullerianHormone (AMH)present in human serum with the help of a Graphene (oxide) based chemilumincscence and/or ELISA diagnostic technology.

The preferred method is initiated with three activation steps such as alkaline activation of Multi Well Plate (MWP), conjugation of graphene (oxide) with (3-Aminopropyl)triethoxysilane(APTES), conjugation ofprimary capturing antibody with 1-Ethyl-3-(3-dimethyl aminopropyl)-carbodiimide (EDC).
Initially, the procedure is started by treating the surface of MWP with KOH for5-7 minutes at 37°C, and subsequently washing the wells for five times with ultrapure water. The organic moiety of polystyrene of MWP is oxidized and hydroxyl groups (-OH) are generated on the surface of MWP.Further, the graphene (oxide) is mixed with APTES and is transferred to a single well of MWP. The mixture is then taken out, and the wells are washed with ultrapure water which is followed by dispensing the mixture into the alkaline treated MWP and incubating them. As a result, the wells are occupied with APTES conjugated with graphene (oxide) and they can readily accept electrons from amino group of the antibodies later.
EDC is then mixed with primary capturing antibodies IgG, against Anti-Mtillerian Hormone and incubated for 15 minutes. This EDC conjugated capturing antibodies are added to each wells of MWP and incubated for 1 hour followed by washing the wells with wash buffer. An amide bond between the amino groups of APTES and EDC-conjugated primary capturing antibodies bind them together and alkoxy functional groups ofAPTES is bonded with hydroxyl groups present on the surface of MWP by hydrolysis.
After conjugation with EDC-activated primary capturing antibodies (anti-AMH), a surface bound APTES conjugate may not form a siloxane with its neighbouring surface bound conjugate owing to its stereo conformation. The amino groups of APTES are covalently bound to the EDC-activated primary capturing IgG (polyclonal IgG, anti-AMH) which are no longer available to form hydrogen bonds with hydroxyl groups on the surface of alkaline treated MWP. The MWP is subsequently blocked by incubating with membrane filtered fresh 1% Bovine Serum Albumin (BSA) for 30 minutes at 37°C, and then the plate is washed with PBS (100 mM).

Finally, sandwich ELISA is developed by involving sequential dispensing of varying concentration of analyte (AMH), followed by the addition of Horse Radish Peroxidase (HRP) conjugated secondary monoclonal antibodies on each well. Then, the plate is incubated for 1 hourwhich is followed by washing each well three times with PBST (Phosphate Buffer Saline with tween 20) and further washed thrice by PBS.
In one . embodiment of the present invention, for ELISA, TMB (3, 3', 5, 5'-Tetramethylbenzidine)-H202mixtureis added to each well and incubated at 37°C.
In another embodiment of the present invention, for chemiluminescence in ELISA, 100 (il of 2,4,5-Triphenylimidazole-H202 mixtureis added to each well and incubated at 37°Cfor 20 minutes.
The enzyme reaction can be stopped by adding 2N H2SO40 each well and results in the formation of sandwich immune complex after incubation for 15 minutes.Finally, the absorbance and luminescence are measured at 450 nm and 545 nm, respectively.

BRIEF DESCRIPTION OF THE DRAWINGS:
Further objects and advantages of this invention will be more apparent from the ensuing description when read in conjunction with the accompanying drawings and wherein:
Figure 1 illustrates the schematic representation of development of graphene (oxide) based ELISA/Chemiluminescence kit for determining the concentration of Anti-Mullerian Hormone.

DETAILED DESCRIPTION OF THE INVENTION:
Aspects of the present invention described herein, discloses a diagnostic technology to determine the precise concentration of Anti-Mullerian Hormone with the help of Graphene (oxide) based chemiluminescence /ELISA diagnostic kit. The features of the invention will be more readily understood from the following detailed description of the various aspects of the invention taken in conjunction with the accompanying drawing that depict various embodiments of the invention.

Graphene (oxide) has the ability to retain enormous amount of capturing antibodies (polyclonal IgG; anti-AMH) through APTES. Hence, using graphene (oxide) in an immunodiagnostic technology can enhance the sensitivity drastically. Thus, the present invention uses graphene oxide to develop a kit to determine the precise concentration of Anti-Mullerian hormone present in human serum.
According to the present invention, the development of Graphene (oxide) based chemiluminescence/ELISA diagnostic kit(s) includes the following procedure as described in Figure 1. Initially, the procedure is initiated with three activation steps as described below:
(a) alkaline (NaOH, KOH, NH4OH) activation of Multi Well Plate (MWP)
(b) Conjugation of graphene (oxide) with (3-Aminopropyl)triethoxysilane(APTES)
(c) Conjugation of primary capturing antibody ■ with l-Ethyl-3-(3- dimethyl aminopropy!)-carbodimide (EDC).
With reference to Figure 1, the ELISA procedure is started by treating the surface of MWP with 100 \x\ of an alkaline KOH for 5 - 7 minutes at 37°C. The wells are subsequently washed with 300 |.il of ullrapure water nearly for five times. As a result of alkaline treatment of MWP, the organic moiety of polystyrene of the MWP is oxidized and hydroxyl (-OH) groups are generated on the surface of MWP.
In addition, 100 |il of graphene (oxide)(l mg ml_1)is mixed with 1 ml of (0.25 %) APTES and this mixture is transferred to a single well of Micro WellPlate (MWP) or Micro Titre Plate (MTP). This mixture is then taken out and the wells of MWP are washed with ultrapure water for 1 hour at room temperature.
The above said process is followed by dispensing the above mentioned mixture of graphene (oxide) and APTES into the alkaline treated MWP. Subsequently, the plates are incubated at 37°C for 30 minutes. Now, the wells of MWP are occupied with APTES conjugated with graphene (oxide) and are ready to accept electrons from the amino group of the antibodies.

10 \i\ of EDC (4 mg ml'1) is mixed with 990 \i\ of anti-AMH (IgG) capturing antibodies (2 mg/ml), and incubated at 37°C for 15 minutes. And, this mixture of EDC-conjugated capturing antibodies is added (100 \i\) to each well, and incubated at 37°C for nearly 1 hour. Then, the wells are washed with wash buffer (phosphate buffer saline; 100 mM, pH 7.2) for five times (300 ^il/time).
The above procedure is followed by the simultaneous binding of amino groups of APTES with the EDC-conjugated capturing antibodies by the formation of amide bond and the binding of alkoxy functional group of APTES with the hydroxyl groups present on the surface of MWP by hydrolysis. Then, the APTES reacts with the free hydroxyls of an oxidized substrate (here Graphene (oxide) by nucleophilic exchange with loss of ethanol. After conjugation with EDC-activated capturing antibodies (anti-AMH), a surface bound APTES conjugate may not form a siloxane with its neighbouring surface-bound conjugate owing to its stereo conformation.
The amino groups of APTES are covalently bound to the EDC-activated capturing IgG (polyclonal IgG; anti-AiVlH) which are no longer available to form a hydrogen bond with the hydroxyl groups on the surface of alkaline treated MWP. The MWP is subsequently blocked by incubating with membrane filtered fresh 1% bovine serum albumin (BSA) for 30 minutes at 37°C (300 jilAvell). Then, the plate was washed with PBS three times.

Accompanied by this, sandwich ELISA is developed by involving the sequential dispensing of varying concentration of analyte (AMH), followed by the addition of 100 |il of secondary monoclonal antibodies (Anti-Rabbit monoclonal IgG; detection antibody, working dilution -1:10000, Arista Biologicals, US) on each well. Previously, the secondary monoclonal antibodies are conjugated with HRP.
Thispolyclonal IgG (anti-AMH) in Rabbits (New Zealand white strain) are generated by the process explained below:
Initially, 100 ng of AMH is dissolved in 300 \i\ of phosphate buffer saline (pH - 7.0) and mixed thoroughly with 300 |il of TiterMax (Sigma Aldrich, Germany). This colloidal mixture is then injected into a rabbit through subcutaneous injection on five different spots (80-100 (il/spot). Prior to injection, bleed was done to fix the cut-off value of unspecific antibodies. After fifteen days, Blood (~1 ml) is withdrawn intravenously and the titre between blood of unspecific antibodies and the blood after injecting the colloidal mixture are checked. Based on the response of rabbits, two booster doses are given to increase the titre. Finally, on the 70thday; 1:3600 is obtained. The total IgG (300 jig mL-l)is then purified by using affinity chromatography with protein A columns. AMH-specific IgG isalso separated by using immuno-affinity chromatography with the ligand embedded with AMH (550 |.ig mL-1).
After adding the desired secondary antibodies on each well, the plate is incubated at 37°C for I hour. Then, 100 [il ofHRP conjugated secondary antibodiesareadded to each well andincubated for 30 minutes at 37°C which is followed by washing MWP with 300 |il of the above described wash buffer for five times.
In one embodiment of the present invention, for ELISA,100 (il of TMB (3, 3', 5, 5'-Tetramethylbenzidinc)-H202 mixture is added to each wells and incubated at 37°C for 20 minutes.
In another embodiment of the present invention, for chemiluminescence in ELISA, 100 |il of TriphenylimidazoIe-H;>0:: rnixtureis added to each wells, and incubated at 37°C for 20 minutes.
When HRP is added, enzyme reaction takes place which produces color change that indicates the proper binding of biomolecules. Then, the enzyme reaction is stopped by the addition of 50 pi of 2N H2SO4 to each well, which results in the formation of a sandwich immune complex after incubation at 37°C for 15 minutes.
Finally, the absorbance and luminescence are measured at 450 nm and 545 nm, respectively by TECAN, multimode reader infinite M200 PRO to determine the precise concentration of Anti-Mullerian hormone present in human serum.

Thus, the present invention provides an improved diagnostic technology and kit to enhance the sensitivity and accuracy of the process of determination of precise concentration of Anti-Mullerian hormone present in human serum.

WE CLAIM:
1. A graphene (oxide)-based chemiluminescence and/orEnzyme Linked Immuno Sorbent Assay(ELISA) diagnostic kit for determining the precise concentration of anti-Mullerian hormone (AMH)present in human serum comprising: (i) Alkaline activated Multi Well Plate (MWP); (ii) Graphene (oxide)conjugated with (3-Aminopropyl)triethoxysilane (APTES); and (iii) primary capturing antibody conjugated with l-Ethyl-3-(3-dimethyl aminopropyl)-carbodiimide (EDC).

2. The diagnostic kit according to claim 01, wherein said activated MWP contain hydroxyl groups generated on the surface.

3. The diagnostic kit according to claim 01, wherein said alkaline is selected from NaOH, KOHandNH4OH.

4. The diagnostic kit according to claim 01, wherein said primary capturing antibody is anti-AMH Polyclonal IgG .

5. A method for determining the precise concentration of anti-Mullerian hormone (AMH) present in human serum using graphene (oxide)-based Enzyme Linked Immuno Sorbent Assay (ELISA) diagnostic kit comprising: (i) activating Multi Well Plate (MWP) with alkaline treatment to generate hydroxyl groups on the surface, wherein said alkaline is selected from NaOH, KOH and NH4OH; (ii) washing the wells with ultrapure water for five times; (iii) mixing graphene (oxide) with APTES; (iv) dispersing said APTES conjugated graphene (oxide) into the said alkaline treated MWP and incubating at 37°C for 30 min; (v) conjugating EDC with anti-AMH (IgG) primary capturing antibodies and incubating at 37°C for 15 min; (vi) adding said EDC conjugated capturing antibodies to each wells of said MWP and incubating at 37°C for 1 hour; (vii) washing said MWP with wash buffer; (viii) blocking said MWP by incubating with membrane filtered fresh 1% bovine serum albuminat 37°C for 30 min; (ix) developing said ELISA by involving the sequential dispensing of varying concentration of analyte; (x) adding HRP conjugated secondary monoclonal antibodies on each well of said MWP and incubating at 37°C for 1 hour; (xi) washing said MWPwith said wash buffer; (xii) adding TMB (3,3',5,5'- Telramethylbenzidine)-H202 mixture to each well of said MWP and incubating at 37°C for 20 min; (xiii) terminating the enzyme reaction by adding 2N H2SO4 to each well of said MWP to form sandwich immune complex after incubationat 37°C for 15 min;and (xiv) measuring the absorbance at 450 nm.

6. Amethod fordetermining the precise concentration of anti-Mullerian hormone (AMH) present in human serum using graphene (oxide)-based chemiluminescence in Enzyme Linked Tmmuno Sorbent Assay (ELISA) diagnostic kit comprising: (i) activating Multi Well Plate (MWP) with alkaline treatment to generate hydroxyl groups on the surface, wherein said alkaline is selected from NaOH, KOH and NH4OH; (ii) washing the wells of said MWP with ultrapure water for five times; (iii) mixing graphene (oxide) with APTES; (iv) dispersing said APTES conjugated graphene (oxide) into the said alkaline treated MWP and incubatingat 37°C for 30 min; (v) adding EDC with primary capturing antibodiesanti-AMH polyclonal IgGand incubatingat 37°C for 15 min; (vi) adding said EDC conjugated capturing antibodies to each wells of said MWP and incubatingat 37°C for 1 hour; (vii) washing said wells for five times with wash buffer; (viii) blocking said MWP by incubating with membrane filtered fresh 1% bovine serum albuminat 37°C for 30 min; (ix) developing said ELISA by involving the sequential dispensing of varying concentration of AMH; (x) adding HRP conjugated secondary monoclonal antibodies on each well of said MWP and incubatingat 37°C for lhour; (xi) washing said MWP for five times with said wash buffer; (xii) addingTriphenylimidazole-H202 mixture to each wells of said MWP and incubatingat 37°C for 20 min; (xiii) terminating the Enzyme reaction by adding 2N H2SO4 to each well of said MWP to form sandwich immune complex after incubationat 37°C for 15 min;and (xiv) measuring the luminescence at 545 nm.

7. The method according to claim 05, wherein said secondary monoclonal antibody is Anti-Rabbit monoclonal IgG.

8. The method according to claim 05, wherein said APTES conjugated with graphene (oxide) accepts electrons from the amino group of said antibody.

9. The method according to claim 07, wherein said APTES amino groups are covalently bonded with said EDC-conjugated capturing antibodies by an amide bond.

10. The method according to claim 05, wherein said APTES alkoxy functional groups are bonded with said hydroxy! groups on the surface of said MWP by hydrolysis.