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
A SOLID PHASE IMMUNOASSAY KIT FOR QUANTITATIVE ESTIMATION 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 : -

A solid phase immunoassay kit for quantitative estimation of C-reactive protein
in serum and/or plasma
FIELD OF THE INVENTION
The present invention relates to a quantitative estimation of substances, particularly antigenic proteins in human serum and/or plasma based on specific solid phase immunoassay techniques. More particularly, the invention relates to the estimation quantity of antigens or hapten like small chemical substances, most particularly C-reactive protein (CRP), based on the solid phase immunoassay techniques, which utilizes labeled reagents, such as enzyme-labeled reagents for detecting and estimating said antigenic proteins or hapten. The present invention most specifically provides a solid phase immunoassay kit for quantitative estimation of CRP in the human serum and/or plasma as an acute phase protein for diagnosis of inflammatory processes. The invention also provides a process for making the immunoassay kit and an in vitro process for the quantitative estimation of CRP in the human serum and/ plasma.
BACKGROUND OF THE INVENTION
The CRP is an alpha globulin with a molecular mass of approximately 110000 to 140000 daltons and is composed of five identical subunits, which are noncovalently assembled as a cyclic pentamer. CRP is one of the acute phase proteins. Normally CRP levels of CRP in serum and plasma are less than 3 ng/ml. However, the level rises in a wide variety of diseases. Although the detection of elevated levels of CRP in the serum and/or plasma is not specific 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), which may also be influenced by physiological changes not associated with an inflammatory process. It is therefore, beneficial to provide a simple and sensitive solid phase immunoassay kit to estimate the quantity of CRP in the human serum and/or plasma samples and an in vitro process for quantitative estimation of said


protein (CRP). The most reliable and sensitive method or technique for estimating the quantity of CRP in the human serum and/or plasma is the solid phase immunoassay method or technique, which utilizes one antibody either covalently bonded or physically adsorbed onto an insoluble solid support or matrix and another 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 glow reagent.
The living system promptly responds to the presence of foreign antigen, such as 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, which 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, such as radioisotopes, co-enzymes, 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 discussed techniques consist of 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 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 concentration of unlabeled antigen, it becomes possible to establish an assay technique for estimating the unknown quantity 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 separating and detecting free unbound antigen or hapten from the antigen-antibody complex. One such method or technique is chromatoelectrophoresis, which is a combination of a paper chromatography and paper electrophoresis. The paper with a high affinity for free antigen, such as Whatman Filter paper is used as carriers. The method is discriminative and has been used in the assay of chemical substances like insulin, growth hormone, glucagons, parathyroid hormone, thyroid stimulating hormone and other peptide hormones. However, this method has number of disadvantages, like limited amount of material can be loaded to the absorbent material and the separation method is laborious and time-consuming that limits its use.
Another such known method is precipitation of antigen-antibody complex that involves use of salts, organic material or solvents under the conditions that do not affect free antigens. Among these, salts, materials and solvents used are ethanol, acetone, sodium sulfate, ammonium sulfate, dioxane, trichloroacetic acid, polyethylene glycol, etc. The use of salts, solvents or organic materials has advantage that the separation is immediate and a second incubation is not required. But, the

chemical precipitation also causes co-precipitation of other proteins molecules that causes incomplete separation of two fractions.
Still another known method is double antibody technique that is widely used for the separation of bound and free antigen. By using this method, 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.
Also, ion exchange and other resins are known to be used 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 yet another 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 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 sieves, which 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 techniques known for the separation of free and bound antigen utilizes antibodies covalently bound or physically adsorbed to an insoluble solid

support or matrix. 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. In view of above, the inventors of the present invention proposed a diagnostic kit based on the solid phase immunoassay technique for quantitative estimation of CRP in the human serum and/or plasma by utilizing one group of monoclonal antibodies covalently bonded with a solid support or matrix and another is conjugated with an enzyme label. Importantly, the advantage of using the solid support or matrix is that no additional centrifugation or filtration step is required for the separation of solid and liquid phases during immunoassay.
OBJECT OF THE INVENTION
Therefore, a main object of the present invention is to provide a solid phase immunoassay kit for quantitative estimation of CRP in human serum and/or plasma.
Therefore, further object of the present invention is to provide a solid phase immunoassay kit for the quantitative estimation of CRP in the human serum and/or plasma that comprises monoclonal antibodies specific to CRP and are covalently attached to a solid support or matrix and a conjugated enzyme.
Therefore, still further object of the present invention is to provide a solid phase immunoassay kit for estimating quantity of CRP in the human serum and/ or plasma samples as an acute phase protein for diagnosis of inflammatory processes.
Therefore, yet further object of the present invention is to produce a simple, sensitive and cost effective immunoassay kit for quantitatively estimating CRP in the human serum and/or plasma for detecting inflammatory processes.
Therefore, another object of the present invention is to provide a process for making a solid phase immunoassay kit for quantitative estimation of CRP in the human serum and/or plasma samples, which is based on the solid phase immunoassay technique for the separation and detection of antigenic proteins.

Therefore, yet another object of the present invention is to provide an in vitro process for quantitative estimation of CRP in the human serum and/or plasma for detecting the inflammatory processes.
STATEMENT OF THE INVENTION
Therefore, based on a solid phase immunoassay technique for separating antigenic proteins, a solid phase immunoassay kit for quantitative estimation of CRP in the human serum and/or plasma is provided, which mainly comprises an insoluble solid support or matrix having multiple microwells that are coated with monoclonal antibodies to CRP, a conjugate material having number of enzyme labels that are bound with monoclonal antibodies to CRP and immunoassay reagents for performing the assay for quantitative estimation of CRP in the human serum and/or plasma.
Therefore, based on a solid phase immunoassay technique for separating antigenic proteins, a process for making a solid phase immunoassay kit for quantitative estimation of CRP in the human serum and/or plasma is provided, which comprises making an insoluble solid support or matrix having multiple microwells coated using monoclonal antibodies to CRP by coating said monoclonal antibodies onto the microwells, making a conjugated material having number of enzyme labels bound to monoclonal antibodies to CRP and making immunoassay reagents for performing immunoassay for quantitative estimation of CRP in the human serum and/or plasma.
Therefore, based on a solid phase immunoassay technique for separating antigenic proteins, an in vitro process for quantitative estimation of CRP in the human serum and/or plasma for the diagnosis of inflammatory processes comprises estimating quantity of CRP in the human serum and/or plasma by contacting test and standard samples with monoclonal antibodies to CRP coated on and bound to an insoluble solid support or matrix and conjugate material, whereby producing antigen-

antibody complex and estimating the quantity of CRP in said test and standard samples by detecting said antigen-antibody complex using glow reagent.
DESCRIPTION OF THE INVENTION
Accordingly, the solid phase immunoassay kit of instant invention for quantitative estimation of CRP in the human serum and/or plasma samples comprises an insoluble solid support or matrix having multiple microwells that are coated with covalently bonded or physically attached anti-CRP monoclonal antibodies; a conjugate material having number of enzyme labels that are conjugated with covalently linked anti-CRP monoclonal antibodies; and immunoassay reagents for performing the solid phase immunoassay for quantitative estimation of CRP in the human serum and/or plasma.
Accordingly, the process for making a solid phase immunoassay kit of instant invention for quantitative estimation of CRP in the human serum and/or plasma samples comprises: making an insoluble solid support or matrix having multiple microwells coated using anti-CRP monoclonal antibodies, which comprises the steps of coating said monoclonal antibodies onto the surface of microwells by covalently bonding or physically attaching them, blocking said monoclonal antibodies onto the surface of microwells by fixing them using blocking solution of phosphate buffer, BSA and Trans-001, and stabilizing the said antibodies onto the microwells by layering them using stabilising solution of PBS, Trans-002 and Tran-003 (bovine immunoglobulin); making a conjugated material by covalently linking enzyme labels with anti-CRP monoclonal antibodies; and making immunoassay reagents by preparing the solutions of required strengths.
Accordingly, the in vitro process for quantitative estimation of CRP in the human serum and/or plasma for the diagnosis of inflammatory processes comprises contacting CRP standards and CRP containing test samples with anti-CRP monoclonal antibodies that are coated onto and bound to an insoluble solid support or matrix and conjugate material, whereby producing stable antigen-antibody

complex; and estimating the quantity of CRP in the standards and test samples by detecting said antigen-antibody complex using glow reagent by reading relative light units (RLU) using standard plot of RLU Vs CRP.
DETAILED DESCRIPTION OF THE INVENTION
The instant 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 preferred embodiment of the invention, the solid phase immunoassay kit for the quantitative estimation of CRP in the human serum and/or plasma samples comprises a microtitre plate having multiple microwells as an insoluble solid support or matrix, in which the surface of said microwells are coated with covalently bonded or physically attached anti-CRP monoclonal antibodies for detecting CRP molecules into the samples.
In another preferred embodiment of the invention, the solid phase immunoassay kit for the quantitative estimation of CRP in the human serum and/or plasma samples comprises the conjugate material containing enzyme labels, in which the enzymes are conjugated with covalently linked anti-CRP monoclonal antibodies for separating CRP molecules from the samples.
In more preferred embodiment of the invention, the conjugated enzyme covalently liked with anti-CRP monoclonal antibodies is Horse Reddish Peroxidase (HRPO).
In still another preferred embodiment of the invention, the solid phase immunoassay kit for the quantitative estimation of CRP in the human serum and/or plasma samples comprises the immunoassay reagents for performing the solid phase


immunoassay for quantitative estimation of CRP in the human serum and/or plasma, which include sample diluent, CRP standards (A to F), glow reagents A and B, and washing solution.
In yet another preferred embodiment of the invention, the microtitre plates of the solid phase immunoassay kit for quantitatively estimating CRP in the human serum and/or plasma samples comprises either 48 or 96 microwells coated with covalently bonded or physically attached anti-CRP monoclonal antibodies for testing 48 or 96 test samples, respectively.
In separate preferred embodiment of the invention, the sample diluent as an immunoassay reagent for diluting the standards and test samples comprises a mixture of phosphate buffer, BSA and Tween-20 along with thimerosal and gentamycin in appropriate proportions.
In still separate embodiment of the invention, the PSA standards as immunoassay reagents for comparing CRP quantity in the test samples comprises solutions namely A, B, C, D, E and F, which contain CRP standards in the concentrations of 0, 0.5, 1, 2.5, 5, 7.5, and 10 u.g per millilitre, respectively, along with thimerosal and gentamycin as preservatives.
In yet separate embodiment of the invention, the glow reagents A and B as immunoassay reagents for providing working glow reagent comprises HRP substrate A and B respectively.
In more preferred embodiment of the invention, the working glow reagent, as an immunoassay reagent comprises a mixture of glow reagent A and B in equal proportions for immediate use that is within 100 minutes of its preparation.
In different preferred embodiment of the invention, the washing solution as an immunoassay reagent for washing microtitre plate and removing unbound fraction of conjugate material comprises a mixture of TRIS buffer, NaCl and Tween-20 in deionized water in appropriate proportions.


In an important embodiment of the invention, the solid phase immunoassay kit for the quantitative estimation of CRP in the human serum and/or plasma samples is stored at temperature between 2°C and 8 °C.
In distinct preferred embodiment of the invention, the process for making a solid phase immunoassay kit for quantitative estimation of CRP in the human serum and/or plasma samples comprises preparing the microtitre plate comprising either 48 or 96 microwells coated using anti-CRP monoclonal antibodies, which comprises subsequent steps of (i) coating anti-CRP monoclonal antibodies onto the surface of microwells by either covalently bonding or physically attaching them, (ii) blocking said monoclonal antibodies onto the surface of microwells by bocking them with blocking solution comprising mixture of phosphate buffer, BSA and Trans-001 and (iii) stabilizing the antibodies onto the surface of microwells by stabilizing them with stabilising solution comprising mixture of PBS, Trans-002 and Tran-003 (bovine immunoglobulin).
In another preferred embodiment of the invention, the process for making a solid phase immunoassay kit for quantitative estimation of CRP in the human serum and/or plasma samples comprises preparing a conjugated enzyme by covalently linking HRPO as enzyme labels with anti-CRP monoclonal antibodies
In still another preferred embodiment of the invention, the process for making a solid phase immunoassay kit for quantitative estimation of CRP in the human serum and/or plasma samples comprises preparing the solutions of sample diluent, CRP standards (A to F), glow reagents A and B, and washing solution of required strengths.
In more preferred embodiment, the process comprises preparing solution of sample diluent by mixing phosphate buffer, BSA and Tween-20 in deionized water sterilized by thimerosal and gentamycin in appropriate proportions.

In another preferred embodiment, the process comprises preparing solutions of CRP standards A, B, C, C, D, E and F by mixing 0, 0.5,1, 2.5, 5, 7.5 and 10 mg per millilitre CRP standards in deionized water sterilized by thimerosal and gentamycin.
In still another preferred embodiment, the process comprises preparing solutions of glow reagents A and B by mixing HRP substrate components A and B in deionized water.
In most preferred embodiment of the process, preparing of working glow reagent comprises mixing glow reagent A and B in equal proportions of for use within 100 minutes.
In yet another preferred embodiment, the process comprises preparing washing solution by mixing TRIS buffer, NaCl and Tween-20 in deionized water in appropriate proportions.
In different preferred embodiment of the invention, the in vitro process for quantitative estimation of CRP in the human serum and/or plasma for the diagnosis of inflammatory processes comprises contacting CRP standards and CRP containing test samples with microwells (microtitre plate) coated with anti-CRP monoclonal antibodies; subsequently contacting said standards and test samples with HRPO conjugated with anti-CRP monoclonal antibodies (anti-CRP-HRPO), whereby producing stable antigen-antibody complex; removing unbound fraction of anti-CRP-HRPO and adding working glow reagent, whereby producing RLU; and estimating quantity of CRP in test samples by reading RLU values using standard plot of RLU Vs CRP.
In more preferred embodiment of the invention, the process for quantitative estimation of CRP in the human serum and/or plasma for the diagnosis of inflammatory processes comprises contacting PSA standards and PSA containing samples with anti-CRP monoclonal antibodies coated onto microwells and conjugated with HRPO, whereby producing stable antigen-antibody complexes and


separating CRP molecules in the samples and estimating quantity of CRP by adding glow reagent and thereafter reading RLU values on standard plot of RLU Vs CRP.
In most preferred embodiment of the invention, the process for quantitative estimation of CRP in the human serum and/or plasma for the diagnosis of inflammatory processes comprises separating antigen-antibody complex so formed due to interaction between CRP molecules and anti-CRP monoclonal antibody by adding glow reagent, whereby producing RLU due to catalytic activity of HRPO on its substrate and reading RLU generated on standard plot, which are proportional to the quantity of CRP present 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 estimation of CRP in the test samples
Putting measured amount of CRP standards and CRP containing samples into the microwells of plate; subsequently putting measured amount of conjugated material into the said microwells and incubating the plate for 60 minutes at room temperature between 20°C and 40°C, whereby producing stable antigen-antibody complex; removing the unbound fraction of conjugated material by washing the plate with washing solution; putting measured amount of working glow reagent containing HRP substrate component to the microwells containing the said antigen-antibody complex, whereby producing RLU in CRP standards and test samples containing microwells; and estimating the quantity of CRP by reading RLU values on standard
Plot of RLU Vs CRP (mg/ml).

Example: 2 - Test procedure for quantitative estimation of CRP in the samples
Bring all the reagents and test samples at room temperature before use; put 50ml of sample diluent to each well; maintain one blank (l00ml sample diluent + 50ml conjugate) and six standards in each run; put 50ml of standards and test samples to respective microwells; put 50ml of 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; put 50ml of working glow reagent to each microwell; and read RLU vales after 1 minutes and before 20 minutes of glow reagent addition.
Example: 3- Interpretation of results obtained using a solid phase immunoassay kit
The results of typical standard immunoassay performed using the solid phase immunoassay kit of the invention that is based on the solid phase immunoassay technique are depicted 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 concentrations of test specimens for testing.
Table 1: Profile of RLU produced as a function of CRP concentration

Sr. No. CRP concentration (mg/ml) RLU
1 0 1234
2 1 34267
3 2.5 99765
4 5 186754
5 7.5 274526
6 10 370752
Any further modifications in and/or improvements in any aspect of the embodiments of instant 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. A solid phase immunoassay kit for quantitative estimation of CRP in the
human serum and/or plasma samples comprises:
(a) an insoluble solid support or matrix having multiple microwells that are coated with covalently bonded or physically attached anti-CRP monoclonal antibodies;
(b) a conjugate material having number of enzyme labels that are conjugated with covalently linked anti-CRP monoclonal antibodies; and
(c) immunoassay reagents for performing the solid phase immunoassay for quantitative estimation of CRP in the human serum and/or plasma.
2. The solid phase immunoassay kit of claim 1, characterized in that the insoluble solid support or matrix comprises a microtitre plate having multiple microwells in which the surface of said microwells is coated using covalently bonded or physically attached anti-CRP monoclonal antibodies for detecting CRP molecules into the samples.
3. The solid phase immunoassay kit of claim 1 or 2, characterized in that the microtitre plate comprises either 48 or 96 microwells coated using covalently bonded or physically attached anti-CRP monoclonal antibodies.
4. The solid phase immunoassay kit of claim 1, characterized in that the coiijugate material comprises enzyme labels in which the enzyme is conjugated with covalently linked anti-CRP monoclonal antibody for separating CRP molecules into the samples.
5. The solid phase immunoassay kit of claim 1 or 4, characterized in that the conjugated enzyme covalently liked with anti-CRP monoclonal antibody is Horse Reddish Peroxidase (HRPO).


6. The solid phase immunoassay kit of claim 1, characterized in that the immunoassay reagents comprise a sample diluent, CRP standards (A to F), glow reagents A and B, and a washing solution.
7. The solid phase immunoassay kit of claim 1 or 6, characterized in that the sample diluent comprises a mixture of phosphate buffer, BSA and Tween-20 along with thimerosal and gentamycin in appropriate proportions.
8. The solid phase immunoassay kit of claim 1 or 6, characterized in that the PSA standards comprises solutions A, B, C, D, E and F that contain CRP standards in the concentrations of 0, 0.5,1, 2.5, 5, 7.5, and 10 (ig per millilitre respectively along with thimerosal and gentamycin as preservatives.
9. The solid phase immunoassay kit of claim 1 or 6, characterized in that the glow reagents A and B comprise HRP substrates A and B respectively in appropriate proportions.
10. The solid phase immunoassay kit of claim 9, characterized in that the glow reagents A and B are mixed in equal proportions for making a working glow reagent solution that is for immediate use within 100 minutes of its preparation.
11. The solid phase immunoassay kit of claim 1 or 6, characterized in that the washing solution comprises a mixture of TRIS buffer, NaCl and Tween-20 in deionized water in appropriate proportions.
12. A process for making a solid phase immunoassay kit for quantitative estimation of CRP in the human serum and/or plasma samples comprises:
(a) making an insoluble solid support or matrix having multiple microwells coated using anti-CRP monoclonal antibodies, which comprises the steps of: (i) coating said monoclonal antibodies onto the surface of microwells by covalently bonding or physically attaching


them, (ii) blocking said monoclonal antibodies onto the surface of microwells by fixing them using blocking solution of phosphate buffer, BSA and Trans-001 and (iii) stabilizing the said antibodies onto the microwells by layering them using stabilising solution of PBS, Trans-002 and Tran-003 (bovine immunoglobulin);
(b) making a conjugated material by covalently linking enzyme labels with anti-CRP monoclonal antibodies; and
(c) making immunoassay reagents by preparing the solutions of required strengths.

13. An in vitro process for quantitative estimation of CRP in the human serum and/or plasma for the diagnosis of inflammatory processes comprises contacting CRP standards and CRP containing test samples with anti-CRP monoclonal antibodies that are coated onto and bound to an insoluble solid support or matrix and conjugate material, whereby producing stable antigen-antibody complex; and estimating the quantity of CRP in the standards and test samples by detecting said antigen-antibody complex using glow reagent by reading relative light units (RLU) using standard plot of RLU Vs CRP.
14. A solid phase immunoassay kit for quantitative estimation of CRP in human serum and/or plasma samples and a process for making the same such as herein described.