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 KIT FOR DETECTION OF HBV SURFACE ANTIGEN BIOLOGICAL SAMPLES

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 t invention and the manner in which it is to be performed : -

A solid phase kit for detection of HBV surface antigen in biological samples
Field of the invention
The present invention relates to a solid phase immunoassay kit for the detection of hepatitis B surface antigens (HBsAg). It also relates to a method for the preparation of said kit and a process for detection of HBsAg in human and/or plasma.
Background of the invention
Hepatitis B virus (HBV) is responsible for both hepatitis B and hepatocellular carcinoma. HBV affects about 5% of world population. One out of every ten persons infected with Hepatitis B infection that develops some form of chronic liver disease and becomes a long-term carrier of HBV. At present various stages after HBV infection; presence of Hepatitis B surface antigen (HBsAg) in serum and plasma is identified. HBsAg is a set of lipoprotein, having molecular weight ranging from 22 kD to 96 kD, which constitute the envelop of the virus. The HBsAg is the first detectable marker in HBV infected serum and/or plasma and being datable during the whole jaundice phase and becomes undetectable after the appearance of anti-HBsAg antibodies in the serum and/or plasma.
Prior art of the invention
In the prior art, number of immunological kits and methods have been developed for the determination of antigens as well as antibodies, including methods for the qualitative and quantitative detection of hepatitis B surface antigen (HBsAg) in human and plasma, a component of hepatitis B virus and its associated antibodies.
However, in the field of diagnostic what is important is method that is used for detection, which should be sensitive and reliable, as the incorrect diagnosis can have serious consequences. This is especially true in the case of the determination of hepatitis virus antigens and their associated antibodies because the transmission of the hepatitis virus via blood donors constitutes a significant public health risk.

At present there are several radio-immunoassay (RIA) method in its various forms are available. However, these methods have several problems, such as, requirement of special equipment, trained staff, need for extra safety measures to protect against harmful radiation, and short half-life span of the radioactive labelling element. The possibility of replacing the radioactive label with an enzyme label was proposed in long lack in 1968 by Miles and Hales (Nature, Vol. 219, pages 186-189), but no procedural details were provided in said article, which left it for future workers to develop an immunoassay kit and method for detecting hepatitis B surface antigens (HBsAg). Further to that an important pioneering work on enzyme-immunoassay (EIA) methodology has been done by Schuurs and Coworkers that is evident form number of US patents awarded (US 3,654,090, US 3,791,932, US 3,850,752, US 3,839,153, and US 3,879,262).
On infection with HBV, large quantities of virus and associated particles are present in the serum. These particles may contain DNA, but are largely empty viral envelopes that have HBsAg on their surface. The appearance of antibodies to the HBsAg is usually not observed until approximately two months following the disappearance of circulating HBsAg. During this period, a person is highly infectious, but may be clinically diagnosed as non-infectious due to undetectable levels of HBsAg or antibodies to HBsAg.
The viral particles present in the serum are known to shed their surface coat exposing the nucleocapsid. Both IgM and IgG class antibodies are produced to a protein of the nucleocapsid core. This protein is known as the hepatitis B core antigen (HBcAg). Early in infection, IgM anti-HBcAg antibodies are produced and their titres rise as circulating HBsAg titres fall. The titre of IgG anti-HBcAg antibodies increases as the titres of the IgM class antibodies fall and before the anti-HBsAg antibody titres rise significantly. It is therefore advantageous to test for the presence of IgM and IgG class antibodies to HBcAg in diagnosing HBV immune status.
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Immunoassays have been developed for the detection of IgM anti-HBcAg antibodies and total anti-HBcAg antibodies. The commercially available assays for total anti-HBcAg antibodies are generally competitive assays using labelled human polyclonal antibody to HBcAg collected from infected donors to compete with unlabeled antibodies from the patient sample for binding to a solid phase coated with HBcAg. An example of a commercially available assay of this type is Corzyme™ (Abbott Laboratories). The most common commercially available assays for IgM class antibodies can be called multi-layer sandwich assays. A solid phase anti-IgM antibody is used to capture all IgM antibodies from a patient's sample. HBcAg is then added and binds to the specific IgM class anti-HBcAg from the same. Labelled human polyclonal anti-HBcAg is then used to detect any HBcAg bound to the support thus indicating the presence of IgM class anti-HBcAg in the sample. An example of a commercially available assay of this type is Corzyme™ (Abbott Laboratories). Both assays use large quantities of Human polyclonal anti-HBcAg.
The large amounts of human polyclonal anti-HBcAg antibodies needed for such assays must be collected, preferably from the same donor population each time, isolated, purified and labelled with a radioisotope or enzyme. There is significant health hazard and expense associated with this extensive processing of contaminated blood. It would be advantageous to obtain a constant and consistent supply of antibody to HBcAg without exposure to such a health risk.
Therefore, it is highly desirable to have ready use reagent kit for detection of HBsAg in human serum and/or plasma that is easy to handle and ready to use within the reasonable time. Accordingly, the inventors of the present invention have developed the enzyme linked immunosorbent assay that uses polyclonal antibodies to HBsAg as coating materials and monoclonal antibodies to HBsAg as conjugate materials.
Object of the invention
The principle object of the invention is, therefore, to provide a solid phase immunoassay kit for detection of HBsAg in human serum and/or plasma.

Another object of the present invention is to develop a solid phase immunoassay kit for detection of HBsAg in human serum and/or plasma by utilizing polyclonal antibodies against HBsAg antigens.
Still another object of the present invention is to design and develop a solid phase immunoassay kit for detection of HBsAg in human serum and/or plasma that is free of risks and problems/drawbacks associated with the prior art.
One another object of the present invention is to provide a method for preparation of a solid phase immunoassay kit for detection of HBsAg in human serum and/or plasma by using polyclonal antibodies against HBsAg antigens and monoclonal antibodies to HBsAg antigens as a conjugate.
One more object of the present invention is a method for producing a solid phase immunoassay kit for detecting HBsAg by utilizing polyclonal antibody against HBsAg antigen, monoclonal antibody to HBsAg as a conjugate and immunochemically acceptable reagents for detection of HBsAg antigens in human serum and/or plasma.
Still another object of the present invention is to provide an immunoassay method for detecting HBsAg antigens in human serum and/or plasma.
Summary of the invention

According to the principle objects of the present invention, there is provided a solid phase immunoassay kit, which comprises antibodies to HBsAg that are coated on to a solid support and immunochemically acceptable reagents required for detecting said antigens in human serum and/or plasma. Also, provided is a method for preparing said solid phase immunoassay kit, which comprises immobilising over solid support antibodies against HBsAg antigens and providing immunochemically acceptable reagents required for detecting said antigens. Also provided is an immunoassay method for detection of HBsAg antigens in human serum and/or plasma.
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Description of the invention

The solid phase immunoassay kit of the present invention comprises a polyclonal goat anti-HBsAg antibody, a monoclonal anti-HBsAg antibody, a positive control, a negative control, a colour reagent, a sample diluent S, a stopping solution and a washing solution (20X).
In preferred embodiment of the invention, the polyclonal goat anti-HBsAg antibody is immobilised on to the solid support by way of coating.
In another preferred embodiment of the invention, the monoclonal anti-HBsAg antibody is linked to an enzyme as a conjugate for detection of said HBsAg antigen in human serum and/or plasma.
In still preferred embodiment, the enzyme conjugated with the monoclonal anti-HBsAg antibody is horse reddish peroxidase provided with substrate.
In another embodiment of the invention, the positive control is an inactivated HBsAg containing human serum along with thimerosal and gentamycin.
In one more preferred embodiment, the negative control is an inactivated normal human serum along with thimerosal and gentamycin.
In still one more preferred embodiment of the invention, the colour reagent is 3,3', 5,5'-tetramethyl benzidine, dimethyl sulfoxide (DMSO) along with H2O2, thimerosal and gentamycin.
In another preferred embodiment, said sample diluent S is a TRIS buffer with BSA, Bovine IgG, NaCl, Tween-20, thimerosal and gentamycin.
In other preferred embodiment of the invention, the stopping solution is a concentrated phosphoric acid and deionized water.

In still another preferred embodiment, the washing solution (20X) is a TRIS buffer with NaCl, Tween-20 and deionized water.
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As per another object of the present invention, there is provided a method for preparing said solid phase immunoassay kit, which comprises immobilising on to a solid support antibodies against HBsAg antigens, providing conjugated anti-HBsAg antibodies and preparing solutions of immunochemically acceptable reagent required for detecting said antigens in human serum and/or plasma.
In preferred aspect of said process, the goat anti-HBsAg antibodies are dissolved in bicarbonate buffer, which are blocked by blocking solution, comprising phosphate buffer with BSA and Trans-001, and stabilised by PBA with Trans-002 and Tran-003 (bovine IgG).
In one embodiment of the process, the immobilised antibody on to the solid support is a polyclonal anti-HBsAg, preferably, a goat polyclonal anti-HBsAg.
In second embodiment, the conjugated antibody provided is a monoclonal anti-HBsAg, preferably, a monoclonal anti-HBsAg.
In another preferred aspect of the invention, the method of preparing solid phase immunoassay kit also comprises preparing the solutions of an inactivated HBsAg containing human serum as a positive control and an inactivated normal human serum as a negative control.
In still another preferred aspect of the process, it comprises preparing the solution of 3,3', 5,5'-tetramethyl benzidine, dimethyl sulfoxide (DMSO), H2O2 as a substrate and a colouring reagent.
In other preferred aspect, the method further comprises preparing the solutions of a sample diluent S, a stopping solution and a washing solution.
In more preferred features, the solution of sample diluent S contains a TRIS buffer, BSA, bovine IgG, Tween-20, thimerosal, gentamycin and Tran-003.

In another preferred features, the solution of stopping solution contains a concentrated phosphoric acid and deionized water.
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In still another preferred features, the solution of washing solution (20X) contains a TRIS buffer, NaCl, Tween-20 and deionized water.
In most preferred aspect, the solid phase immunoassay kit for detecting HBsAg comprises a polyclonal goat anti-HBsAg antibody coated over the solid support, a conjugated monoclonal anti-HBsAg antibody linked to an enzyme by covalently binding and immunologically acceptable reagent, such as, HBsAg positive control, HBsAg negative control, colour reagent, sample diluent S, stopping solution and washing solution (20X).
In another most preferred aspect, the method for preparing solid phase immunoassay kit for the detection HBsAg comprises coating a polyclonal goat anti-HBsAg antibody on to the solid support, blocking said polyclonal antibody by blocking solution containing phosphate buffer with BSA and Trans-001, stabilizing said antibody by stabilizing solution containing PBS with Trans-002 and Tran-003 (bovine IgG), coupling immunochemically equivalent monoclonal anti-HBsAg antibody by covalently binding with enzyme and providing solutions of HBsAg positive control, HBsAg negative control, colour agent, sample diluent S, stopping solution and washing solution (20X).
As per another object, there is also provided an immunoassay method for detecting HBsAg in human serum and/or plasma comprises contacting a sample from a patient with an antibody against said HBsAg antigen on to the surface of a solid support and an antibody linked with enzyme of a conjugate, incubating the sample with the said solid support, washing the said incubated solid support with washing solution, adding a colour reagent, thereby developing a blue colour in a positive control and HBsAg containing sample, adding stopping solution to the positive control and HBsAg containing sample, thereby changing the blue colour to yellow and measuring an intensity of the yellow colour spectrophotometrically for detection of presence of HBsAg antigens in the sample.

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In the most preferred aspect of the method, it comprises contacting the sample from the patient with the polyclonal goat anti-HBsAg antibody, which is attached to the solid support and directed against HBsAg antigen, and the monoclonal anti-HBsAg-HRPO antibody, which is immunochemical equivalent with first polyclonal antibody; incubating the sample with the said solid support, thereby forming the stable antibody-antigen complex, washing the said incubated solid support with the washing solution, thereby removing the unbound fraction of said reaction, adding the colour reagent to the bound fraction of the said reaction, thereby developing a blue colour in the positive control and the HBsAg containing sample, adding the stopping solution to the said reaction mixture, thereby changing the blue colour to yellow and measuring an intensity of the yellow colour spectrophotometrically, which is directly proportional to the amount of bound HBsAg in the sample, for detection of HBsAg in the sample.
In preferred aspect, the incubation period usually ranges from 0.5 to 25 hours and incubation temperature is in between 4°C and 5°C, and for the detection of HBsAg antigens, the foregoing reagents are employed in predetermined amounts.
In another preferred aspect of the invention, the solid support to which goat anti-HBsAg antibody is bound is any water-insoluble solid support. An example of suitable water-insoluble solid support is microtitre plate. The immunological components bound to the solid support by covalent bonds or by adsorption. An advantage of using the solid support is no centrifugation required for separation of solid and liquid phases.
The enzyme conjugate consists of immunological components covalently linked to the enzyme molecule that is achieved either by direct condensation or by using external bridging molecules, which are known to those skilled in the art. Thus, the enzyme coupling products is produced by employing the covalent bond that is effected by reagents, such as, carbodiimides, diisocyanates, glutaric aldehyde and bis-diazobenzidine.

The choice of the enzyme that is to form a part of the coupling product is determined by properties, such as, specific binding activity, high conversion rate and simplicity of detection. The determination of the enzyme activity in which coloured reaction components are involved is simple, which can be determined spectrophotometrically or fluorimetrically. These determinations are also suitable for automation that is an additional advantage. The suitable enzyme used in the kit and methods hereinbefore described is horseradish peroxidase.
The solid phase immunoassay kit of the present invention and method for detection do not possess the disadvantages of a radioimmunoassay (RIA) method and are simple to perform, and surprisingly are as sensitive as the RIA. One advantage of the kit and methods of the present invention is that by using the enzyme as a labelling means the immunological reaction is measured by way of colour changes either naked eye or by spectrophotometrica measurement.
Examples
The following examples serve to illustrate the invention by way of best method of performing and not to be regarded as limitations.
Example: 1 - Detection of HBsAg antigens
Adding positive control or HBsAg containing human serum and/or plasma to micro-wells of the microtitre plate, thereby forming the stable complex with bound polyclonal goat anti-HBsAg antibodies of the microtitre plate and monoclonal anti-HBsAg-HRPO antibodies of the conjugate; incubating the microtitre plate containing reaction mixture for 0.5 to 25 hours at temperature between 4°C and 5°C, thereby forming stable antibody-antigen complex, thereafter washing the microtitre plate using washing solution, thereby removing unbound fraction of conjugate, adding the colour reagent to the wells of microtitre plate, thereby developing a blue colour in the wells containing positive control and HBsAg containing sample, adding the stopping solution to the wells, thereby changing the blue colour to yellow and

finally measuring an intensity of the yellow colour spectrophotometrically for detection of HBs Ag.
Example: 2 - Test procedure for detection of HBs Ag antigens
Any one of three test procedures can be used for regular screening of HBsAg in human serum and/or plasma.
A. Test procedure I:
(a) Bring all the reagents and test specimens at room temperature before use; (b) add 25μl of sample diluent to each well. In each run, there will be one blank (l00μl sample diluent plus 50μl conjugate), three negative controls and one positive control. Add 75μl of control and test specimens to the respective wells. Add 50μl of conjugate to each well. Cover the plate with black cover and incubate 60 minutes at room temperature (20 - 25°C); (c) wash the plate as per microplate washing procedure known to person skilled in the art; (d) add 50ul of colour reagent and cover the plate with black cover and incubate for 15 minutes in dark at 20 - 30°C; (e) add lOOul of stopping buffer to each well; and (f) read absorbance at 450 nm and deduct the blank absorbance from the control and test wells.
B. Test procedure II:
(a) Bring all the reagents and test specimens at room temperature before use; (b) add 25ul of sample diluent to each well. In each run, there will be one blank (100[j.l sample diluent plus 50JJ1 conjugate), three negative controls and one positive control. Add 75|al of control and test specimens to the respective wells. Add 50ul of conjugate to each well. Cover the plate with black cover and incubate 15 minutes at 37°C; (c) wash the plate as per microplate washing procedure known to person skilled in the art; (d) add 50ul of colour reagent and cover the plate with black cover and incubate for 15 minutes in dark at 20 - 30°C; (e) add lOOul of stopping buffer to each well; and (f) read absorbance at 450 nm and deduct the blank absorbance from the control and test wells.

C. Test procedure III:

(a) Bring all the reagents and test specimens at room temperature before use; (b) add 25ul of sample diluent to each well. In each run, there will be one blank (lOOul sample diluent plus 50ul conjugate), three negative controls and one positive control. Add 75ul of control and test specimens to the respective wells. Add 50fil of conjugate to each well. Cover the plate with black cover and incubate 60 minutes at 37°C; (c) wash the plate as per microplate washing procedure known to person skilled in the art; (d) add 50ul of colour reagent and cover the plate with black cover and incubate for 15 minutes in dark at 20 - 30°C; (e) add lOOul of stopping buffer to each well; and (f) read absorbance at 450 nm and deduct the blank absorbance from the control and test wells.
Example: 3 - Calculation for cut-off value determination
Blank value: Absorbance of blank values should be less than 0.2. Positive Control: Absorbance of individual positive control should be grater than 1.0. Negative Control: Absorbance of individual negative control should be less than 1.0.
NCx: Average value of negative controls.
Calculation of NCx:
For example:
NC Absorbance
1 0.022
2 0.020
3 0.024
NCx: (0.22+0.020+0.024)/3=0.022

Cut-off value formula: 0.15+NCx
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Cut-off vale: 0.15+0.022=0.172
Interpretation of results:
Non-reactive: If the absorbance of the test serum and/or plasma is less than the cutoff value, then it is considered as non-reactive.
Reactive: If the absorbance of the test serum and/or plasma is equal or greater than the cut-off value, then it is considered as initial reactive. This sample should be retested as duplicate. If the absorbance of duplicate retests result is less than cut-off value, then the specimen is considered as non-reactive. If both of duplicate retest results are found reactive, then the specimen is considered as repeatedly reactive.

WE CLAIM :
1. A solid phase immunoassay kit for detecting HBsAg antigen in human serum and/or plasma comprises an first antibody to HBsAg antigen, which is immobilised onto a solid support; an immunochemically equivalent conjugated second antibody, which is linked to an enzyme by covalently bonding; and immunologically acceptable reagent, preferably, HBsAg positive control, HBsAg negative control, colour reagent, sample diluent, stopping solution and washing solution, which are required for detecting HBsAg antigens in said sample, wherein the first antibody that is immobilized onto the said support is a polyclonal goat anti-HBsAg antibody and the second antibody that is covalently linked to the enzyme is monoclonal anti- HBsAg-HRPO.
2. The solid phase immunoassay kit as claimed in claim 1, wherein the enzyme linked to conjugated monoclonal antibody is horse reddish peroxidase.
3. The solid phase immunoassay kit as claimed in claim 1, wherein the positive control is an inactivated HBsAg antigen containing human serum.
4. The solid phase immunoassay kit as claimed in claim 1, wherein the negative control is an inactivated normal human serum.
5. The solid phase immunoassay kit as claimed in claim 1, wherein the colour reagent is 3,3', 5,5'-tetra methyl benzidine dimethyl sulfoxide with H2O2.
6. The solid phase immunoassay kit as claimed in claim 1, wherein the sample diluent is solution S, which contains a TRIS buffer, BSA, bovine IgG, Tween-20, thimerosal, gentamycin and Tran-003.
7. A diagnostic kit as claimed in claim 1, wherein the stopping solution is solution of a concentrated phosphoric acid and deionized water.

8. A diagnostic kit as claimed in claim 1, wherein the washing solution is a solution of TRIS buffer, NaCl, Tween-20 and deionized water.
9. A method for preparing a solid phase immunoassay kit for detection of HBsAg antigens of claims 1 to 8 comprises immobilising an first polyclonal goat anti-HBsAg antibody onto a solid support; blocking said polyclonal antibody using blocking solution, which contains phosphate buffer, BSA and Trans-001; stabilizing further using stabilizing solution, which contains PBS, Trans-002 and Tran-003 (bovine IgG); preparing an immunochemically equivalent monoclonal anti-HBsAg-HRPO by covalently binding to the enzyme, which is horse reddish peroxidase; and preparing solutions of HBsAg positive control, HBsAg negative control, colour reagent, sample diluent, stopping solution and washing solution as herein described.
10. An immunoassay method for detecting HBsAg in human serum and/or plasma comprises contacting a sample from a patient with a polyclonal goat anti-HBs Ag antibody of HBsAg antigen and a monoclonal anti-HBsAg-HRPO antibody linked with enzyme; incubating the sample with the said solid support; washing the said incubated solid support using washing solution; adding a colour reagent, thereby developing a blue colour in a positive control and HBsAg containing sample; adding stopping solution, thereby changing the blue colour to yellow and measuring an intensity of the yellow colour spectrophotometrically for detection of HBsAg antigens.