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 SERODIAGNOSTIC KIT FOR DETECTION OF ANTIBODIES TO TREPONEMA PALLIDUM ;

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 SERODIAGNOSTIC KIT FOR DETECTION OF ANTIBODIES TO TREPONEMA PALLIDIUM
FIELD OF THE INVENTION
The present invention relates to the fields of microbial immunology. More specifically, it relates to a serodiagnostic kit for detecting antibodies to Treponema pallidum proteins in human serum and plasma, a method of preparing the serodiagnostic kit for detecting antibodies to T. pallidum proteins and a serodiagnosis method for detecting antibodies to T. pallidum proteins in human serum and plasma.
BACKGROUND OF THE INVENTION
T. pallidum is the microaerophilic spirochete that is causative agent for syphilis, which is a systemic venereal disease with multiple clinical presentations. Pinta (T. carateum), yaws (T. pallidum subspecies pertenue), and bejel (T. pallidum subspecies endemicum) are other closely related treponemas. The initial infection causes an ulcer at the site of infection; however, the bacteria move throughout the body, damaging many organs over the time. Although treatment with penicillin in the early stages may be successful, the early symptoms of syphilis can be very mild, and many people do not seek treatment when they first become infected. This delay in seeking treatment is harmful because the damage to the organs in late syphilis cannot be reversed. Further, the risk of transmitting and acquiring HIV that causes AIDS via open ulcers caused by syphilis is major concern now days.

The scientists have divided the syphilis into primary, secondary, latent, and tertiary stages. An infected person who has not received the treatment may infect others in the first two stages that usually last one to two years. The bacteria spread from the initial ulcer of an infected person to the skin or mucous membranes of the genital area, the mouth, or the anus of a sexual partner. The bacteria can also pass through broken skin on other parts of the body. In its tertiary stage, untreated syphilis, although not contagious, can cause serious heart abnormalities, mental disorders, blindness, other neurologic problems, and even death.
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In primary syphilis, the first symptom is an ulcer known as a chancre, which may appear within 10 days to three months after exposure, but it generally appears within two to six weeks. The secondary syphilis is often marked by a skin rash, brown sores about the size of a penny, which appears anywhere from three to six weeks after the chancre appears. In the tertiary stage of syphilis, bacteria damage the heart, eyes, brain, nervous system, bones, joints, or almost any other part of the body that may last for years, or even decades. Late syphilis can result in mental illness, blindness, other neurologic problems, heart disease, and even death.
The syphilis bacteria also frequently invade the nervous system in the early stage of infection that may develop neurosyphilis. However, development of the neurosyphilis can take up to twenty years and some persons with neurosyphilis never develop any symptoms. Those who show the symptoms may experience headaches, stiff necks, and fever, inflammation of brain lining, weakness and visual problems. It can be treated, but the treatment may be more difficult and its course may be different in persons infected with HIV. The syphilis in pregnant women is particularly compelling as it causes consequential effects on unborn child. An untreated pregnant woman with active syphilis will pass on the infection to her unborn child.

Due to serious and life threatening effects of syphilis infection, and risk of transmitting or contracting HIV, specific and early diagnosis of the infection is important. However, it has been called the great imitator, as its early symptoms are similar to those of many other diseases. Therefore, a doctor usually does not rely upon recognition of the signs and symptoms of syphilis, but performs both microscopic identification of syphilis bacteria and blood tests. To diagnose syphilis by a microscopic identification of the bacterium, the physician may take a scraping from the surface of the ulcer or chancre and examine it under a dark-field microscope to detect the organism. However, the dark-field microscopy requires considerable skill and is prone to misinterpretation. For these reasons, most syphilis cases are diagnosed serologically.
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The blood tests, such as, Venereal Disease Research Laboratory (VDRL) test and rapid plasma reagent (RPR) test are often used to detect evidence of syphilis, which employ natural lipids, cardiolipin and lecithin, to detect antibodies against nonspecific antigens. However, these tests lack specificity in comparison to the treponemal tests due to occurrence of false positives and false negatives, which usually require more than one blood test. The treponemal-based tests, such as, fluorescent treponemal antibody-absorption (FTA-ABS) and T. pallidum hemagglutination assay (TPHA) can be used to confirm a positive test. But these tests are more expensive and more difficult to use than non-treponemal tests.
Recent treponemal tests in use detect the proteins anchored in the T. pallidum cytoplasmic membrane that is particularly difficult because of the unusual structure of T. pallidum membrane, which predominantly consists lipids that tend to hide these proteins from detection. This hiding of proteins often delays host's immune response frequently resulting in false negative results.
Though some treatments for treponemal infections are available, control of treponemal diseases is managed by eliminating person-to-person spread. Therefore, the early detection of treponemal infection is vital for reducing widespread dissemination of related diseases. Thus, there remains a high need for accurate and improved serodiagnostic kit and method for detecting antibodies to T. pallidum proteins for effective, accurate and early diagnosis of syphilis.
OBJECT OF THE INVENTION

It is therefore an object of the present invention is to provide a serodiagnostic kit for the detection of antibodies to T. pallidum proteins by utilising a mixture of recombinant antigens of T. pallidum for the detection of T. pallidum antibodies present in human serum and/ or plasma.

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Another object of instant invention is to develop a serodiagnostic kit, which is solid phase immunoassay kit, for the detection of T. pallidum antibodies in human serum and/or plasma.
Still another object of the instant invention is to design a serodiagnostic kit for the detection of antibodies to T. pallidum proteins in human serum and/ or plasma, which is free of drawbacks and limitations of the prior art kits and methods.
Another object of the present invention is also to provide a method for preparation of a serodiagnostic kit for the detection of antibodies to T. pallidum proteins in human serum and/or plasma by using the mixture of recombinant antigens.
Further object of the present invention is a method for producing a serodiagnostic kit for the detection of antibodies to T. pallidum proteins in human serum and/or plasma comprising a mixture of recombinant antigens, conjugate and immunochemically acceptable reagents required for detection of antibodies to T. pallidum proteins.
Yet another object of the instant invention is to provide a serodiagnosis method for the detection of antibodies to T. pallidum proteins in human serum and/or plasma.
SUMMARY OF THE INVENTION
Pursuant to main object of the present invention, it provides a serodiagnostic kit, for the detection of antibodies to T. pallidum proteins, based on the principles of the solid phase immunoassay methods. The serodiagnostic kit comprises a microplate coated with treponemal antigens, conjugate and immunochemically acceptable reagents required for detecting antibodies to T. pallidum proteins in human serum and/or plasma. There is also provided a method for preparing said serodiagnostic kit by utilizing a mixture of recombinant antigens of T. pallidum. The method comprises coating on to a microplate the said antigens, preparing a conjugate for forming a stable complex of antibody-antigen, and preparing solutions of immunochemically acceptable reagents for detecting said antibodies. Further, it
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is also provided a serodiagnosis method for the detection of antibodies to T. pallidum proteins in human serum and/or plasma by employing the kit of the instant invention.
DETAILED DESCRIPTION OF THE INVENTION
The instant invention can be understood more readily by reference to following detailed description of specific embodiments. Although, the present invention has been described with reference to particular and specific details of certain embodiments, it is not intended that such details should be regarded as limitations to the scope of the invention. Unless otherwise depicted, all the technical and scientific terms used have the same meaning as commonly understood by the person skilled in the art to which this invention belongs.
The serodiagnostic kit of the present invention comprises a microplate plate containing multiplicity of microwells, which comprises a mixture of recombinant T. pallidum antigens, a conjugate containing recombinant T. pallidum antigens-HRPO, a syphilis positive control, a syphilis negative control, a colour reagent, a sample diluent, a stopping solution and a washing solution (20X).
In preferred embodiment of the instant invention, the said recombinant antigens to antibodies of T. pallidum proteins are coated on to the microwell of the microplate.
In another preferred embodiment, the mixture of said T. pallidum recombinant antigens to antibodies of T. pallidum proteins comprises pl5, pl7, p41 and p47 antigens.
In still another preferred embodiment, the microplate of the present invention comprises at least 24 microwells.
In still one more preferred embodiment, the microplate of the present invention comprises at least 96 microwells.

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In yet another embodiment of the present invention, the enzyme of the conjugate is linked to the recombinant antigens of T. pallidum for the detection of said antibodies in human serum and/or plasma.
In yet another preferred embodiment, the mixture of said T. pallidum recombinant antigens of the conjugate comprises p15-HRPO, p17-HRPO, p41-HRPO and p47-HRPO antigens.
In yet one more preferred embodiment, the said enzyme linked to the recombinant antigens is horse reddish peroxidase.
In another embodiment of the invention, the syphilis positive control is an inactivated anti-syphilis containing serum with thimerosal and gentamycin as preservatives.
In one more embodiment of the instant invention, the syphilis negative control is an inactivated normal human serum with thimerosal and gentamycin as preservatives.
In yet another embodiment of the invention, the colour reagent is 3,3',5, 5'-tetramethyl benzidine, dimethyl sulfoxide (DMSO) with H2O2, thimerosal and gentamycin.
In still another embodiment of the present invention, the sample diluent is a TRIS buffer with animal serum, Tween-20, thimerosal and gentamycin.

In an additional embodiment of the invention, the stopping solution is a concentrated phosphoric acid and deionized water.
In further embodiment of the invention, the washing solution (20X) is a TRIS buffer along with NaCl, Tween-20 and deionized water.

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In an important embodiment, the serodiagnostic kit of the present invention is stored at temperature between 2°C and 8 °C.
Pursuant to another object, it is provided herewith is a process for preparing the serodiagnostic kit of the present invention. The process of preparation comprises preparing a coated microplate, containing multiplicity of microwells, by coating a mixture of recombinant antigens to antibodies of T. pallidum proteins; preparing a conjugate by covalently linking an enzyme with recombinant antigens to antibodies of T. pallidum proteins; and preparing immunochemically acceptable reagent by preparing the solutions of said reagents, which are required for detecting said antibodies.
In a preferred embodiment of the said process, the solution of a mixture of the said recombinant antigens of T. pallidum are dissolved in bicarbonate buffer, which is used for coating on to the microwells of the plate.
In a further preferred embodiment, the coated mixture of recombinant antigens is blocked using a blocking solution, which comprises phosphate buffer, BSA and Trans-001.
In yet further embodiment of the coating process, the blocked recombinant antigens
are stabilised using a stabilising solution, which comprises PBA, Trans-002 and Tran-
003.
In another preferred embodiment, the mixture of said T. pallidum recombinant
antigens comprises p15, p17, p41 and p47 antigens.
In still another preferred embodiment of the coating process, the said microplate comprises at least 24 microwells.
In most preferred embodiment of the coating process, the said microplate comprises at least 96 microwells.

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In another preferred embodiment of the process of preparing the conjugate, the mixture of the said recombinant antigens of T. pallidum are covalently linked with an enzyme, such as, horse reddish peroxidase.
In yet another preferred embodiment, the mixture of the said recombinant antigens of the conjugate comprises p15-HRPO, p17-HRPO, p41-HRPO and p47-HRPO.
In further preferred embodiment of the present invention, the method for preparing a serodiagnostic kit comprises preparing a solution of an inactivated anti-syphilis containing human serum as a syphilis positive control.
In another further preferred embodiment, the method comprises preparing a solution of an inactivated normal human serum as a syphilis negative control.
In still further embodiment, the method of the instant invention comprises preparing a solution of colouring reagent comprising 3,3', 5,5'-tetramethyl benzidine, dimethyl sulfoxide (DMSO) and H2O2 with along thimerosal and gentamycin.
In yet further preferred embodiment, the method comprises preparing a solution of sample diluent, which contains a TRIS buffer, animal serum, Tween-20, thimerosal and gentamycin.
In one further feature of the method, it comprises preparing a solution of stopping reagent that contains a concentrated phosphoric acid and deionized water.
In last further embodiment, the method comprises preparing a solution of washing solution (20X), which contains a TRIS buffer, NaCl, Tween-20 and deionized water.
Pursuant to one more object of the present invention, there is provided a serodiagnosis method for detecting antibodies to T. pallidum in human serum and/or plasma. The method comprises contacting a biological sample obtained from a


patient with a microplate plate, which contains a mixture of recombinant T. pallidum antigens and a conjugate, which contains a mixture of recombinant T. pallidum antigens-HRPO; incubating the said microplate; after incubation, washing the incubated microplate with washing solution; adding a colour reagent, which contains a substrate for HRPO, thereby developing a blue colour in a positive control and anti- T. pallidum antibodies containing wells; immediately adding stopping solution to the positive control and anti- T. pallidum antibodies containing wells, thereby changing the blue colour to yellow; and spectrophotometrically reading the colour intensity for the detection of presence of anti- T. pallidum antibodies in the sample.
In preferred embodiment, the method for serodiagnosis of the present invention comprises contacting the human serum and/or plasma with a mixture of recombinant antigens to T. pallidum proteins, which contains the antigens, such as p15, p17, p41 and p47, and a conjugate, which contains antigens linked to an enzyme, such as p15-HRPO, p17-HRPO, p41-HRPO and p47-HRPO; incubating the microwells containing plate, thereby forming stable antigen-antibody complex; washing the incubated plate with the washing solution, thereby removing the unbound fraction of said reaction; adding the colour reagent to the microwells, thereby developing a blue colour in the positive control and anti- T. pallidum antibodies containing wells, adding the stopping solution to the said microwells, thereby changing the blue colour to yellow and spectrophotometrically reading the colour intensity for the detection of presence of anti- T. pallidum antibodies in the sample.
In another preferred embodiment of the method, 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 anti- T. pallidum antibodies, the foregoing reagents are employed in predetermined amounts.

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In yet another preferred embodiment of the present invention, the immunological components are bound to the microwells containing plate by covalent bonds or by adsorption. An advantage of using said microwells containing solid plate is that no centrifugation required for separation of solid and liquid phases.
In still another preferred embodiment of the present invention, the enzyme conjugate consists of immunological components that covalently linked to the enzyme molecule, which is achieved either by direct condensation or by using external bridging molecules. Thus, the enzyme coupling products is produced by employing the covalent bond, which is effected by using the reagents, for example, 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.
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 ANTIBODIES TO T. PALLIDUM PROTEINS
Adding positive control or anti-T. pallidum antibodies containing human serum and/or plasma to microwells of the plate, incubating the plate containing reaction mixture for 0.5 to 25 hours at temperature between 4°C and 37°C, thereby forming stable antigen-antibody complex with recombinant antigens of the T. pallidum that

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are coated onto the microwells and with recombinant antigens of the T. pallidum that are linked to the HRPO of the conjugate; washing the microplate using washing solution, thereby removing unbound fraction of the conjugate; adding the colour reagent, which contains the substrate of HRPO to the microwells, thereby developing a blue colour in the wells containing positive control and anti-T. pallidum antibodies containing wells; 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 the detection of antibodies to T. pallidum proteins.
EXAMPLE: 2 - TEST PROCEDURE FOR DETECTION OF ANTIBODIES
(1) Bring all the reagents and test specimens at room temperature before use.
(2) Except blank (100 ml sample diluent + 50 ml conjugate), add 50 ml of sample diluent to each well. In each run, there will be three negative controls and one positive control. Add 50 ml of control and test specimens to the respective wells. Add 50ul of conjugate to the respective well. Mix properly the overall mixture. Cover the plate with black cover and incubate 15 minutes at room 37°C.
(3) Wash the plate as per microplate washing procedure known to person skilled in the art.
(4) Add 50ml of colour reagent to each well. Cover the plate with black cover and incubate for 15 minutes in dark at 20 - 30°C.
(5) Add 100 ml of stopping buffer to each well.
(6) Read absorbance at 450 nm and deduct the blank absorbance from the control and test wells.

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EXAMPLE: 3 - CALCULATION FOR CUT-OFF VALUE DETERMINATION
Blank value: Absorbance of blank values should be less than 0.1.
Positive Control: Absorbance of individual positive control should be grater than 0.5.
Negative Control: Absorbance of individual negative control should be less than
0.05.
NCx: Average value of negative controls.
Calculation of NCx:
For example:
NC Absorbance
1 0.04
2 0.044
3 0.042
NCx: (0.04+0.044+0.042)/3=0.042 Cut-off value formula: 0.05+NCx Cut-off vale: 0.05+0.042=0.092
INTERPRETATION OF RESULTS:
Non-reactive: If the absorbance of the test serum and/or plasma is less than the cutoff value, then the sample 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.
Repeatedly reactive specimens found by using the serodiagnostic kit of the present invention, these must be confirmed with other test such as Dark Filed Microscopy for confirmation.


Limitation of the test performed by using the serodiagnostic kit of the present invention is that the non-reactive result does not preclude the possibility of the T. pallidum infection.

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WE CLAIM:
1. A serodiagnostic kit for detecting antibodies to Treponema pallidum proteins in human serum and/or plasma comprises a microplate plate containing multiplicity of microwells, which comprises a mixture of recombinant antigens of T. pallidum; a conjugate, which comprises a mixture of recombinant antigens of T. pallidum linked to an enzyme; and immunochemically acceptable reagents for detecting T. pallidum proteins in human serum and/or plasma, wherein the recombinant antigens of microplate are coated onto the microwells, and the recombinant antigens of conjugate are linked with enzyme.
2. The serodiagnostic kit of claim 1, wherein the mixture of recombinant antigens coated onto the microwells for detection of anti- T. pallidum antibodies comprises p15, p17, p41 and p47.
3. The serodiagnostic kit of claim 1, wherein the mixture of recombinant antigens of conjugate linked with enzyme for detection of anti- T. pallidum antibodies comprises p15-HRPO, p17-HRPO, p41-HRPO and p47-HRPO.
4. The serodiagnostic kit of claim 1, wherein the reagents used for detection of anti- T. pallidum antibodies comprise syphilis positive control, syphilis negative control, colour reagent, sample diluent, stopping solution and washing solution.
5. The serodiagnostic kit of claim 1, wherein the syphilis positive control comprises an inactivated anti-syphilis containing serum with thimerosal and gentamycin.

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6. The serodiagnostic kit of claim 1, wherein the syphilis negative control comprises an inactivated normal human serum with thimerosal and gentamycin.
7. The serodiagnostic kit of claim 1, wherein the colour reagent comprises 3,3',5, 5'-tetramethyl benzidine, dimethyl sulfoxide (DMSO) with H2O2, thimerosal and gentamycin.
8. The serodiagnostic kit of claim 1, wherein the sample diluent comprises a TRIS buffer with animal serum, Tween-20, thimerosal and gentamycin.
9. A serodiagnostic kit of claim 1, wherein the stopping solution comprises a concentrated phosphoric acid and deionized water.
10. A serodiagnostic kit of claim 1, wherein the washing solution comprises a TRIS buffer along with NaCl, Tween-20 and deionized water.
11. A method for preparing a serodiagnosis kit for detection of anti- T. pallidum antibodies in human serum and/or plasma according to claims 1 to 10, comprises preparing a coated microplate, containing multiplicity of microwells, by coating a mixture of recombinant antigens to antibodies of T. pallidum proteins, wherein said mixture of antigens comprises p15, p17, p41 and p47 antigens; preparing a conjugate by covalently linking an enzyme with a mixture of recombinant antigens to antibodies of T. pallidum proteins, wherein said mixture of antigens linked with enzyme comprises p15-HRPO, p17-HRPO, p41-HRPO and p47-HRPO antigens; and preparing immunochemically acceptable reagents by preparing the solutions of said reagents.

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12. A serodiagnostic method for detecting antibodies to T. pallidum in human serum and/or plasma obtained by using a serodiagnostic kit according to claims 1 to 11 comprises contacting a biological sample obtained from a patient with a microplate plate, which comprises a mixture of recombinant T. pallidum antigens and a conjugate, which comprise a mixture of recombinant T. pallidum antigens-HRPO and incubating the said microplate; after incubation, washing the incubated microplate with washing solution; adding a colour reagent, which comprises a substrate for HRPO, thereby developing a blue colour in a positive control and anti- T. pallidum antibodies containing wells; immediately adding stopping solution to the positive control and anti-T. pallidum antibodies containing wells, thereby changing the blue colour to yellow; and spectrophotometrically reading the colour intensity for the detection of anti- T. pallidum antibodies in the sample.
Dated this 25th day of May, 2007

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