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 IMMUNOASSAY KIT FOR QUANTITATIVE DETECTION OF C-REACTIVE PROTEIN 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 - 400 072
MAHARASHTRA

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

An immunoassay kit for quantitative detection of C-reactive protein in serum
and/or plasma
FIELD OF THE INVENTION
The instant invention relates to a quantitative detection of substances, particularly antigenic proteins in human serum and/or plasma based on specific solid phase immunoassay techniques. More preferably, the instant invention relates to detection of amount of antigens or hapten like small chemical substances, most preferably C-reactive protein (CRP), based on the solid phase immunoassay techniques that employs labeled reagents like enzyme-labeled reagents for detecting said antigenic proteins or hapten. The instant invention specifically relates to an immunoassay kit for quantitative detection of CRP in the human serum and/or plasma for diagnosis of inflammatory processes as an acute phase protein. The instant application also relates to a process for manufacturing the immunoassay kit and an in vitro process for the quantitative detection of CRP in the human serum and/plasma.
BACKGROUND OF THE INVENTION
The CRP is an alpha globulin protein with a molecular weight of approximately 110000 to 140000 Daltons and is composed of five identical subunits that are non-covalently assembled as a cyclic pentameric polypeptide. CRP is one of the acute phase proteins and normally CRP levels of CRP in serum and plasma are less than 3 ng/ml. However, the level of CRP rises in a wide variety of diseases. Although the detection of elevated levels of CRP in the serum and/or plasma is not specific indicator for any particular disease, it is a useful indicator of inflammatory processes. The detection of CRP is a more reliable and sensitive indicator of the inflammatory process than Erythrocyte Sedimentation Rate (ESR) that may also be influenced by physiological changes not associated with an inflammatory process. It is, therefore highly desirable to provide a simple and sensitive immunoassay kit to detect the amount of CRP in the human serum and/or plasma and an in vitro process for quantitative detection of said protein. The most reliable and sensitive method for estimating the amount of CRP in the human serum and/or plasma is the solid phase




immunoassay method that employs first antibody either covalently bonded or physically adsorbed onto an insoluble solid support or matrix and second antibody covalently attached to an enzyme conjugate material. The antigen-antibody complex so formed is then held by the solid phase immunoassay technique and bound fraction can easily be separated and detected using colour reagent.
The living system promptly responds to the presence of foreign antigen like protein, virus, bacteria, etc. by producing specific antibodies against that particular antigen. Then, there is a specific interaction between said antibody and antigen to form a stable antigen-antibody complex. An antibody once produced is also capable of binding to a hapten, which is relatively a small and simple molecule that may be determinant group for the given antigen and that hapten is capable of binding with said specific antibody but incapable of inducing an antibody production, unless it is bound to an antigenic carrier. The interaction between said antigen and antibody is very specific and sensitive. Other materials that show similar specific and sensitive binding interactions are enzymes and their substrates; hormones; vitamins; metabolites; pharmacological agents and their receptors or binding substances; and such other substances known in the prior art. Such specific and sensitive binding interactions have given rise to a rapidly emerging analytical technique known as specific binding immunoassay. In one such immunoassay, the substance or group of substances to be determined that may be referred as a ligand, in a liquid sample is placed in competition with a labeled form of the ligand or binding analog thereof for binding to the reagent. Where an enzyme label is used and the binding reagent is an antibody, the method is known as an enzyme-immunoassay method. Several other labeling reagents are also known for replacing the enzymes like radioisotopes, coenzymes, enzyme substrates, enzyme-modulators-inhibitors and allosteric effectors, fluorescent molecules and luminescent molecules. However, these substitutes have several disadvantages of handling and require sophisticated instruments, trained manpower and special care for using.
The above techniques include antigen labeled with a enzyme marker, unlabeled native antigen in test sample and specific antibodies in which there is competition

between unlabeled antigen and labeled antigen for binding to limited amount of antibodies present. Therefore, greater the amount of unlabeled antigen from the test sample less the labeled antigen will be bound to the antibodies. If the amount of labeled antigen and antibody is fixed and the only variable is amount of unlabeled antigen, it becomes possible to establish an assay technique for detecting the unknown amount of unlabeled antigen by physically separating the antigen-antibody complex from the remaining free antigen. The enzyme activity of the unknowns sample 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 techniques are known in the prior art for detecting free unbound antigen or hapten from the antigen-antibody complex. One such known technique is chromatoelectrophoresis that is a combination of a paper chromatography and paper electrophoresis techniques. The paper with a high affinity for free antigen like Whatman's Filter paper is used as carriers. The technique is discriminative and has been used in the assay of chemical substances, such as insulin, growth hormone, glucagons, parathyroid hormone, thyroid stimulating hormone and other peptide hormones. However, this technique has number of disadvantages, such as limited amount of material can be loaded to the absorbent material and the separation technique is laborious and time-consuming, which limits its use.
Another known technique is precipitation of antigen-antibody complex, which 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 required. But, the chemical precipitation also causes co-precipitation of other proteins molecules, which causes incomplete separation of two fractions.



Still another known technique is double antibody technique, which is widely used for the separation of bound and free antigen. By using this technique, a second antibody that was raised against the first antibody is used to precipitate primary antigen-antibody complex. More precisely, if the first antibody is raised in rabbit then the second antibody may be an antiserum to rabbit gammaglobulin raised in goat. But the disadvantage of this technique is that use of second antibody requires an additional incubation time.
Yet another known technique is use of ion exchange and other resins for binding free antigens by electrostatic forces and mainly used for determination of small molecules like thyroid hormones. One such technique of this type used for the separation of the antigen-antibody complex from free antigen employs a column packed with material that preferably adsorbs either free antigen or antigen-antibody complex. The incubated aqueous reaction mixture is charged onto 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 material is calculated from the count.
In one another known method, free unbound antigens adsorbed onto adsorbent and then precipitated by the centrifugation. Powdered talc such as 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 sieves that allows the smaller molecules of free antigen to pass and 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 method known for the separation of free and bound antigen employs antibodies covalently bound or physically adsorbed to an insoluble solid support. The formed antibody-antigen complex is held by the solid phase and the bound fraction can easily be separated from the free fraction by filtration. Therefore, the inventors of the instant invention have proposed an immunoassay kit based on

the solid phase immunoassay technique for the quantitative detection of CRP in the human serum and/or plasma by employing one monoclonal antibody covalently bonded with a solid support and another is conjugated with an enzyme. Importantly, the advantage of using the solid support is that no additional centrifugation or filtration step is required for the separation of solid and liquid phases during immunoassay.
OBJECT OF THE INVENTION
Importantly, the principle object of instant invention is to provide an immunoassay kit for quantitatively detecting amount of CRP in human serum and/or plasma.
Importantly, the further object of the instant invention is to provide an immunoassay kit for the quantitative estimation of CRP in the human serum and/or plasma by employing monoclonal antibodies raised against CRP that are covalently linked to a solid support and a conjugated enzyme.
Importantly, the still further object of the instant invention is to provide an immunoassay kit for detecting amount of CRP in the human serum and/ or plasma samples as an indicator of inflammatory processes.
Importantly, the yet further object of the instant invention is to produce a simple, sensitive and cost-effective immunoassay kit for quantitatively detecting CRP as an acute phase protein in the human serum and/or plasma for diagnosis of inflammatory processes.
Importantly, another object of the instant invention is to provide a process for preparing the immunoassay kit for quantitatively detecting CRP in the human serum and/or plasma samples that is based on a solid phase immunoassay technique for the detection of antigenic proteins.
Importantly, the yet another object of the instant invention is to provide an in vitro method for the quantitative detection of CRP in the human serum and/or plasma for the diagnosis of inflammatory processes.


STATEMENT OF THE INVENTION
In view of main object of instant invention, an immunoassay kit for the quantitative detection of amount CRP in the human serum and/or plasma is provided that comprises an insoluble solid support, which comprises number of microwells coated with monoclonal antibodies raised against CRP, an enzyme conjugate comprising another monoclonal antibodies raised against CRP, which are linked with an enzyme tag and reagents for quantitative detection of CRP in the human serum and/or plasma.
In view of another object of instant invention, a process for preparing the immunoassay kit for the quantitative detection of CRP in the human serum and/or plasma is provided that comprises preparing an insoluble solid support comprising number of microwells, which are coated with monoclonal antibodies raised against CRP, preparing an enzyme conjugate comprising another monoclonal antibodies raised against CRP, which are linked with an enzyme tag, and preparing reagents for quantitative detection of CRP in the human serum and/or plasma.
In view of another object of instant invention, an in vitro process for quantitatively detecting CRP in the human serum and/or plasma for diagnosis of inflammatory processes comprises detecting amount of CRP in the human serum and/or plasma by reacting test and standard samples with monoclonal antibodies raised against CRP that are applied onto and linked to insoluble solid support and conjugated enzyme, thereby forming antigen-antibody complex and detecting the amount of CRP in test and standard samples by estimating amount of said antigen-antibody complex using colour reagent.
DESCRIPTION OF THE INVENTION
Therefore, an immunoassay kit for the quantitative detection of CRP in the human serum and/or plasma comprises an insoluble solid support comprising number of microwells, wherein monoclonal antibodies raised against CRP are applied onto surface of said microwells; an enzyme conjugate, wherein monoclonal antibodies



raised against CRP are linked with an enzyme tags; and immunochemical reagents for the quantitative detection of CRP in the human serum and/or plasma.
Therefore, a process for preparing an immunoassay kit for quantitatively detecting CRP in the human serum and/or plasma comprises preparing an insoluble solid support comprising number of microweils, wherein monoclonal antibodies raised against CRP are applied onto said microweils by covalently or physically adhering, blocking said adhered antibodies by blocking them using blocking solution, and stabilizing said blocked antibodies by stabilizing them using stabilising solution; preparing an enzyme conjugate, wherein monoclonal antibodies raised against CRP are linked with an enzyme tag by covalently linking; and preparing immunochemical reagents for quantitative detection of CRP in the human serum and/or plasma by preparing their solutions.
Therefore, an in vitro process for quantitatively detecting CRP in the human serum and/or plasma for diagnosis of inflammatory processes comprises detecting amount of CRP in the human serum and/or plasma by reacting test and standard samples with monoclonal antibodies raised against CRP that are applied onto and linked with insoluble solid support and conjugated enzyme, thereby forming antigen-antibody complex and detecting the amount of CRP in test and standard samples by estimating amount of said antigen-antibody complex using colour reagent.
DETAILED DESCRIPTION OF THE INVENTION
The present invention can be understood with reference to the following particular and specific embodiments of the invention. Although, the features have been disclosed with reference to particular and specific embodiments, it is not intended that such details should be regarded as limitations to the scope of the invention. Besides, unless otherwise presented, 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 a specific embodiment of the invention, an immunoassay kit for the quantitative detection of CRP in the human serum and/or plasma comprises a microtitre plate that comprises number of microwells, which are coated with monoclonal antibodies raised against CRP using covalently bonding or physical adsorption for detecting CRP in the human serum and/or plasma.
In another specific embodiment of the invention, an immunoassay kit for the quantitative detection of CRP in the human serum and/or plasma samples comprises an enzyme conjugate that comprises enzyme tags, which are linked with monoclonal antibodies raised against CRP using covalently bonding for detecting CRP in the human serum and/or plasma.
In more specific embodiment of the invention, the conjugated enzyme tag liked with monoclonal antibodies raised against CRP is Horse Reddish Peroxidase (HRPO).
In still specific embodiment of the invention, an immunoassay kit for the quantitative detection of CRP in the human serum and/or plasma samples comprises sample diluent, CRP standards (A to F), colour reagent, stopping solution and washing solution required for the quantitative detection of CRP in the human serum and/or plasma.
In yet another specific embodiment of the invention, the microtitre plate of the immunoassay kit for quantitative detection of CRP in the human serum and/or plasma comprises sets of 48 and 96 microwells in one plate, which are coated with covalently bonded or physically adsorbed monoclonal antibodies raised against CRP for testing 48 and 96 test samples, respectively.
In different specific embodiment of the invention, the sample diluent provided for dilution of standard and test samples comprises an admixture of phosphate buffer, BSA and Tween-20 along with thimerosal and gentamycin in required proportions.
In still different embodiment of the invention, the CRP standards provided for
comparing amounts of CRP in test samples comprises solutions namely A, B, C, D, E
and F that comprise CRP standards in the concentrations of 0, 0.5, 1, 2.5, 5, 7.5, and


10 mg per millilitre, respectively, along with thimerosal and gentamycin as preservatives.
In yet different embodiment of the invention, the colour reagent provided for the qualitative detection of CRP in the standard and test samples comprises 3,3',5,5'-tetramethyl benzidine, dimethyl sulfoxide and hydrogen peroxide along with thimerosal and gentamycin.
In separate specific embodiment of the invention, the stopping solution provided for stopping a catalytic reaction of enzyme tags and detecting the CRP in the samples comprises an admixture of concentrated phosphoric and deionized water.
In still separate embodiment of the invention, the washing solution provided for washing microtitre plate and removing unbound fraction of enzyme conjugate comprises an admixture of TRIS buffer, NaCl and Tween-20 in deionized water in required proportions.
In an important embodiment of the invention, an immunoassay kit for the quantitative detection of CRP in the human serum and/or plasma samples is stored at temperature between 2°C and 8 °C.
In another embodiment of the invention, the process for preparing an immunoassay kit for the quantitative detection of CRP in the human serum and/or plasma comprises preparing a microtitre plate with number of microwells coated with monoclonal antibodies raised against CRP in which the said monoclonal antibodies are applied onto said microwells by covalently or physically adhering; subsequently blocking said antibodies using blocking solution comprising phosphate buffer, BSA and Trans-001; lastly stabilizing said antibodies using stabilising solution comprising PBS, Trans-002 and Tran-003 (bovine immunoglobulin); preparing an enzyme conjugate in which monoclonal antibodies raised against CRP are linked with an enzyme tags by covalently bonding; and preparing immunochemical reagents required for the quantitative detection of CRP in the human serum and/or plasma.

In more specific embodiment of the invention, the process for preparing an immunoassay kit for the quantitative detection of CRP in the human serum and/or plasma comprises preparing a conjugated enzyme by covalently linking HRPO as enzyme tag with monoclonal antibodies raised against CRP.
In still specific embodiment of the invention, the process for preparing an immunoassay kit for the quantitative detection of CRP in the human serum and/ or plasma comprises preparing solutions of sample diluent, CRP standards (A to F), colour reagent, stopping solution and washing solution in required concentrations.
In yet specific embodiment of the invention, the process comprises preparing solution of sample diluent by admixing phosphate buffer, BSA, Tween-20 and deionized water along with thimerosal and gentamycin in required proportions.
In yet another embodiment of the invention, the process comprises preparing solutions of CRP standards A, B, C, C, D, E and F by admixing 0, 0.5,1, 2.5, 5, 7.5 and 10 f^g of CRP standard in per millilitre deionized water along with thimerosal and gentamycin.
In still another embodiment of the invention, the process comprises preparing the colour reagent by admixing S/S'^S'-tetramethyl benzidine, dimethyl sulfoxide and hydrogen peroxide along with thimerosal and gentamycin in required proportions.
In yet specific embodiment of the invention, the process comprises preparing the stopping solution by admixing concentrated phosphoric and deionized water in required proportions.
In still specific embodiment of the invention, the process comprises preparing washing solution by admixing TRIS buffer, NaCl, Tween-20 and deionized water in required proportions.
In different embodiment of the invention, an in vitro process for quantitatively detecting CRP in the human serum and/or plasma for diagnosis of inflammatory processes comprises detecting amount of CRP in the human serum and/or plasma

by reacting test and standard samples with monoclonal antibodies raised against CRP that are applied onto the microwells of microtitre plate and linked with enzyme tag, such as HRPO of enzyme conjugate, thereby forming the stable antigen-antibody complex; detecting the amount of the said antigen-antibody complex, thereby detecting the amount of CRP in test and standard samples using colour reagent.
In more specific embodiment of the invention, the process for the quantitative detection of CRP in the human serum and/or plasma for the diagnosis of inflammatory processes comprises detecting amount of CRP in the human serum and/ or plasma by reacting test and standard samples with monoclonal antibodies raised against CRP, which are coated onto the microwells of microtitre plate, thereby forming the stable antigen-antibody complex; subsequently reacting the said antigen-antibody complex with HRPO of enzyme conjugate, thereby detecting and separating the bound CRP in the microwells and finally detecting the amount of CRP by adding colour reagent and reading amount of CRP spectrophotometrically.
In most specific embodiment of the invention, the process for the quantitative detection of CRP in the human serum and/or plasma for the diagnosis of inflammatory processes comprises separating the stable antigen-antibody complex formed as a function of interaction between CRP and monoclonal antibodies raised against CRP, thereby producing a blue colour product due to catalytic activity of HRPO and reading intensity of a yellow colour obtained as a function of addition of stopping solution spectrophotometrically that is directly proportional to the amount of CRP in the samples.
EXAMPLES
The following examples are served just to illustrate the invention by way of the best method of performing the invention and should not to be regarded as limitations to the scope of the invention.



Example: 1 - Quantitative detection of amount of CRP in the samples
Adding dispensed amount of CRP standards and CRP containing samples into the microwells of microtitre plate; immediately adding dispensed amount of HRPO conjugate into the microwells, followed by incubating the plate for 60 minutes at room temperature between 20°C and 40°C, thereby forming stable antigen-antibody complex; after incubation, removing the unbound fraction of enzyme conjugate by washing the microtitre plate with washing solution; after washing, adding dispensed amount of colour reagent to the microwells containing said antigen-antibody complex, thereby developing blue colour into the microwells containing CRP standards and test samples; adding a stopping solution into the said microwells, thereby changing the blue colour to yellow and finally reading intensity of said yellow colour spectrophotometrically.
Example: 2 - Test procedure for quantitative detection of CRP in the samples
Bring all the reagents and test samples at room temperature before use; add 50ml of sample diluent to each well; keep one blank (l00ml sample diluent+50ml enzyme conjugate) and six standards in each run; add 50ml of standard and test samples to respective microwells; add 50ml of enzyme conjugate to each well and incubate for 60 minutes at room temperature ranging from 20 to 40°C, preferably at 37°C; wash the microplate as per known washing procedure; add 50ml of colour reagent to each microwell and cover the plate with black cover and incubate for 15 minutes in dark at 20 to 30°C; add 100ml of stopping solution to each microwell; and read absorbance of yellow reaction mixture at 450nm and deduct the absorbance of blank well from the absorbance of control and test wells.
Example: 3- Interpretation of results
The results of typical standard immunoassay performed using the immunoassay kit of the invention, which is based on the solid phase immunoassay technique are illustrated in Table 1 and Figure 1. The results illustrated in Table 1 and Figure 1 are



for just illustration purpose only and should not be used to calculate the amount of test specimens for testing.
Table 1: Profile of absorbance as a function of amount of CRP in the wells

Sr. No. CRP concentration (ng/ml) Absorbance (450 nm)
1 0 0.076
2 1 0.412
3 2.5 0.973
4 5 1.729
5 7.5 2.476
6 10 3.264
Any further modifications in and/ or improvements in any aspect of the embodiments of the present invention will also fall under the scope of the 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 the invention. Various features of the invention hereinbefore described are set forth in the following claims.

WE CLAIM:
1. An immunoassay kit for the quantitative detection of CRP in the human
serum and/or plasma comprises:
(i) an insoluble solid support comprising number of microwells, wherein
monoclonal antibodies raised against CRP are applied onto surface of
said microwells;
(ii) an enzyme conjugate, wherein monoclonal antibodies raised against
CRP are linked with an enzyme tags; and
(iii) immunochemical reagents for the quantitative detection of CRP in the
human serum and/or plasma.
2. The immunoassay kit as claimed in claim 1, wherein the insoluble solid support comprises a microtitre plate comprising number of microwells and monoclonal antibodies raised against CRP are applied onto surface of said microwells using covalent bonding or physical adsorption.
3. The immunoassay kit as claimed in claim 2, wherein each microtitre plate comprises sets of either 48 or 96 microwells that contain applied thereto covalently bonded or physically adsorbed monoclonal antibodies raised against CRP.
4. The immunoassay kit as claimed in claim 1, wherein the enzyme conjugate comprises enzyme tags and monoclonal antibodies raised against CRP are linked with said enzyme tags.
5. The immunoassay kit as claimed in claim 4, wherein the Horse Reddish Peroxidase (HRPO) is used enzyme tag for covalently liking to monoclonal antibodies raised against CRP.
6. The immunoassay kit as claimed in claim 1, which comprises sample diluent, CRP standards (A to F), colour reagent, stopping solution and washing solution as immunochemical reagents.



7. The immunoassay kit as claimed in claim 6, wherein the sample diluent phosphate buffer, BSA and Tween-20 along with thimerosal and gentamycin in required proportions.
8. The immunoassay kit as claimed in claim 6, wherein the CRP standards provided for comparing amount of CRP comprises solutions namely A, B, C, D, E and F that comprise CRP standards in the concentrations of 0, 0.5,1, 2.5, 5, 7.5, and 10 mg per millilitre, respectively along with thimerosal and gentamycin.
9. The immunoassay kit as claimed in claim 6, wherein the colour reagent provided for qualitative detection of CRP comprises 3,3',5,5'-tetramethyl benzidine, dimethyl sulfoxide and hydrogen peroxide along with thimerosal and gentamycin.
10. The immunoassay kit as claimed in claim 6, wherein the stopping solution provided for stopping a catalytic reaction of enzyme tags and detecting the CRP in the samples comprises an admixture of concentrated phosphoric and deionized water.
11. The immunoassay kit as claimed in claim 6, wherein the washing solution provided for washing microtitre plate and removing unbound fraction of enzyme conjugate comprises an admixture of TRIS buffer, NaCl and Tween-20 in deionized water in required proportions.
12. A process for preparing an immunoassay kit for quantitatively detecting CRP in the human serum and/or plasma comprises:
(i) preparing an insoluble solid support comprising number of microwells, wherein monoclonal antibodies raised against CRP are applied onto said microwells by covalent bonding or physically adhering, blocking said applied antibodies using blocking solution comprising phosphate buffer, BSA and Trans-001 and stabilizing said

blocked antibodies using stabilising solution comprising PBS, Trans-
002 and Tran-003 (bovine immunoglobulin); and
(ii) preparing an enzyme conjugate, wherein monoclonal antibodies raised
against CRP are linked with an enzyme tag by covalent linking; and
(iii) preparing immunochemical reagents for quantitative detection of CRP
in the human serum and/or plasma by preparing their solutions.
13. An in vitro process for quantitatively detecting CRP in the human serum and/or plasma for diagnosis of inflammatory processes comprises detecting amount of CRP in the human serum and/or plasma by reacting test and standard samples with monoclonal antibodies raised against CRP that are applied onto the microwells of microtitre plate and linked with the enzyme tags of enzyme conjugate, thereby forming the stable antigen-antibody complex; detecting the amount of the said antigen-antibody complex, thereby detecting the amount of CRP in test and standard samples using colour reagent.
14. An immunoassay kit for the quantitative detection of CRP in human serum and/or plasma samples and a process for preparing the same such as herein described.