Title of Invention

A PROCESS FOR THE COVALENT BINDING OF HAPTEN MOLECULES TO POLYSTYRENE SURFACE

Abstract

The present invention relates to a process for the covalent binding of hapten molecules to polystyrene. More particularly the process of this invention is based on covalent attachment of hapten molecules to pplystyrene surface via an aliphatic primary amine generated on polystyrene with silanizaiton treatment using 3-aminopropyltriethoxysilane (APTES). The usefulness of this process is in affinity purification and enzyme immunoassay (EIA) based assay development.

Full Text

A PROCESS FOR THE COVALENT BINDING OF HAPTEN MOLECULES TO POLYSTYRENE Field of Invention The present invention relates to a novel process for the covalent binding of hapten molecules to polystyrene support. More particularly, the process of this invention is based on covalent attachment of hapten molecules to polystyrene surface via an aliphatic primary amine generated on polystyrene with silanization treatment using 3-aminopropyltriethoxysilane (APTES). The usefulness of this process is in affinity purification and enzyme immunoassay (EIA) based assay development. Polystyrene supports (microtiter plates, strips,, or beads) are widely used for enzyme linked immunosorbent assay (ELISA) and affinity purification applications. Biological macromolecules, such as proteins, are immobilized on polystyrene surface by passive adsorption. The adsorption of these molecules to polystyrene surface is due to intermolecular attraction forces (van der Waals forces), which are based on intramolecular electric polarities. It has been demonstrated that the protein molecules immobilized on hydrophobic polystyrene surface by passive adsorption loose their activity and suffer considerable denaturation [Butler, et al., Mol. Immunol., 30 (1993): 1165]. However, biological macromolecules are found to retain better functional activity when immobilized through extended hydrophilic spacer arms, as the sorption on surface is substantially reduced. Polystyrene surface can be modified to improve its hydrophilicity by incorporating various functional groups such as hydroxyl, amino, carbonyl, carboxyl etc. on its surface [Zammatteo et al., Anal. Biochem., 236 (1996): 85]. Direct amine grafting on polystyrene for immunoaffinity and assay applications has been reported by various groups [Barrett, US Patent 4001583; Hobbs, J. Immunol. Methods, 63 (1983): 359; Klin et al., Allergy, 48 (1993): 151; Matson et al., Anal. Biochem. 217 (1994): 306]. However, in case of small organic molecules like haptens, the direct attachment of these molecules on polystyrene surface is not possible due to the lack of available functional groups on polystyrene. The main objective of the present invention relates to a process for covalent binding of hapten molecules to polystyrene. Another objective of the present invention is to develop a process to provide an amino derivatized polystyrene surface for covalent attachment of hapten to it. Yet another objective of the present invention is to employ the process for affinity purification and immunological determination of biological substances employing such bound haptens. The present invention is based on fictionalization of polystyrene surface for covalent attachment of molecules, including biological substances without loosing their activity. Statement of invention Accordingly the present invention relates to a process for the covalent binding of hapten molecules to polystyrene surface which consists of: a) reacting pretreated polystyrene with inorganic acid with aminopropyltriethoxy silane at room temperature to generate amino groups on its surface, washing the polystyrene surface with water followed by curing at a temperature ranging 70-75°C, b) adding a solution of hapten molecule containing activated carboxyl groups on hapten molecule to the salinated polystyrene obtained in step (i) followed by incubation at a temperature in the range of 2-6°C for a period of 0-12 hours to obtain the covalently binded hapten polystyrene. In an embodiment of the present invention wherein the activation of carboxy group on hapten molecule may be carried out by reacting hapten molecule with N-hydrxy-succinimide and dicyclohexyl carbodiimide at a temperature ranging between 10-40°C for a period of about 20hrs. In yet another embodiment of the invention wherein the covalent binding of hapten to polystyrene surface produce a carboxylated hapten (2,4-dichlorophenoxy acetic acid). In yet another embodiment of the invention wherein the polystyrene surface used may be modified to couple covalently a hapten for the immunopurification and immunoassay applications. In an embodiment of the invention wherein the inorganic acid used may be about 40% in HNO3 in H2SO4. The present invention relates to a novel process for the covalent binding of the hapten molecules to polystyrene support comprising microtiter ELISA plates and beads. The process of this invention thus provides a method of immunopurification and immunoassay of biological substances from samples. The stepwise details of the present invention are: i. derivatization of polystyrene surface by aminopropyltriethoxy silane to generate amino groups on its surface, ii. activation of carboxyl groups on hapten for covalent coupling with modified polystyrene surface, iii. optimization of developed hapten-bound polystyrene surface for immuno¬purification and immunoassay applications. The present invention describes a method for grafting aliphatic primary amines on polystyrene surface using aqueous silanization chemistry. The polystyrene carrier support (microtiter wells and beads) is first treated with diluted APTES solution in distilled water (acidic pH lowered with HCl) and kept in a hot air oven at temperature ranging between 70 and 75°C for 2-3 hrs. After incubation, the surface is washed with distilled water and cured in an oven to enhance the binding of APTES with the polystyrene surface. The generated amine groups are utilized for binding of carboxylated hapten on the polystyrene surface. Carboxyl groups of the hapten 2,4-dichlorophenoxy acetic acid (2,4-D) are activated using carbodiimide activation method so as to link with amino groups of proteins or carrier surface. A small quantity of 2,4-D was dissolved in dioxane along with N-hydroxy-succinimide (NHS) and dicyclohexyl carboddimide (DCC). The mixture was kept for incubation for 20 hrs at room temperature, and then passed through a Wattman filter to remove solid substrates, mainly dicyclourea. The solvent was removed under reduced pressure at 35C in a rotavapour apparatus. The method represents a well-controlled methodology for binding small molecules, proteins etc., also applied to bind other macromolecules, immunoglobulins and albumin to polystyrene supports (microbeads and plates) via aldehyde groups on silane coated polystyrene surface. A further description of the invention is given in examples below, which should not however, be construed to limit the scope of the present invention unless otherwise stated. Example 1: Binding of hapten to the polystyrene surface (micro-titer wells) for antibody capture assay was carried out using a procedure in which 300 µl solution of 47% HNO3 in concentrated. H2SO4 was dispensed into each well of an ELISA plate and incubated at room temperature with mild shaking. After decanting the acid the wells were washed with double distilled water. To these wells, 200 µl of 5% aminopropyltriethoxy silane solution was added and the plates were kept for incubation at room temperature. Thereafter the plates were washed thoroughly with double distilled water followed by curing at 75°C in a dry air oven. After silanization, 100 µl activated 2,4-D solution (1000 nmoles/ml) in carbonate buffer was added to each well and left to incubate overnight. Subsequently the plates were washed with phosphate buffer saline (PBS). Unreacted sites were blocked by adding 200 µl/well of glycine (0.1 M in PBS) in each well and incubating at 37°C. This was followed by three washes with PBS containing 0.5% Tween-20 (PBST), and then one wash with PBS. After washing, 100 µl primary antibody solution (rabbit anti-2,4-D) in different concentrations was added into all the wells and kept for incubation at 37°C. This was followed by washing first with PBST, and then with PBS. Into the wells were now added 100 µl of secondary antibody solution (1:5000; goat-anti-rabbit) and then kept for incubation at 37°C followed by washing three times with PBST and then once with PBS. To this, 100 µl of substrate (tetramethylbenzidine-H2O2 solution) was added and incubated for 30 minutes at 37°C. The reaction was stopped with IN H2SO4. Optical density was measured with ELISA reader to determine the concentration of antibodies in the serum. Example 2: Binding of hapten to the polystyrene surface (micro-beads) for protein purification was carried out by dispensing 300 µl solution of HNO3 in con. H2SO4 into each well of Elisa plates. The plates were kept for incubation at room temperature imder mild shaking. After incubation, acid was decanted out and the wells were washed with double distilled water. To each wells were then added 200 µl of 5% aminopropyltriethoxy silane solution made in acidified double distilled water and the plates were kept for incubation at room temperature. The unbound APTES was removed from the wells, washed thoroughly and then plates were cured at 75°C in a dry air oven. After curing, 100 µl activated 2,4-D solution (1000 nmoles/ml) in carbonate buffer was added into each well, incubated overnight at 4°C, and then plates washed with phosphate buffer saline. We Claim: 1. A process for the covalent binding of hapten molecules such as herein described to polystyrene surface which consists of: a) reacting pretreated polystyrene with inorganic acid such as herein described with aminopropyltriethoxy silane at room temperature to generate amino groups on its surface, washing the polystyrene surface with water followed by curing at a temperature ranging from 70-75°C, b) adding a solution of hapten molecule containing activated carboxyl groups on hapten molecule to the salinated polystyrene obtained in step (i) followed by incubation at a temperature in the range of 2-6°C for a period of 0-12 hours to obtain the covalently binded hapten polystyrene. 2. A process as claimed in claim 1, wherein the hapten molecule is a carboxylated hapten (2,4-dichlorophenoxy acetic acid). 3. A process as claimed in claims 1 to 2, wherein covalently binded hapten polystyrene surface is used for the immunopurificaiton and immunoassay applications. 4. A process as claimed in claims 1 to 3, wherein the inorganic acid used is 40-50% HNO3 in concentrated H2SO4. 5. A process for the covalent binding of hapten molecules to polystyrene surface substantially as herein described with reference to the examples accompanying this specification.

Documents:

150-DEL-2003-Abstract-(19-02-2004).pdf

150-DEL-2003-Abstract-(27-10-2008).pdf

150-del-2003-abstract.pdf

150-DEL-2003-Claims-(10-12-2008).pdf

150-DEL-2003-Claims-(19-02-2004).pdf

150-DEL-2003-Claims-(27-10-2008).pdf

150-del-2003-complete specification (granted).pdf

150-DEL-2003-Correspondence-Others-(10-12-2008).pdf

150-DEL-2003-Correspondence-Others-(27-10-2008).pdf

150-del-2003-correspondence-others.pdf

150-DEL-2003-Description (Complete)-(19-02-2004).pdf

150-DEL-2003-Description (Complete)-(27-10-2008).pdf

150-DEL-2003-Description (Complete)-10-12-2008.pdf

150-del-2003-description (provisional).pdf

150-DEL-2003-Form-1-(27-10-2008).pdf

150-del-2003-form-1.pdf

150-del-2003-form-18.pdf

150-DEL-2003-Form-2-(19-02-2004).pdf

150-DEL-2003-Form-2-(27-10-2008).pdf

150-del-2003-form-2.pdf

150-del-2003-form-3.pdf

150-del-2003-form-5.pdf

150-DEL-2003-Others-Document-(10-12-2008).pdf