FORM 2
THE PATENT ACT 1970 (39 of 1970)
&
The Patents Rules, 2003 COMPLETE SPECIFICATION
(See Section 10, and rule 13)
TITLE OF INVENTION
AN IMMUNOASSAY KIT FOR QUANTITATIVE DETECTION OF PSA 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

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 PSA in serum and/or plasma
FIELD OF THE INVENTION
The present invention relates to quantitative detection of substances, preferably antigens, in human serum and/or plasma based on a specific solid phase immunoassay technique. More preferably, the invention relates to qualitative detection of antigens or hapten like small chemical substances, preferably prostate specific antigen (PSA), based on solid phase immunoassay technique that specifically utilizes labeled reagents, such as enzyme-labeled reagents for detecting the said antigens in the human serum and/or plasma samples. The present invention most preferably provides an immunoassay kit for qualitative detection of PSA in the human serum and/ or plasma samples for the diagnosis of prostate cancer, benign prostatic hypertrophy or inflammatory conditions of other adjacent genitourinary tissues. It also provides a process for the preparation said immunoassay kit and in vitro process for the quantitative detection of the PSA in the human serum and/or plasma samples.
BACKGROUND OF THE INVENTION
The PSA is a serine protease, a single chain glycoprotein and its molecular weight is approximately 30000 daltons. Elevated serum concentrations have been reported in patients suffering with prostate cancer, benign prostatic hypertrophy or inflammatory conditions of other adjacent genitourinary tissues. Reports have suggested that PSA is one of the most useful tumor makers in oncology. It is therefore highly desirable to provide a simple and sensitive immunoassay kit to detect a concentration of PSA in the human serum and/or plasma samples and an in vitro process for the quantitative detection of the said antigen using the kit of the present invention. The most reliable and user-friendly method or technique for detecting the amount of PSA in the human serum and/or plasma is the solid phase immunoassay method or technique that particularly uses one antibody either covalently bonded or physically adsorbed onto an insoluble solid support or matrix

and another antibody covalent linked to 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 antigens or substances like protein, virus, bacteria, etc. by producing specific antibodies against that particular antigens or substances. Then, there is a specific interaction between said antibodies and antigens to form a complex. An antibody once produced is also capable of binding a hapten that is relatively a small and simple molecule, which 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 of 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 mentioned methods 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 system for measuring the unknown concentration 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 methods are known in the prior art for separating free unbound antigen or hapten from the antigen-antibody complex. One method is chromato-electrophoresis that combines 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 insulin, growth hormone, glucagons, parathyroid hormone, thyroid stimulating hormone and other peptide hormones. However, this method has number of disadvantages, such as limited amount of material can be applied to the absorbent and the separation method is laborious and time-consuming that limits its use.
One another 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 particularly, 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.
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 such as 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 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 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 technique known for the separation of free and bound antigen utilizes antibodies covalently bound or physically adsorbed to an insoluble matrix. The formed antibody-antigen complex is then 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 have surprisingly proposed a diagnostic kit based on the solid phase immunoassay technique for qualitative detection of PSA in the human serum and/or plasma by utilizing the monoclonal antibodies that are covalently bonded with a solid support or matrix and conjugated with enzyme labels. The advantage of utilizing the solid support or matrix is that unlike the prior art methods, no additional centrifugation or filtration is required for the separation of solid and liquid phases during immunoassay.
OBJECT OF THE INVENTION
Therefore, an object of the present invention is to provide an immunoassay kit for the quantitative detection of PSA in the human serum and/or plasma samples that is based on the solid phase immunoassay technique.
Therefore, further object of the present invention is to provide an immunoassay kit for the quantitative detection of PSA in the human serum and/or plasma that comprises anti-PSA monoclonal antibodies of which some are covalently attached to a solid support or matrix and remaining are linked to the enzymes.
Therefore, still further object of the present invention is to provide an immunoassay kit for detecting the concentration of PSA in the human serum and/or plasma samples for the diagnosis of prostate cancer and benign prostatic hypertrophy.
Therefore, yet further object of the present invention is to prepare a simple, cost effective and reliable immunoassay kit for detecting the amount of PSA in human serum and/or plasma for diagnosis of inflammatory conditions of other adjacent genitourinary tissues.
Therefore, another object of the present invention is to provide a process for the preparation of immunoassay kit for the quantitative detection of PSA in the human serum and/or plasma samples that is based on the solid phase immunoassay technique for the separation of antigens.

Therefore, still another object of the present invention is to provide a process for the quantitative detection of PSA in the human serum and/or plasma for diagnosis of conditions, such as prostate cancer, benign prostatic hypertrophy or inflammatory conditions of other adjacent genitourinary tissues.
SUMMARY OF THE INVENTION
Accordingly, an immunoassay kit for the quantitative detection of PSA in the human serum and/or plasma is provided that is based on the solid phase immunoassay technique for separating the antigens. The immunoassay kit comprises a solid support or matrix comprising monoclonal antibodies to PSA, a conjugate comprising conjugated monoclonal antibodies to PSA and enzyme label; and reagents for performing immunoassay for the qualitative detection of PSA in the human serum and/or plasma samples.
Accordingly, a process for the preparation of an immunoassay kit for the quantitative detection of PSA in the human serum and/or plasma samples is also provided. The process of the preparation comprises preparing the solid support or matrix comprising monoclonal antibodies to PSA, the conjugate comprising monoclonal antibodies to PSA and enzyme label and reagents for performing immunoassay.
Accordingly, a process for the qualitative detection of PSA in the human serum and/or plasma for diagnosis of conditions, like prostate cancer, benign prostatic hypertrophy or inflammatory conditions of other adjacent genitourinary tissues is provided. The process of the detection comprises detecting the concentration of PSA in the human serum and/or plasma samples by contacting test and standard samples comprising PSA with the solid support or matrix and conjugate comprising monoclonal antibodies to PSA and enzyme that forms stable antigen-antibody complex and detecting the said antigen-antibody complex using colour reagent.

DESCRIPTION OF THE INVENTION
In one embodiment of the present invention, an immunoassay kit for the quantitative detection of PSA in the human serum and/or plasma comprises a solid support as an insoluble matrix comprising number of microwells that are coated with covalently bound or physically adsorbed monoclonal antibodies to PSA; a conjugate comprising number of enzyme labels that are conjugated with covalently bound monoclonal antibodies to PSA; and reagents for performing the immunoassay for quantitative detection of PSA in the human serum and/or plasma.
In another embodiment of the present invention, the process for the preparation immunoassay kit for the qualitative detection of PSA in the human serum and/or plasma comprises preparing an insoluble matrix or solid support comprising number of microwells coated with monoclonal antibodies to PSA that comprises coating the said microwells with monoclonal antibodies to PSA by either a covalent bonding or physical adsorption, blocking the coated monoclonal antibodies by blocking them using blocking solution comprising phosphate buffer, BSA and Trans-001, and stabilizing the blocked monoclonal antibodies by stabilizing them using stabilising solution comprising PBS, Trans-002 and Tran-003 (bovine immunoglobulin); preparing conjugate comprising number of enzyme labels conjugated with monoclonal antibodies to PSA that comprises conjugating the said enzyme labels with monoclonal antibodies to PSA by using covalent bonding; and preparing reagents for performing the immunoassay and detecting the PSA in the samples by preparing the solutions of required strengths.
In still another embodiment of the present invention, the process for quantitative detection of PSA in the human serum and/or plasma for the diagnosis of conditions, like prostate cancer, benign prostatic hypertrophy or inflammatory conditions of other adjacent genitourinary tissues comprises contacting PSA standard and PSA containing test samples with a solid support or matrix comprising covalently bound or physically adsorbed monoclonal antibodies PSA by adding dispensed quantities of PSA standard and PSA containing test samples into the microwells of solid

support; contacting further with a conjugate comprising enzyme labels conjugated with covalently bound monoclonal antibodies to PSA by adding dispensed quantity of conjugate into the microwells of solid support, thereby forming antigen-antibody complex; removing an unbound fraction of conjugate by washing the microwells using washing solution; detecting said antigen-antibody complex by adding dispensed quantity of colour reagent that contains a substrate for labelled enzyme of conjugate to the microwells, thereby developing a blue colour into the microwells containing PSA standard and test samples, which changes to yellow upon addition of stoping solution; and measuring the concentrations of PSA in the standard and test samples by reading an intensity of yellow colour spectrophotometrically.
DETAILED DESCRIPTION OF THE INVENTION
The present invention can be understood with reference to the following particular and preferable embodiments of the present invention. Although, the features have been disclosed with reference to particular and preferable 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 preferable embodiment of the invention, the immunoassay kit for the quantitative detection of PSA in the human serum and/or plasma comprises a microtitre plate as an insoluble solid support or matrix that comprises number of microwells coated with monoclonal antibodies specific to PSA, wherein said monoclonal antibodies are coated using a covalent bonding or physically adsorbing the antibodies onto the microwells without affecting structure and functional activity of the antibodies for separating PSA in the standard and test samples.
In another preferable embodiment of the invention, the immunoassay kit for the qualitative detection of PSA in the human serum and/or plasma comprises a conjugate that contains enzyme labels conjugated with monoclonal antibodies

specific to PSA, wherein said monoclonal antibodies are conjugated using a covalent bonding with enzyme labels without affecting structure and functional activity of the antibodies for separating PSA in the standard and test samples.
In more preferable embodiment of the invention, the enzyme label used for the preparation of conjugate that is conjugated with monoclonal antibodies specific to PSA is Horse Reddish Peroxidase (HRPO) and the substrate for the said enzyme is present in the solution of colour reagent.
In still another preferable embodiment of the invention, the immunoassay kit for the qualitative detection of PSA in the human serum and/or plasma that is based on solid phase immunoassay technique comprises reagents for performing said immunoassay for the quantitative detection of PSA in the human serum and/or plasma.
In more preferable embodiment of the invention, the reagents for performing the immunoassay for the quantitative detection of PSA in the human serum and/or plasma comprises a sample diluent, PSA standard solutions (A to F), a colour reagent, a stopping solution and a washing solution.
In yet another preferable embodiment of the invention, the microtitre plate as an insoluble support or matrix for the solid phase immunoassay is mainly available in two different capacities as per as number of microwells are present and provided in 48 and 96 microwells capacities.
In still further embodiment of the invention, the PSA standards provided for comparatively estimating amounts of PSA in the test samples comprises solutions A, B, C, D, E and F comprising PSA standards in the concentrations of 0, 0.5,1, 2.5, 5,15 and 25 ng per millilitre along with thimerosal and gentamycin.
In yet further embodiment of the invention, the colour reagent provided for qualitatively detecting the PSA in the standard and test samples comprises 3,3',5,5'-

tetramethyl benzidine, dimethyl sulfoxide and hydrogen peroxide along with thimerosal and gentamycin.
In a separate preferable embodiment of the invention, the stopping solution provided for stopping a catalytic reaction of enzyme label and detecting the PSA in the samples comprises an admixture of concentrated phosphoric and deionized water.
In another preferable embodiment of the invention, the washing solution provided for washing the microplate and removing the unbound fraction of conjugate comprises an admixture of TRIS buffer, NaCl and Tween-20 in deionized water.
In an important embodiment of the invention, the immunoassay kit for the quantitative detection of PSA in the human serum and/or plasma is required to be stored at temperature in the range of 2°C to 8 °C.
In another preferable embodiment of the invention, the process for the preparation immunoassay kit for the quantitative detection of PSA in the human serum and/or plasma comprises preparing a microtitre plate as an insoluble solid support or matrix that comprises number of microwells coated with monoclonal antibodies specific to PSA, and comprises the steps of coating surface of microwells with monoclonal antibodies specific to PSA either by a covalent bonding or physical adsorption; blocking the said monoclonal antibodies by blocking them using blocking solution comprising an admixture of phosphate buffer, BSA and Trans-001; and stabilizing the blocked monoclonal antibodies by stabilizing them using stabilising solution comprising an admixture of PBS, Trans-002 and Tran-003 (bovine immunoglobulin).
In still another preferable embodiment of the invention, the process for the preparation immunoassay kit for the quantitative detection of PSA in the human serum and/or plasma comprises preparing conjugate comprising enzyme labels conjugated with monoclonal antibodies specific to PSA and the process comprises

conjugating the enzyme labels, such as HRPO with monoclonal antibodies specific to PSA by covalent bonding.
In yet another preferable embodiment of the invention, the process for the preparation immunoassay kit for the quantitative detection of PSA in the human serum and/or plasma comprises preparing solutions of appropriate strengths of reagents,, PSA standard solutions (A to F), colour reagent, stopping solution and washing solution.
In another particular embodiment of the process, it comprises preparing the solutions of PSA standards A, B, C, C, D, E and F by admixing 0, 0.5,1, 2.5, 5,15 and 25 ng per millilitre PSA standards in deionized water sterilized with thimerosal and gentamycin.
In yet another particular embodiment of the process, it comprises preparing the solutions of colour reagent by admixing dispensed quantities of 3,3',5,5'-tetramethyl benzidine, dimethyl sulfoxide and hydrogen peroxide along with thimerosal and gentamycin.
In one another particular embodiment of the process, it comprises preparing the stopping solution by admixing dispensed quantities of concentrated phosphoric and deionized water.
In still another particular embodiment of the process, it comprises preparing the washing solution by admixing dispensed quantities of TRIS buffer, NaCl and Tween-20 in deionized water.
In another preferable embodiment of the invention, the process for the qualitative detection of PSA in the human serum and/ or plasma for the diagnosis of conditions, like prostate cancer, benign prostatic hypertrophy or inflammatory conditions of other adjacent genitourinary tissues comprises contacting PSA standard and PSA containing test samples with a microtitre plate comprising covalently bound or physically adsorbed monoclonal antibodies specific to PSA by adding the dispensed

quantities of PSA standard and PSA containing test samples into the wells of plate, thereby forming antigen-antibody complex; again contacting said standard and test samples with a conjugate comprising enzyme labels conjugated with covalently bound monoclonal antibodies specific to PSA by adding the dispensed quantity of conjugate into the wells of plate, thereby forming antigen-antibody complex; removing the unbound fraction of conjugate by washing the wells using washing solution; detecting said antigen-antibody complex by adding the dispensed quantity of colour reagent that contains a substrate for catalytic reaction of labelled enzyme to the wells, thereby developing a blue colour into the wells containing PSA standard and test samples; stopping the reaction catalysed by labelled enzyme by adding the stopping solution, thereby changing the blue colour to yellow; and measuring the concentrations of PSA in the standard and test samples by reading an intensity of yellow colour at 450 nm spectrophotometrically that is directly proportional to the concentration of PSA in the samples.
In more particular embodiment of the process, it comprises contacting PSA standard and PSA containing test samples with monoclonal antibodies to PSA coated onto the wells of plate and conjugated with enzyme label, thereby forming stable antigen-antibody complexes that separate PSA molecules in the standard and test samples and detecting the concentration of PSA by adding colour reagent, thereby developing blue colour that is directly proportional to the concentration of PSA in the samples.
In most particular embodiment of the process, it comprises separating the antigen-antibody complexes so formed due to interaction of PSA molecules and monoclonal antibodies to PSA by addition of colour reagent, thereby generating a blue colour due to catalytic activity of HRPO on its substrate and reading an intensity of yellow colour developed upon addition of stopping solution, which is directly proportional to the amount of PSA present in standard and test sample.

EXAMPLES
The following examples are served just to illustrate the present invention by way of the best method of performing the invention and should not to be regarded as limitations to the scope of the present invention.
Example: 1 - Quantitative detection of PSA in the test samples
Adding dispensed quantities of PSA standard and PSA containing samples to the microwells of the plate; thereafter adding dispensed quantity of conjugate into the microwells and incubating the plate for 60 minutes at room temperature (20 - 40°C), thereby forming stable antigen-antibody complex; removing away the unbound fraction of conjugate by washing the plate using washing solution; adding further dispensed quantity of colour reagent comprising substrate for enzyme label of the conjugate to the microwells containing said antigen-antibody complex, thereby developing blue colour in the PSA standard and test samples containing microwells; then adding stopping solution, thereby changing blue colour to yellow; and subsequently measuring an intensity of yellow colour spectrophotometrically, which is directly proportional to the concentration of PSA in the standard and test samples.
Example: 2 - Test procedure for quantitative detection of PSA in the samples
Bringing all the reagents and test samples at room temperature before use; keep six standards in each run; add 50ul of standard and test samples to the respective wells; adding 50ul of conjugate to each well and incubating for 60 minutes at room temperature (20 - 40°C), preferably 37°C; washing the microplate as per known washing procedure and adding 50ul of colour reagent to each well; covering the plates with black cover and incubating for 15 minutes in dark at 20 to 30°C; and finally reading an absorbance at 450 nm and deducting the absorbance of blank from the control and test wells.

Example: 3- Interpretation of results
The results of typical standard run of the assay performed using the kit of the present invention, which is based on the solid phase immunoassay technique, are shown in Table 1 and Figure 1.
Table 1: Profile of Absorbance as function of PSA concentration

Sr. No. PSA concentration (ng/ml) Absorbance 450 nm)
1 0 0.036
2 1 0.176
3 2.5 0.453
4 5 0.924
5 15 2.012
6 25 3.012

The results illustrated (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.
Any further modifications in and/or improvements in any aspect of the embodiments of this invention will also fall under the scope of this invention. In view of the foregoing description and example, it will become apparent to those of ordinary skill in the art that equivalent modification thereof may be made without departing from the spirit and scope of this invention. Various features of the invention hereinbefore described are set forth in the following claims.

WE CLAIM :
1. An immunoassay kit for quantitative detection of PSA in human serum
and/or plasma comprises:
(a) a solid support as an insoluble matrix comprising number of microwells that are coated with covalently bound or physically adsorbed monoclonal antibodies to PSA;
(b) a conjugate comprising number of enzyme labels that are conjugated with covalently bound monoclonal antibodies to PSA; and
(c) reagents for performing the immunoassay for qualitative detection of PSA in the human serum and/or plasma.

2. The immunoassay kit of claim 1, in which said insoluble solid support is a microtitre plate comprising number of microwells coated with covalently bound or physically adsorbed monoclonal antibodies specific to PSA.
3. The immunoassay kit of claim 2, in which the coating of monoclonal antibodies onto the microwells is accomplished by covalent bonding and/or physical adsorption without affecting integrity and activity of said antibodies.
4. The immunoassay kit of claim 1, in which said conjugate comprises an enzyme label conjugated with monoclonal antibodies specific to PSA and wherein said enzyme label is Horse Reddish Peroxidase (HRPO).
5. The immunoassay kit of claim 4, in which the conjugations of monoclonal antibodies to the enzyme label is accomplished by using covalent bonding without affecting integrity and activity of said antibodies.
6. The immunoassay kit of claim 1, in which said microtitre plate as an insoluble solid support or matrix comprises minimum 48 and maximum 96 microwells.

7. The immunoassay kit of claim 1, in which said reagents for performing immunoassay for the detection of PSA in human serum and/ or plasma are selected from the group consisting of PSA standard solutions (A to F), colour reagent, stopping solution and washing solution.
8. The immunoassay kit of claim 1, in which said PSA standards comprises solutions A, B, C, D, E and F comprising PSA standards in the concentrations of 0, 0.5, 1, 2.5, 5, 15 and 25 ng per millilitre along with thimerosal and gentamycin.
9. The immunoassay kit of claim 1, in which said colour reagent comprises 3,3',5,5'-tetramethyl benzidine, dimethyl sulfoxide and hydrogen peroxide along with thimerosal and gentamycin.
10. The immunoassay kit of claim 1, in which said stopping solution comprises an admixture of concentrated phosphoric and deionized water.
11. The immunoassay kit of claim 1, in which said washing solution comprises an admixture of TRIS buffer, NaCl and Tween-20 in deionized water.
12. A process for the preparation immunoassay kit for qualitative detection of PSA in human serum and/or plasma comprises:
(a) preparing an insoluble matrix or solid support comprising number of microwells coated with monoclonal antibodies to PSA that comprises (i) coating the said microwells with monoclonal antibodies to PSA by either a covalent bonding or physical adsorption, (ii) blocking the coated monoclonal antibodies by blocking them using blocking solution comprising phosphate buffer, BSA and Trans-001, and (iii) stabilizing the blocked monoclonal antibodies by stabilizing them using stabilising solution comprising PBS, Trans-002 and Tran-003 (bovine immunoglobulin);

(b) preparing conjugate comprising number of enzyme labels conjugated with monoclonal antibodies to PSA that comprises conjugating the said enzyme labels with monoclonal antibodies to PSA by using covalent bonding; and
(c) preparing reagents for performing the immunoassay and detecting the PSA in the samples by preparing the solutions of required strengths.
13. A process for quantitative detection of PSA in human serum and/or plasma comprises:
(a) contacting PSA standard and PSA containing test samples with a solid support or matrix comprising covalently bound or physically adsorbed monoclonal antibodies PSA by adding dispensed quantities of PSA standard and PSA containing test samples into the microwells of solid support;
(b) contacting further with a conjugate comprising enzyme labels conjugated with covalently bound monoclonal antibodies to PSA by adding dispensed quantity of conjugate into the microwells of solid support, thereby forming antigen-antibody complex;
(c) removing an unbound fraction of conjugate by washing the microwells using washing solution;
(d) detecting said antigen-antibody complex by adding dispensed quantity of colour reagent that contains a substrate for labelled enzyme of conjugate to the microwells, thereby developing a blue colour into the microwells containing PSA standard and test samples, which changes to yellow upon addition of stoping solution; and
(e) measuring the concentrations of PSA in the standard and test samples by reading an intensity of yellow colour spectrophotometrically.

14. An immunoassay kit for quantitative detection of PSA in human serum and/or plasma and a process for preparing the same described herein with reference to description.