Title of Invention	"A STABILIZED IMMUNO-REACTIVE SUBSTANCE COATED ON A SUBSTRATE AND A METHOD FOR PREPARATION THEREOF"
Abstract	Present invention relates to a composition for stabilization of immunoreactive substances and the stabilized composition is capable of storage even at room temperature. The invention also provides a process for the preparation of reagents which can be used for measuring physiologically active substances using immobilized immunoreactive substance stabilized by he process of the invention.

Full Text

FIELD OF THE INVENTION The present invention provides a stabilized antibody coated substrate and a method for stabilizing immunoreactive substances on a solid matrix. The invention also provides a process for the preparation of reagents which can be used for measuring physiologically active substances using immobilized immunoreactive substances stabilized by the process of this invention. BACKGROUND AND PRIOR ART REFERENCES Recently in the field of research, immuno-diagnosis, measurement of various substances utilizing an immunoreagent/ immunoreactant immobilized to solid support is widely performed. Antibodies represent a specific class of proteins generated by the immune system. These protein molecules are capable of binding or complexing with invading molecules, often termed as "antigens". Antibody molecules recognize the antigen by complexing its antigen-binding sites with areas called 'epitopes'. The epitopes fit into the antigen-binding sites of the antibody, thus enabling the antibody to bind to the antigen. Antibodies are used in immuno-assays. In immuno-assays, antibodies are widely employed for detection or determination of a substances in biological samples. These assays involve the binding of an antigen with an antibody. Such binding occurs with high specificity even at low concentration. Immobilization of antigen or antibody to solid surface is the primary and most important step for solid phase immunoassay. The success of this method depends on the quality of the carrier and the stability of the immunoreactive substances coated on the solid substrate. Thus, it is important in immuno-assays to ensure that the coated antigen/antibody is stable. Immuno-reagent (antigen or antibody) and biological binder (receptor, carrier proteins, avidin-biotin, protein, A, G and L) immobilized to solid support in requisite amount needed for assay often have poor stability when stored for long time. Hence, selection of right kind of carrier, the binding method of the immuno-reactive substance and the storage method of the immuno-reactive substance are areas of active research. GB2 016687 A describes a method for stabilizing an immuno reactant immobilized onto solid support protected / stabilized by employing a sugar coating. Combined effect of sugars has not been demonstrated. WO 98/58259 describes the method for the stabilization of polypetide or proteins attached to solid support stabilized by employing a sucrose coating. WO 94 00594 describes the use of 5% sucrose for stabilizing polypeptide or protein attached to solid support. EP 0140489 describes the use of 5% sucrose for stabilizing polypeptide or protein attached to solid support. EP 0140489 describes the use of 5% either sucrose/lactose/mannose/dextrin for stabilizing the protein or polypeptide attached to solid support. Combine effect of sugars on stability of immobilized antibody has not been shown/demonstrated. EP 0805 353 describes the use of 10% water soluble gelatin for lyophilized state stabilization of polypeptide or protein for solid surface. US 5273 908 describes the use of 5% either sucrose/lactose/mannose/dextrin for stabilization of protein attached to solid support. Combined effect of sugars on stability of immobilized antibody has not been shown/demonstrated. These attempts in the prior art are directed towards the common objective of providing stabilized immuno substances on solid substrates. However, the approach adopted and taught by each of the above patents is distinct and different. In countries like India, where, the supply of electricity is quite irregular, leads to frequent freeze-thaw cycles of stored immunoreagents are adversely affecting the stability of such compositions/reagents. Hence, it is important to develop reagents capable of being stored even under difficult conditions for a very long period of time. To fulfil this objective, the applicant has conducted a detailed study and developed a method for stabilization of immunoreactive substances on solid substrates. OBJECTS OF THE INVENTION The main object of this invention is to provide stabilized antibody coated substrates. Another object is to provide a method for stabilization of immuno-reactive substances on solid substrates. Another object of the invention is to provide a process for the preparation of reagent which is used for measuring physiologically active substances. SUMMARY OF THE INVENTION Accordingly, the invention provides stabilized antibody coated substrates and a method for stabilization of immobilized immuno-reactive substances on solid substrates. DETAILED DESCRIPTION OF THE INVENTION Accordingly, the invention provides a method for stabilization of an immuno-reactive substance immobilized on a substrate, which comprises the steps of immersing carrier/substrate bound immuno-reactive substances in a solution consisting of sugars, melatonin and suitable buffer system. This invention also provides an assay reagent for measuring a physiologically active substance as a component of an immuno-reactive substance immobilized on a substrate and stabilized by immersing the carrier/substrate bound immuno-reactive substance in a solution consisting of sugars, melatonin and suitable buffer system. Any conventional insoluble carrier or substrate, especially those that are water-insoluble and which allow easy solid-liquid separation without conducting centrifugational separation may be used in the invention. Examples of such insoluble carriers are synthetic polymer compounds such as polystyrene, polypropylene, polyvinylchloride, polyethylene, polychlorocarbonate, silicone resin, silicone rubber, etc.; inorganic materials such as porous glass, ground glass, alumina, silica gel, activated charcoal, metal oxides, etc. These materials can be used in any forms of tubes, beads, disk, flakes, fine particles (latex or magnetic particles), microwells strips/plates, etc. The immuno-reactive substance to be immobilized on the carrier or substrate, may be an antigen, an antibody or even a hapten (drugs, etc.). Examples of the antigen are hormones such as insulin, glucagon, growth hormone, human chorionic gonadotropin, adrenocortical hormone, thyroid stimulating hormone, etc.; proteins such as IgG, IgM, IgA, IgE, IgD, α-fetoprotein, ferritin, ß2-microglobulin, CEA, etc.; and viral antigens such as HBs antigen, rubella virus antigen, etc. Examples of the antibody are those obtained by immunizing foreign material in an animal. Examples of the haptens are steroid and thyroid hormones, catecholamines and vitamins. As said earlier, the immunoreactive substance coated on substrate is dipped or immersed, in a solution of sugars, melatonin and a suitable buffer system. It is applicant's finding that a combination of reducing and non-reducing sugars along with melatonin and a suitable buffer system provides a stabilizing solution which if contacted with the immuno-reactive substance will enable formation of a stabilized composition. It is important that the sugars selected comprise a combination of reducing and non-reducing sugars. The reducing sugars may be present in equimolar amount. Equimolar denotes concentration of the ingredients taken in equal amount in terms of molarity. More specifically, the sugar combination (i.e. reducing and non-reducing) must be present in an isotonic strength i.e. 0.52 freezing point depression of 0.9% NaCl solution. By 'isotonic', it is meant that the composition has essentially the same osmotic pressure as human blood. Sugars act as cryothermostabilizers and as antioxidants. A combination of reducing and non-reducing sugars along with melatonin has the potential not only to stabilize immuno-reagent at room temperature, but also at temperatures which may be higher or lower than room temperature, thereby preserving the true state and properties of the immuno-reagent or immuno-reactive substance in question. A reducing sugar is one which contains a hemiacetal group that can reduce metal ions or react covalently with amino groups in proteins. Examples of reducing sugars are fructose, mannose, arabinose, xylose, ribose, galactose maltose, lactose and glucose. All other sugars that do not possess the properties of the reducing sugars are termed as 'non-reducing sugars'. Examples of such sugars include sucrose, trehalose, sorbose, and raffinose, etc. Any of sugars may be used, the only condition being that the combination used should essentially comprise a reducing and non-reducing sugar such as glucose, fructose and sucrose. Sugars prevents oxidative degradation, strengthen the hydrophobic bonds and prevents denaturation. Invert sugar being a equimolar mixture of glucose and fructose acts as antioxidant, preventing the immuno-reagent from oxidation (as oxygen scavengers). Sucrose being an example of a non-reducing polyhydric sugar strengthens the hydrophobic bond and such sugars are believed to have a positive effect on hydration of immuno-reagent. It is the applicants finding that the ingredients of the composition exhibit synergistic effect which is unexpected and surprising. The applicants have found that the immunoreactive substance coated on a substrate remains stable and active for extended periods, say upto about 2 years on account of the synergistic effect exhibited by the ingredients of the composition. Further, the applicants have noticed to their surprise that the immunoreactive substance does not lose its activity even when stored at room temperature such as 20 to 35°C or at elevated temperatures upto 40°C. The composition containing the immunoreactive substance, the reducing and non-reducing sugars along with melatonin and appropriate buffer system stabilizes the substance, helps to retain its activity for extended periods and thus remain stable. The sugars such as non-reducing sugars strengthen the hydrophobic bond and these sugars are believed to exhibit a positive effect on hydration of the immuno-reactive substance. The sugars also act as cryothermostabilizers and effective antioxidants. Melatonin being a strong antioxidant prevents oxidation of the immunoreactive substance at higher temperature. The buffer system retains molecular structure of the immunoreactive substance and thus prevents its denaturation even at elevated temperatures. Thus, all the ingredients of the composition exhibit synergistic effect, which effect is surprising and unexpected. Melatonin is a hormone secreted by the pineal gland. It is a strong antioxidant and prevents the oxidation of enzyme/enzyme conjugate or any other proteins at higher temperature. Melatonin is sparingly soluble in water. It may be dissolved in any solvent that does not affect the enzyme/protein of interest. For example, ethylene glycol and dimethyl sulphoxide (DMSO) etc. The amount of melatonin that may be used in the composition depends on various factors. The recommended amount is 5 - 50 µg/ml. The applicants have also found that existence of an appropriate buffer system adds to the stability of the composition. This buffer system should comprise an alkaline component and ammonium sulfate. The alkaline component i.e. conjugate base or salt of an acid, selected from acetate buffer, barbital buffer, borate buffer, citrate buffer, cocodylate buffer, phosphate buffer, tris buffer and the like. It is to be ensured that, the alkaline component selected does not affect molecule of interest. The amount of the alkaline component may be about 5 to 100 mM. This alkaline component is combined with ammonium sulfate because it is a protein precipitant and is capable of replacing the essential structural water molecule hydrogen bonded to an antigen / antibody i.e. initiating a salting out reaction, thereby maintaining the molecular structure of an immobilized immuno-reactant and preventing its denaturation. Therefore, ammonium sulphate is also an important ingredient in the stabilizing composition. The amount of ammonium sulfate may be present in the solution is up to 0.5% w/v i.e. 0.5gm per 100 ml. As to the solvent for dissolving a sugars along with melatonin and ammonium sulfates there can be used purified water or a buffer solution. Examples of the buffer solution are those having buffering effect at near neutral pH such as a phosphate buffer solution, a Tris-HCl buffer solution, acetate citrate buffer solution and the like. Among them, phosphate buffer solution is particularly preferred. It is believed and it is a basis of the present invention that, the immobilized immunoreactant or biological binder when coated with isotonic solution of mixture reducing, non-reducing sugars along with melatonin and ammonium sulfate in combination, that is equal to 0.52% freezing point depression of 0.9% NaCl solution (blocking buffer) would stabilize them by preventing oxidation and denaturation. It is probably the unique combination of the properties (additive effect) of water replacement of ammonium sulfate (salting out), antioxidant of invert sugar (equimolar concentration of glucose and fructose) and melatonin and strengthening hydrophobic bonds and hydration of sucrose explain the stabilizing properties for the production of stable antibody, immobilized to solid surface. In order to stabilize the immuno-reactive material immobilized on a carrier in the dried state, the immuno-reactive coated carrier material is first immersed in the solution of ammonium sulfate, sugars and melatonin, for example, for 20 to 40 minutes at room temperature, and dried, for example, by placing the thus treated carrier on a filter paper for a sufficient time to allow air drying. The dried carrier/substrate with stabilized immuno-reactive substance can be used as a reagent. More preferably, the thus dried carrier is stored in a vessel sealed and capped under nitrogen gas or reduced pressure. By subjecting the carrier bound antibody/antigen to immersing treatment in the solution of ammonium sulphate sugars and melatonin , decrease of the antigen or antibody activity caused during air drying procedure of the carrier can be prevented effectively. The stabilized immuno-reactive substances immobilized on a carrier is useful as a reagent for measuring physiologically active substances. Needless to say, the application of the present invention is not limited to the typical measuring systems in RIA and EIA mentioned above. It also can be applied to various modified systems which utilize the immuno-reactive substance immobilized on a carrier. This invention is illustrated in detail by way of the following Examples, wherein all percents are by weight unless otherwise specified. The following examples are given for the purpose of illustrating the present invention. EXAMPLES Example 1 Immobilization of antibody to solid support (Immunobridge Method) Murthy et al. J. Biosci 14:9-20 (1989) and Parson Jr. G.H. Methods. Enzymol. 73:224-239(1981) An antibody immobilized to solid support following the method of Murthy et al. (1989) and Parson Jr. G.H. (1981) with the modification of the procedures. The 250 µl of 1:100 diluted normal rabbit serum (pretreated with 18% ammonium sulfate) in water containing 0.1% glutaraldehyde is added to the polystrene microwell and incubated for overnight at 4°C followed by washing five to six times with running tap water. Where after 250 µl of 1:4000 diluted second antibody in 10mM phosphate buffer saline (PBS) were added to each well and again incubated for overnight at 4°C followed by washing five to six times with running tap water. Where after 200 µl of 1:8000 diluted primary antibody in 10mM PBS were added to each well and again incubated for overnight at 4°C followed by washing five to six times with running tap water. Where after 300 µl of blocking buffer were added to each well and incubated for 1 hour at 37°C followed by decanting the blocking buffer and drying of the well at room temperature. Composition of blocking buffer 5mM sodium acetate 1.85% Sucrose 1.5% Glucose 1.5% Fructose 0.336% Ammonium sulfate 0.2% gelatin, 5-50 µg/ml melatonin, 0.1% Bovine serum albumin and thimerosel each Example 2 Carrying out of an immunological determination process The test was carried out according to the enzyme linked immunosorbent assay (ELISA) test principle as a competitive inhibition by means of solid phase bound cortisol primary antibody and coated by blocking buffer as described in example 1. 25 µl of sample and 100 ul of cortisol- horse radish peroxidase conjugate were introduced into a polystyrene microwell coated with cortisol primary antibody. Incubation was carried out for 2 hours at 37°C followed by washing five to six times with running tap water, where after 100 ul of substrate solution were added thereto. Substrate Solution 100 µg/ml Tetramethyl Benzidine (TMB) 0.003% H2O2 In 100mM citrate acetate buffer pH 3.5 After incubation for 20 minutes at 37°C, the reaction was stopped by adding 50 ul of 4M H2SO4, the color formed was determined at 450 nm in Tecan Spectra ELISA reader. Example 3 Influence of Sucrose (Table Removed) The above example demonstrate that the antibody immobilized to solid support retains its immuno reactivity for longer period when coated with isotonic solution of sucrose in 5 mM sodium acetate as compared to diabasic sodium phosphate. This means, phosphate is having some inhibitory effect on antibody activity. Example 4 Influence of reducing and non-reducing sugar (Table Removed) The example 4 demonstrate that when immobilized antibody is coated with the reducing sugars along with non-reducing sugar in isotonic strength in 5 mM sodium acetate are dibasic sodium phosphate is able to stabilize immobilized antibody for longer period as compared to isotonic sucrose solution (example 3) at 4-8°C. This shows the additive effect of reducing and non-reducing sugars for stabilizing antibody immobilized on solid surface. Example 5 Influence of Ammonium sulfate (Table Removed) The above example demonstrate that combination of ammonium sulfate reducing and non-reducing sugars into the isotonic strength in 5mM sodium acetate or dibasic sodium phosphate is able to stabilize antibody immobilized to solid support for more than one year at 4-8°C. This may be because of unique combination of the properties (additive effect) of ammonium sulfate and reducing and non-reducing sugars for stabilizing antibody immobilized to solid support. At higher temperature (37°C) and above the solid phase immobilized immuno reactive substances retains the immuno-activity when procted with 5 mM sodium acetate rather than dibasic sodium phosphate. The addition of 5-50 µg/ml of melatonin in sodium acetate solution further enhances its stabilizing effect of immobilized immuno reactant at higher temperature for longer time (more than three months). WE CLAIM A stabilized immuno-reactive substance coated on a substrate useful in immune- assays, and comprising a stabilizing amount of reducing, non-reducing sugars, appropriate buffer system and melatonin. A composition as claimed in claim 1 wherein the substrate is selected from the group comprising synthetic polymer compounds such as polystyrene, polypropylene, polyvinylchloride, polyethylene, polychlorocarbonate, silicone resin, silicone rubber and inorganic materials such as porous glass, ground glass, alumina, silica gel, activated charcoal and metal oxides. A composition as claimed in claim 1 wherein the immuno-reactive substance immobilized on the carrier or substrate is selected from antigen, antibody or hapten (drugs, etc.). A composition as claimed in claim 4 wherein the antigen is selected from the group of hormones comprising insulin, glucagon, growth hormone, human chorionic gonadotropin, adrenocortical hormone, thyroid stimulating hormone and proteins such as IgG, IgM, IgA, IgE, IgD, α-fetoprotein, ferritin, ß2- microglobulin, CEA, and viral antigens such as HBs antigen, rubella virus antigen. A composition as claimed in claim 4 wherein the antibody is obtained by immunizing foreign material in an animal. A composition as claimed in claim 1 wherein the antibody is selected from polyclonal or monoclonal antibodies. A composition as claimed in claim 4 wherein the haptens are steroid and thyroid hormones, catecholamines and vitamins. A composition as claimed in claim 1 wherein the non-reducing sugars are present in equimolar amount. A composition as claimed in claim 1 wherein the reducing sugars are selected from glucose, mannose, fructose, arabinose, xylose, ribose maltose, lactose and galactose. 14 A composition as claimed in claim 1 wherein the non-reducing sugars are selected from sucrose, trehalose, sorbose and raffinose. A composition as claimed in claim 1 wherein the sugar or combination of sugars (reducing and non-reducing) and the buffer are present in isotonic strength i.e. equal to 0.52 freezing point depression of 0.9% NaCl solution. A composition as claimed in claim 1 wherein the melatonin is obtained from natural or synthetic sources. A composition as claimed in claim 1 wherein melatonin is dissolved in suitable solvents like ethylene glycol or dimethyl sulphoxide. A composition as claimed in claim 1 wherein the amount of melatonin is 5 to 50µg/ml. A composition as claimed in claim 1 wherein the buffer system comprises an alkaline component and ammonium sulphate. A composition as claimed in claim 1 wherein the alkaline component is a conjugate base or salt of an acid. A composition as claimed in claim 1 wherein the alkaline component is selected from the group of buffers such as acetate buffer, barbital buffer, borate buffer, citrate buffer, cocodylate buffer, phosphate buffer, tris buffer and the like. A composition as claimed in claim 1 wherein the amount of the alkaline component of the buffer system is 5 to 100 mM. A composition as claimed in claim 1 wherein if the alkaline component is sodium acetate, it is present up to 100 mM in the solution. A composition as claimed in claim 1 wherein the amount of ammonium sulfate in the buffer system is 0.5%w/v. A process for preparing a stabilized immuno-reactive substance coated on a substrate, said process comprising the steps of: preparing a solution comprising reducing, non-reducing sugars and buffer system in isotonic strength along with melatonin, and immersing a substrate containing an immunoreactive substance coated thereon in the solution of step (a). 15 A process as claimed in claim 21 wherein the substrate is selected from the group comprising synthetic polymer compounds such as polystyrene, polypropylene, polyvinylchloride, polyethylene, polychlorocarbonate, silicone resin, silicone rubber and inorganic materials such as porous glass, ground glass, alumina, silica gel, activated charcoal and metal oxides. A process as claimed in claim 21 wherein the immuno-reactive substance immobilized on the carrier or substrate is selected from antigen, antibody or hapten (drugs, etc.). A process as claimed in claim 23 wherein the antigen is selected from the group of hormones comprising insulin, glucagon, growth hormone, human chorionic gonadotropin, adrenocortical hormone, thyroid stimulating hormone and proteins such as IgG, IgM, IgA, IgE, IgD, a-fetoprotein, ferritin, ß2-microglobulin, CEA, and viral antigens such as HBs antigen, rubella virus antigen. A process as claimed in claim 23 wherein the antibody is obtained by immunizing foreign material in an animal. A process as claimed in claim 23 wherein the antibody is selected from polyclonal or monoclonal antibodies. A process as claimed in claim 23 wherein the haptens are steroid and thyroid hormones, catecholamines and vitamins. A process as claimed in claim 21 wherein the non-reducing sugars are present in equimolar amount. A process as claimed in claim 21 wherein the reducing sugars are selected from glucose, mannose, fructose, arabinose, xylose, ribose maltose, lactose and galactose. A process as claimed in claim 21 wherein the non-reducing sugars are selected from sucrose, trehalose, sorbose and raffinose. A process as claimed in claim 21 wherein the sugar or combination of sugars (reducing and non-reducing) and the buffer are present in isotonic strength i.e. equal to 0.52 freezing point depression of 0.9% NaCl solution. 16 A process as claimed in claim 21 wherein the melatonin is obtained from natural or synthetic sources. A process as claimed in claim 21 wherein melatonin is dissolved in suitable solvents like ethylene glycol or dimethyl sulphoxide. A process as claimed in claim 21 wherein the amount of melatonin is 5 to 50µg/ml. A process as claimed in claim 21 wherein the buffer system comprises an alkaline component and ammonium sulphate. A process as claimed in claim 21 wherein the alkaline component is a conjugate base or salt of an acid. A process as claimed in claim 21 wherein the alkaline component is selected from the group of buffers such as acetate buffer, barbital buffer, borate buffer, citrate buffer, cocodylate buffer, phosphate buffer, tris buffer and the like. A process as claimed in claim 21 wherein the amount of the alkaline component of the buffer system is 5 to 100 mM. A process as claimed in claim 21 wherein if the alkaline component is sodium acetate, it is present up to 100 mM in the solution. A process as claimed in claim 21 wherein the amount of ammonium sulfate in the buffer system is 0.5%w/v. A stabilized immunoreactive substance coated on a substrate substantially as herein described and illustrated. A process for preparing an immunoreactive substance coated on a substrate substantially as herein described and illustrated. RAJESHWARI H. OF KUMARAN & SAGAR ATTORNEY FOR THE APPLICANT Dated this 20"1 day of December, 2000. 17

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1189-del-2000-correspondence-others.pdf

1189-del-2000-correspondence-po.pdf

1189-del-2000-description (complete).pdf

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1189-del-2000-form-13.pdf

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1189-del-2000-form-26.pdf

1189-del-2000-form-3.pdf

1189-del-2000-form-5.pdf