FORM 2
THE PATENT ACT 197 0 (39 of 1970)
The Patents Rules, 2003 COMPLETE SPECIFICATION See Section 10, and rule 13)
1. TITLE OF INVENTION
AN IMMUNOASSAY KIT FOR CD4/CD8 T-CELL RATIO AND METHOD FOR MANUFACTURING THE SAME;

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 CD4/CD8 T-cell ratio and method for manufacturing the same
Filed of the invention
The present invention relates to a solid phase immunoassay kit and method for preparing the kit. The invention further relates to an immunoassay method using the kit for the determination of surface antigens characteristic of populations or subpopulations of T cells. Accordingly, the kit and immunoassay method are intended for the determination of the T cells themselves via the determination of their surface antigens (CD4, CD8 and CD4/CD8 ratio). The immunoassay kit and method of determination of surface antigens of characteristic of CD4+ T-cells, CD8+ T-cells and their ratio of the present invention are, therefore, useful in diagnosing HIV infections.
Background of the invention
More than 40 million people in developing world are suffering form HIV infections. Enormous global efforts are undergoing to provide effective antiretroviral treatment. With dramatic developments in the field of antiretroviral therapy, an estimation of human immunodeficiency viral load and CD4+ T-cells counts and CD4/CD8 ratio is required for decision of treatment and analysis of response. The standard markers, CD4, CD8 cell count and viral load, used to determine the time of treatment or therapy, as it is expensive and mostly unavailable in resource-poor settings. Subsequent to HIV infection and after certain incubation, it was generally observed that CD4+ T-cell count decreases and CD8+ T-cell count increases. Therefore, the present invention propose a solid phase immunoassay test kit by utilizing a mixture of Pan T-cell monoclonal antibody (Pan T-cell MAb) for quantitative measurement of CD4/CD8 ratio in human blood.

Prior art of the invention
Knowledge of the antigens or markers on the cell surface has made enormous advances with the development of lymphocyte hybridisation and the discovery of monoclonal antibodies (MAb) by Koehler and Milstein (Nature, 1975, 256, 495-497). In particular, MAb have made it possible to reveal and analyse membrane antigens or surface markers of T-cells. These markers or antigens can be of different kinds: proteins, glycoproteins or glycolipids. A particularly important field of application is the study of the cell lines of hemopoiesis, more particularly lymphocytes.
Thus, the MAb have made it possible to specify the respective surface characteristics of T lymphocytes. The corresponding markers, by themselves or in combination, identify stages of differentiation and functional specialization of the lymphocytes. By international convention, the surface markers or antigens of human leukocytes have been classified in differentiation groups or differentiation classes (CD) defined by the IUIS-WHO subcommittee, 1984, and described in Bulletin of the World Health Organization, 1984, 62(5), 813-815.
The identification of these markers or antigens that has been made possible by MAb has provided access to their structure and biological functions. For example, the molecules of the CD4 and CD8 markers participate in leukocyte adhesion functions and are present on the surface of T lymphocytes with an auxiliary and inductive function (CD4 marker) or, respectively, with a cytotoxic and suppressive function (CD8 marker).
With above said knowledge, it has been made possible to use these markers or antigens, by virtue of the antibodies, which recognize them, for diagnosing and following up of dysfunction of the immune system (acquired immune deficiencies, AIDS). Therefore, MAb have become irreplaceable tools of clinical biology applied to cell analyses.
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Cell counting methods use the marking of surface antigens, but such methods are often lengthy, laborious and difficult to carry out and results obtained are sometimes random. The known methods used for measuring the normal or modified expression of antigens on the surface of the cell are separated into two groups. In the first group, the antigens are measured with the aid of complex and specialized laboratory equipment based on flow cytometry or quantitative microscopy techniques. These methods for the evaluation of cell antigens are based on the measurement of signals provided by anti-cell antibodies coupled directly or indirectly to a reagent labelled with fluorescent substances or with enzymes. The use of these fluorescent or enzyme reagents in association with appropriate washing steps then leads to the appearance of fluorescence or colorations which are strictly limited to the cell membranes and do not diffuse into the surrounding medium. Common use of these methods in the laboratory is still restricted by the need for specialized and expensive equipment. Moreover, the analysis and interpretation of the immuno-labelling of cells demand the competence of a specialist in cytology.
A second group of methods for the measurement of antigens is based on the quantitative evaluation of the markers or antigens of the overall cell population. These methods make it possible to measure antigens either by direct labelling or by indirect labelling, which are being carried out in two to four steps. The reagent employed in the labelling step carries a probe, which is either of isotopic character or enzymes for a determination of the enzyme immunoassay type. The methods are rather inconvenient, laborious and risky to apply because of the need to ash and centrifuge the cell material many times. It is sometimes necessary to take a sample of the coloured medium resulting from the enzyme reaction in order to carry out the final spectrophotometric measurement. Finally, chemical fixation of the cells causes the irreversible destruction of certain antigens, which are particularly sensitive to the customary chemical binding agents such as glutaraldehyde or methanol.

It is well established in the literature that an antigen carrying several epitopes, can be determined by fixing this antigen via one of its epitopes using an antibody immobilized on a solid support, and by binding, to another epitope of the antigen, another antibody carrying an enzyme label enabling the determination to be carried out. This kind of sandwich technique is well known in the art but no one has described use of this technique in determination of CD4+ T-cells or CD8+ T-cells.
The various antigens described in the prior art fall solely protein molecules soluble in water and physiological liquids, such as tumoral markers, enzymes or hormones in the bloodstream. However, the T-cell is not a molecule and differs there from at least by being considerably larger and by the fact that it is not soluble in physiological media. Thus, the sandwich technique has so far never been used in case of T-cells as a whole.
Further, immobilisation as function of immuno-capturing of cells on a solid support is also described in prior art, but the object of which is to remove undesirable cells from samples of bone marrow intended for transplantations. The capturing of the cells is effected on floating microbeads and requires that the antibody used be bound to the solid support by a complex macromolecular structure, called a network-relay, which is capable of ensuring a preferential orientation of the antibody relative to that of the corresponding cell antigen. However, there is no indication of an appUcation of the technique to the quantitative determination of an antigen.
It is also described in the prior art that the immuno-capturing technique could be used for an analytical application. In the said application, the object is to identify the tissue groups to which the examined cells belong, which is generally being known HLA typing. The cells are captured by means of antibodies arranged according to a particular geometry on very specialized supports, for example microscope cover glasses. The results are obtained simply by visual observation of the support and produce all or nothing responses. Thus the cell immuno-capturing systems described above do not lead to analytical applications permitting the quantitative determination of an antigen expressed at the membrane of cells.

Object of the present invention
Therefore, the kit and method of determination of present invention has considerable advantages as compared to all the techniques known so far and used in the prior art, since it permits the quantitative measurement of CD4 and CD8 antigens for determination of CD4/CD8 ratio in a single step. The kit and method of determination according to the present invention has the characteristics of very high specificity which are inherent in the double immunological recognition systems involving tow different monoclonal antibodies specific for two different antigens carried by the same cell. This kit and method of determination using this kit is simple, rapid and reproducible. It is totally suitable for the analysis of a large number of samples, which enables it to be used for diagnostic purposes in clinical biology laboratories.
Description of the invention
Thus, the present invention relates to a solid phase immunoassay kit for the determination of at least one surface antigen (CD4 and/or CD8) of a T-cell, which comprises a solid support to which one or more monoclonal antibodies (MAb) are fixed by covalent bonding or physical adsorption, the said monoclonal antibodies (MAb) being directed against surface antigens (CD4 and/or CD8) of the CD4+ T-cells and /or CD8+ T-cells carrying the antigen to be determined; at least one solution comprising a monoclonal antibody (MAb) specific for the said antigen (CD4) characteristic of the CD4+ T-cells carrying the antigen to be determined that is labelled with an enzyme probe; at least one solution comprising a monoclonal antibody (MAb) specific for the said antigen (CD8) characteristic of the CD8+ T-cells carrying the antigen to be determined that is labelled with an enzyme probe; and a developer for the enzyme containing the substrate for said enzyme and one or more reagents necessary for developing the activity of the enzyme.

In a preferred embodiment of the present invention, the reference to CD4+ T-cells and CD8+ T-cells used in the present specification includes blood cells, preferably T4 lymphocytes and T8 lymphocytes. These cells have not undergone any physical or chemical intervention and they are used in a state of complete physiological integrity.
In another preferred embodiment of the present invention, the solid support is any device suitable for the handling of cell suspensions, and preferably microtitre plates made of polyethylene, polystyrene, polyvinyl chloride or nitrocellulose containing micro-wells. The monoclonal antibodies intended for immobilization of the CD4+ T-cells and/or CD8+ T-cells can be fixed to the solid supports either by covalent chemical bonding or by physical adsorption.
In further preferred embodiment of the present invention, the monoclonal antibody or antibodies fixed to the solid support must permit the immuno- capture of the CD4+ T-cells and CD8+ T-cells, which includes the cell population or populations carrying the antigens to be determined. The monoclonal antibody labelled with an enzyme probe is coupled to an enzyme which, when associated with the use of appropriate reagents, permits quantitative measurement of this monoclonal antibody.
In a preferred embodiment of the present invention, the monoclonal antibody or antibodies fixed to the solid support, which permit the immuno-capturing of the CD4+ T-cells and CD8+ T-cells population of type that specifically carrying CD4 and CD8 antigens on its membrane. The said monoclonal antibodies are preferably Pan T-cell monoclonal antibodies (Pan-T-cell-MAb)
In a more preferred embodiment of the present invention, the monoclonal antibody or antibodies fixed to the solid support, which permit the immuno-capturing of the CD4+ T-cells and CD8+ T-cells population of type that specifically carrying CD4 and CD8 antigens on its membrane. The said monoclonal antibody is preferably anti-CD45 monoclonal antibody (Anti-CD45MAb).

In another more preferred embodiment of the present invention, the monoclonal antibody or antibodies fixed to the solid support, which permit the immuno-capturing of the CD4+ T-cells and CD8+ T-cells population of type that specifically carrying CD4 and CD8 antigens on its membrane. The said monoclonal antibody is preferably anti-CD3 monoclonal antibodies (Anti-CD3MAb).
In still another more preferred embodiment of the present invention, The monoclonal antibody or antibodies fixed to the solid support as a conjugated antibody or antibodies, which permit the immuno-capturing of the CD4+ T-cells and CD8+ T-cells population of type that specifically carrying CD4 and CD8 antigens on its membrane. The said monoclonal antibodies are labelled with an enzyme probe, preferably peroxidase.
In still most preferred embodiment of the present invention, the conjugated monoclonal antibody or antibodies fixed to the solid support and labelled with an enzyme probe, such as peroxidase is most preferably Mouse-Anti-Human-CD4-HRPO.
In still another most preferred embodiment of the present invention, the conjugated monoclonal antibody or antibodies fixed to the solid support and labelled with an enzyme probe, such as peroxidase is most preferably Mouse-Anti-Human-CD8-HRPO.
The substrate and the reagents chosen are so that the final product of the reaction or reaction sequence caused by the enzyme, involving these substances is a coloured substance which diffuses into the liquid medium surrounding the cells and which is the object of the final spectrophotometric measurement.
In another embodiment of the present invention, the solid phase immunoassay kit additionally contains a buffer solution intended for washing the solid support after immobilization and labelling of the cells with the antibody or antibodies carrying probe; standard samples necessary for standardization and quality control of the measurement; a sample diluent required for dilution of sample at the time of
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measuring CD4+T-cells and CD8+ T-cell count, and their ratio a colour reagent for development of colour product of the enzyme-substrate reaction; and stopping solution for controlling and/or stopping the reaction.
In a preferred aspect of the invention, the buffer solution used in the present invention is selected form the phosphate group (phosphate buffered saline, PBS, of pH 7.4) mixed with surfactant, stabilizer, sodium chloride and preservative.
In more preferred aspect of the invention, the stabilizer used in the said buffer solution is a protein stabilizer preferably bovine serum albumin. The surfactant used in the said buffer solution may be selected from the non-ionic, anionic and zwitterionic surfactant; preferably the surfactant used is non-ionic surfactant. The preservative used in the said buffer solution is selected from thimerosal and gentamycin.
In another preferred aspect of the invention, the enzyme linked conjugate comprises Mouse-Anti-Human CD4-HRPO with thimerosal and gentamycin.
In one aspect of the invention, the CD4 standards (3a, 3b, 3f) are lyophilised
human blood along with thimerosal and gentamycin.
In another aspect of the invention, the CD8 standards (4a, 4b, 4f) are lyophilised
human blood along with thimerosal and gentamycin.
In another preferred aspect of the invention, the sample diluent is a TRIS buffer along animal serum, Tween-20, thimerosal and gentamycin.
In further embodiment of the invention, the colour reagent is 3,3', 5,5'-tetra methyl benzidine dimethyl sulfoxide along with H2O2, thimerosal and gentamycin.
In still another further embodiment of the invention, the stopping solution is a concentrated phosphoric acid and deionized water.

In a preferred aspect of the invention, the washing solution concentrate is a TRIS buffer along with NaCl and Tween-20 in deionized water.
According to another object of the present invention, a method for manufacturing a solid phase immunoassay kit for the determination of CD4/CD8 ratio in human blood comprises: immobilizing on to a solid support, by covalent chemical bonding or physical adsorption, a mixture of purified Pan T-cell monoclonal antibodies (Pan-T-cell-MAb), in bicarbonate buffer, blocking using blocking solution, containing phosphate buffer and stabilizing using stabilizing solution; providing at least one coupling product as an enzyme linked conjugate obtained by covalently binding mouse-anti-human-CD4-HRPO; also providing at least one coupling product as an enzyme linked conjugate obtained by covalently binding mouse-anti-human-CD8-HRPO and providing solutions of CD4 and CD8 standard solutions, a sample diluent solution, a colouring reagent, a stopping solution and a washing solution.
In preferred embodiment, the immobilized Pan T-cell monoclonal antibodies are anti-CD45-MAb and anti-CD3-MAb for immuno-capturing of CD4+ T-cells and CD8+ T-cells present in the human blood.
In another preferred embodiment, the enzyme linked conjugate is mouse-anti-human CD4-HRPO for immuno-capturing of CD4+ T-cells present in the human blood.
In still another preferred embodiment, the enzyme linked conjugate is mouse-anti-human CD8-HRPO for immuno-capturing of CD8+ T-cells present in the human blood.
In still preferred embodiment of the invention, the method comprises preparing the
solutions of CD4 and CD8 standards, such as, 3a, 3b, 3f, in thimerosal and
gentamycin.

In still another preferred embodiment, preparing the solution of colouring reagent comprises preparing a solution of 3,3', 5,5'-tetra methyl benzidine dimethyl sulfoxide in H2O2, thimerosal and gentamycin.
In one distinct embodiment, the method comprises preparing a solution of the sample diluent containing a TRIS buffer, animal serum, Tween-20, thimerosal and gentamycin.
In a one separate embodiment, the method comprises preparing a stopping solution containing a concentrated phosphoric acid with deionized water.
In a different embodiment, the method comprises preparing a washing solution concentrate containing a TRIS buffer, NaCl, Tween-20 in deionized water.
In accordance with still another object of the present invention also provided is an in vitro solid phase immunoassay method for measurement of CD4/CD8 ration of surface antigens of T4 and T8 lymphocytes, which comprises a single step for the specific immobilization of CD4 and CD8 antigens of CD4+ T-cells and CD8+ T-cells as a function of immuno-capturing of said antigen present in the human blood. The method comprises providing on to the solid support mixture of one or more Pan T-cell monoclonal antibodies (Pan-T-cell-MAb) fixed to the said support, which are capable of recognizing an antigen present on the surface of targeted T4 and T8 lymphocytes; other than the antigen to be determined by immobilizing T4 lymphocytes, a monoclonal antibody specific for this antigen to be determined wherein said monoclonal antibody carrying an enzyme probe; other than the antigen to be determined by immobilizing T8 lymphocytes, a monoclonal antibody specific for this antigen to be determined wherein said monoclonal antibody carrying an enzyme probe; contacting the human blood sample to be examined for an incubation period to allow the simultaneous immobilization and labelling to take place; the washing of the solid support to remove the non-immobilized cells and the excess of monoclonal antibody carrying the enzyme probe; and actually determining the

antigen to be detected in the labelled cell population by counting after treatment of the medium with the substrate for the enzyme by photometric measurement.
The immunoassay kit and method according to the present invention are preferably useful for the determination of number of CD4 and CD8 antigens present on to membrane of leukocytes and their ratio in human blood sample and more particularly the ratio of T4 lymphocytes and T8 lymphocytes (CD4+ T-cell/CD8+ T-cell).
The immunoassay kit and the method according to the invention make it possible to measure signals which depend both on the number of T-cells present in the cell population examined and on the density of the antigen measured on the surface of these cells. Measurement of these signals permits quantitative evaluation of the total number of molecules of this antigen, which are carried by the cell population.
More preferably, in the case of the ratio T4 and T8 lymphocytes, in the majority of situations in healthy subjects, the mean value of the ratio antigen (CD4/CD8) does not vary substantially between samples for one and the same cell population, so there is a good correlation between the cyt'ological count of the cells carrying the antigen in question and the signal measured according to the invention, which is proportional to the total number of antigen molecules present in the sample examined. However, in some pathological states, the density of the surface antigens (CD4 and CD8) may vary for one and the same cell population without a notable variation in the number or proportion of the positive cells.
In an alternate embodiment of the present invention, the immunoassay kit and the method can be used for the determination, on a single plate, of a series of surface antigens characteristic of various subpopulations making up the cell population examined.

In one of the preferred embodiment, it is possible on the one hand to take ready-to-use microtitre plates to which one or more monoclonal antibodies capable of retaining all cell populations to be examined have been fixed, and on the other hand to have a series of monoclonal antibodies coupled to an appropriate probe and each specific for an antigen characteristic of one of the subpopulations to be evaluated. Thus, in a single manipulation and on one and the same support, it is possible to carry out the quantitative determination of all the antigens necessary for characterization of the chosen subpopulations.
In still another preferred embodiment of the present invention, the human T4 lymphocytes characterized by the presence of the CD4 marker will be called positive T4 lymphocytes, and the lymphocytes characterized by the presence of the CD8 marker will be called positive T8 lymphocytes.
In the most preferred embodiment of the present invention, the measurement of the numerical ratio T4/T8 is of great diagnostic interest in clinical biology. In fact, modifications of the T4/T8 ratio appear in various complaints of the immune system, such as viral infections and, in particular, HIV complaints (AIDS virus).
In another aspect of the present invention, the immunoassay kit and assay process according to the invention can be used for the determination of antigens universally characteristic of the T lymphocyte population or antigens characteristic of the T4 and T8 lymphocytes. In this case, the T lymphocytes of the sample examined are specifically immobilized on a solid support and, simultaneously, the surface antigens of the T4 and T8 lymphocytes are labelled directly with an anti-CD4 and anti-CD8 monoclonal antibody, respectively, carrying an enzyme probe.
In specific embodiment, the immobilization of the T lymphocytes of human blood is preferably carried out using one or more monoclonal antibodies which are capable, by themselves or in combination, of recognizing all the CD4+ T cells and/or CD8+ T-cells of the sample, this being the case of the anti-common leukocyte and called as anti-CD45 monoclonal antibodies or antibodies which recognize the whole of the


CD4+ T cell population called as "pan-T" monoclonal antibodies (Pan-T-MAb), such as the anti-CD3.
In more specific embodiment, the immunoassay method of the invention can advantageously be used for the determination of antigens characteristic of the population of T4 and T8 lymphocytes on several parts of the same solid support. Measurement of the signals by photometric measurement enables the numerical ratio CD4/CD8 to be calculated easily and directly from the standard curve.
When an enzyme probe is used on the monoclonal antibody, the appearance of a coloured product is brought about by adding, to the solid support to which the cell population carrying the antigen to be determined has been fixed, a solution containing the substrate for the enzyme such that the reaction product which is finally obtained is a coloured product soluble in the medium. The light signal coming from the samples treated in this way is then measured with the spectrophotometer.
When the monoclonal antibodies are preferably coupled to peroxidase. The reagents used to develop the peroxidase conjugated with the monoclonal antibodies contain hydrogen peroxide, which is the substrate for the enzyme, and a colour reagent to give coloured reaction product that is soluble in the medium is 3,3',5,5'-tetra methyl benzidine dimethyl sulfoxide.
In the most preferred embodiment, the immunoassay kit according to the present invention for the determination of antigens characteristic of the T4 and T8 lymphocytes comprises a microtitre plate wherein one or more monoclonal antibodies directed against T4 and T8 lymphocytes have been fixed in micro-wells; a solution containing at least one peroxidase-labelled monoclonal antibody directed against CD4 antigen; a solution containing at least one peroxidase-labelled monoclonal antibody directed against CD8 antigen; a solution containing hydrogen peroxide, which is the substrate for the enzyme, in an appropriate buffer; and a

solution containing the colour reagent used to develop the expression of the enzyme's activity.
The results of the determination of antigens according to the invention can be expressed in any form appropriate. More particularly, these results can be expressed either as the total number of CD4 antigen present in a given volume of the sample, for example per microliter of blood, or as the ratio of the number of CD4 antigens to the CD8 antigens.
The number of antigen in the sample will preferably be determined using a standard curve consisting of CD4 and CD8 lyophilised human blood cells carrying the antigen to be determined, which will have been calibrated by a known reference method.
These standards are then treated in exactly the same way as the samples to be examined. The resulting signals are used to build up a standard curve against which the signals measured with the samples to be examined are compared.
To determine the ratio of the numbers of CD4 and CD8 antigens in the sample to be examined, it is possible to use the standard system described above and finally to calculate the desired ratio.
The immunoassay kit and method according to the invention is simple, rapid and reproducible. Its use is totally suitable for the analysis of a large number of samples. The kit and process according to the invention makes it possible to determine the ratio of surface antigens, such as CD4 and CD8 over a wide range of cell concentrations.
The above said preferred embodiments of the present invention are now described in detailed by way of examples, which are no way constructed to limit the scope of the invention.

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Example 1: Preparation of the plate:
The microtitre plate used contains 96 micro-wells; each micro-well receives around 200 ul of a solution containing the purified anti-CD45Mab and anti-CD3Mab to immobilize the T4 and T8 lymphocytes as function of immuno-capturing. These antibodies are used at a concentration of 10 ng/ml in a phosphate buffered saline (PBS) of pH 7.4. The adsorption of the monoclonal antibody is occurred at 4°C for 12 hours and excess antibodies are removed by turning the plate over.
Solutions of 0.1% gelatine and 0.5% BSA in PBS are prepared. 200 ul of these solutions are introduced into each micro-well so as to saturate the wells with protein, which takes 1 hour at 37°C; the plates are washed three times with PBS. The plates prepared are lyophilised and stored at 4 °C in a sealed plastic sachet.
Example 2: Preparation of the solution of monoclonal antibody-peroxidase conjugate:
The coupling of the antibody to the peroxidase is performed as per the known method (M. B. Wilson and P. K. Nakane in Immunofluorescence and Related Techniques, edited by W. Knapp, K. Kolubar and G. Wick, 1978, Elsevier/North Holland, Amsterdam, p. 215-224). The resulting product is coupled to 2 mg of anti-CD4 IgG contained in 500 JJ.1 of carbonate buffer. After treatment with sodium borohydride and dialysis against PBS, the IgG-POD conjugate is sterilized by filtration on a 0.22 urn membrane and kept under sterile conditions at a concentration of 0.5 mg of IgG per ml, at 4°C, in glass tubes. The reagent is stable for at least 1 year. Similar methodology has been used to prepare the anti-CD8 IgG-POD conjugate.

Example 3: Measurement of CD4+ and/or CD8+ T-Cell or ratio thereof (results):
The numbers of T4 and T8 lymphocytes present in the human blood are determined by reference to calibration curves established by using CD4 and CD8 standard solutions. The results are being expressed as molar concentrations ratio of the CD4 and CD8 antigens in the sample examined using the described below.
In the first step of assay of immunoassay method, whole blood to be added on sample diluent. After incubation, well will be washed. In the second step, anti-CD4-MAb-HRPO conjugate and anti-CD8-MAb-HRPO conjugate will be added to each required wells. After incubation at room temperature, the well will be washed. In the third step, colour reagent will be added. After incubation at room temperature, stopping solution will be added. Reading will be taken at 450 nm with reference wavelength around 620 nm. The number of CD4 antigens will be determined from the standard curve. The ratio of CD4/CD8 will be determined from the standard curve.
Example 4: Stepwise procedure for measurement of T-Cell or ratio thereof:
(a) Bring all the reagents and test specimens at room temperature before use. (b) Except blank, add 200 ul of sample diluent to each well, in each run, there will be one blank and twelve standards, add 50 yd of control and test specimens to the respective wells. Cover the plates with black cover and incubate for 15 minutes at 20 - 30°C. (c) Wash the plate as per microplate washing procedure known to person skilled in the art. (d) Add 50ul of conjugate to each well (except blank well), cover the plate with black cover and incubate for 15 minutes at 20-30°C. (e) Repeat step (c). (f) Add 50\xl of colour reagent to each well and cover the plate with black cover and incubate for 10 minutes in dark at 20-30°C. (g) Add 100ul of stopping buffer to each well, (h) Finally read absorbance at 450 nm (using 620/630/650 nm as reference wavelengths) and deduct the blank absorbance and test wells.

Validation: Blank value: Absorbance of blank value should be less than 0.1 and absorbance of standard should be 0.2 - 0.3.
Standard curve: By using absorbance values of six sets of absorbance values of
twelve standards, ie. 3a/4a, 3b/4b, 3f/4f, plot a curve. Based on ratio of
absorbance values of standard sample, determine CD4/CD8 ratio per test specimen.

WE CLAIM :
1. A solid phase immunoassay kit for determination of at least one surface antigen (CD4 and/or CD8) comprises a solid support to which one or more monoclonal antibodies (MAb) are fixed by covalent bonding or physical adsorption, which are directed against surface antigens (CD4 and CD8) characteristic of the CD4+ T-cells and CD8+ T-cells, respectively, carrying the antigen to be determined; at least one solution comprising a monoclonal antibody (MAb) specific for one antigen (CD4) characteristic of the CD4+ T-cells carrying the antigen to be determined, which is labelled with an enzyme probe; at least one solution comprising a monoclonal antibody (MAb) specific for one antigen (CD8) characteristic of the CD8+ T-cells carrying the antigen to be determined, which is labelled with an enzyme probe; and a developer for the enzyme containing the substrate for said enzyme and one or more reagents necessary for developing the activity of the enzyme.
2. The kit as claimed in claim 1, wherein the CD4+ T-cells and CD8+ T-cells of the human blood sample includes T4 lymphocytes and T8 lymphocytes, respectively, which are not undergone any physical or chemical intervention.
3. The kit as claimed in claim 1, wherein the solid support is any device suitable for the handling of human blood cell suspensions, which is preferably a microtitre plate made of polyethylene, polystyrene, polyvinyl chloride or nitrocellulose containing micro-wells..
4. The kit as claimed in claim 1, wherein the monoclonal antibodies are fixed to the microtitre plate for immobilisation as a function of immuno-capturing of the CD4+ T-cells and CD8+ T-cells.

5. The kit as claimed in claims 1 and 4, wherein the monoclonal antibodies are fixed to the microtitre plate for immobilisation of the CD4+ T-cells and/ or CD8+ T-cells populations of type that specifically carrying CD4 and/or CD8 antigens on its membrane.
6. The kit as claimed in claim 5, wherein the monoclonal antibodies fixed to the microtitre plate for immobilisation of the CD4+ T-cells and/or CD8+ T-cells populations of type that specifically carrying CD4 and/or CD8 antigens on its membrane are Pan T-cell monoclonal antibodies (Pan-T-cell-MAb).
7. The kit as claimed in claim 6, wherein at least one said monoclonal antibody is anti-CD45 monoclonal antibody (anti-CD45MAb).
8. The kit as claimed in claim 6, wherein at least one said monoclonal antibody is anti-CD3 monoclonal antibodies (Anti-CD3MAb).
9. The kit as claimed in claim 1, wherein at least one monoclonal antibody is labelled with an enzyme probe as a conjugate for quantitative measurement of CD4+ T-cells and CD8+ T-cells.
10. The kit as claimed in claim 9, wherein the monoclonal antibodies fixed to the microtitre plate for immuno-capturing of the CD4+ T-cells population of type that specifically carrying CD4 antigens on its membrane are labelled with enzyme peroxidase.
11. The kit as claimed in claim 9, wherein the monoclonal antibodies fixed to the microtitre plate for immuno-capturing of the CD8+ T-cells population of type that specifically carrying CD8 antigens on its membrane are labelled with enzyme peroxidase.

12. The kit as claimed in claim 10, wherein the conjugated monoclonal antibodies fixed to the solid support for immobilisation of the CD4+ T-cells are mouse-anti-human-CD4-HRPO.
13. The kit as claimed in claim 11, wherein the conjugated monoclonal antibodies fixed to the solid support for immobilisation of the CD8+ T-cells are mouse-anti-human-CD8-HRPO.
14. The kit as claimed in claim 1, which contains a buffer solution for washing the carrying enzyme probe, six standard samples for each of CD4 and CD8 antigens for standardization and quality control of the measurement, a sample diluent for dilution of sample, a colour reagent for development of colour product and a stopping solution for controlling and/ or stopping the reaction.
15. The kit as claimed in claim 14, wherein the buffer solution is a phosphate chloride and preservative.
16. The kit as claimed in claim 14, wherein the CD4 standards (3a, 3b, 3f) are
lyophilised human blood cells with thimerosal and gentamycin.
17. The kit as claimed in claim 14, wherein the CD8 standards (4a, 4b, 4f) are
lyophilised human blood cells with thimerosal and gentamycin.
18. The kit as claimed in claim 14, wherein the sample diluent is a TRIS buffer with animal serum, Tween-20, thimerosal and gentamycin.
19. The kit as claimed in claim 14, wherein the colour reagent is 3,3', 5,5'-tetra methyl benzidine dimethyl sulfoxide with H2O2, thimerosal and gentamycin.

20. The kit as claimed in claim 14, wherein the stopping solution is a concentrated phosphoric acid in deionized water.
21. The kit as claimed in claim 14, wherein the washing solution is a TRIS buffer with NaCl and Tween-20 in deionized water.
22. A method for manufacturing the sohd phase immunoassay kit for determination of CD4/CD8 ratio in human blood sample of claims 1 to 22 comprises: immobilizing on to a solid support such as herein defined by covalent bonding or physical adsorption, a mixture of purified Pan T-cell monoclonal antibodies (Pan-T-cell-MAb), such as Anti-CD45MAb and Anti-CD3MAb, against CD4 and CD8 antigens in bicarbonate buffer; blocking said antibodies with blocking solution of phosphate buffer saline, and stabilizing with stabilizing solution; providing an enzyme linked conjugated monoclonal antibody obtained by covalently binding of mouse-anti-human-CD4-HRPO; providing an enzyme linked conjugated monoclonal antibody obtained by covalently binding of mouse-anti-human-CD8-HRPO; providing six standard solutions of each of CD4 and CD8, respectively; providing solutions of sample a diluent, a colouring reagent, a stopping solution and a washing solution separately.

23. A solid phase immunoassay for determination of CD4/CD8 antigens ratio in human blood sample, which is characteristics of T4/T8 ratio using the immunoassay kit of claims 1 to 22 in a single step by immobilizing CD4 and CD8 antigens of CD4+ T-cells and CD8+ T-cells, the method comprises the following step steps: (a) providing a microtitre plate containing (al) a mixture of one or more Pan T-cell monoclonal antibodies (Pan-T-cell-MAb), such as Anti-CD45MAb and Anti-CD3MAb, which are capable of immobilising the targeted antigens present on the surface of T4 and/or T8 lymphocytes, (a2) at least one monoclonal antibody specific for CD4 antigen to be determined, which is conjugated with a peroxidase probe, and (a3) at least one monoclonal antibody specific for CD8 antigen to be determined, which is conjugated with a peroxidase probe; (b) contacting the human blood sample to be examined with micro-wells of said microtitre plate for an incubation to allow the immobilization and labelling to occur simultaneously; (c) washing the microtitre plate to remove excess non-immobilized monoclonal antibodies; (d) estimating the antigens to be determined by counting the substrate of the enzyme; and (e) determining the CD4/CD8 ratio using standard curve.
Dated this 28th day of August, 2006.

SURESH VAZIRANI
CHAIRMAN AND MANAGING DIRECTOR
TRANSASIA BIO-MEDICALS LTD.
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