Indian Patents. 236000:CONJUGATE COMPOSITION AND PROCESS OF THE PREPARATION OF THE COMPOSITON

Title of Invention	CONJUGATE COMPOSITION AND PROCESS OF THE PREPARATION OF THE COMPOSITON
Abstract	A colloidal gold conjugate composition comprising gold chloride, Sodium citrate, buffer solution, Stabilizer and other conventional ingredients and the process for the preparation of the same.

Full Text	CONJUGATE COMPOSITION AND PROCESS FOR THE PREPARATION OF THE COMPOSITION The present invention relates to a colloidal gold conjugate composition comprism colloidal particles to the surface of which biomolecules are adsorbed and a process of preparing the same, The composition is used in immunological test methods. It is particularly useful for detection of malarial parasites in the human blood and plasma. Conjugates which are composed of biomolecules such as proteins or nucleic acids and colloidal particles are widely used in diagnostic and therapeutic procedures e.g. as markers in detection methods such as immunoassays or as microprojectiles for gene transfer. Particles composed'of metals and metal compounds such as metal oxides, metal hydroxides, metal salts and polymer cores which are coated with metals or metal compounds can be used as particles (cf. e.g. U.S. Pat No. 4,313,734; Leuvering et al., J. Immunoassay 1 (1980), 77-91; Leuvering Dissertation (1984), Sol Particle Immunoassay (SPIA): The Use of Antibody Coated Particles as Labelled Antibodies in Various Types of Immunoassay; Uda et al., Anal. Biochem. 218 (1994), 259-264, DE-OS 41 32 133, page 3, lines 16-18, for applications as markers and Tang et al., Nature 356 (1992), 152-154; Eisenbraun et al., DNA and Cell Biology 12 (1993), 791-797; Williams et al., Proc. Natl. Acad. Sci. USA 88 (1991), 2726-2730 for applications for gene transfer). Furthermore it is also known that non-metallic colloidal particles such as carbon particles (van Amerongen, Anabiotic '92 (1993), 193-199) can also be used. At present colloidal gold particles are used most frequently. In order to produce biomolecule-gold conjugates gold sols are firstly produced according to generally known processes by reducing tetrachloroauric acid. Subsequently the gold sols are loaded with the desired respective biomolecule e.g. proteins such as antibodies, protein A, protein G, streptavidin etc. The respective loading conditions (pH, buffer, concentration of biomolecules etc.) depend on the isoelectric point of the biomolecule, the MPA (minimal protecting amount) or/and the specific application of the conjugate (cf. e.g. De Mey, The Preparation and Use of Gold Probes, in: Immunocytochemistry, eds: J. M. Polak and S. V. Noorden, pp 115-145, Wright, Bristol 1986 and J E. Beesley, Colloidal Gold: A New Perspective for Cytochemical Marking, Microscopy Handbooks 17, Oxford University Press, 1989, in particular pp 1-14). Explicit reference is made to the disclosure of these documents. After the colloidal particles have been loaded with the respective desired biomolecule it is necessary to stabilize the conjugates. This stabilization is intended to minimize an aggregation of the particles and to saturate remaining free surfaces accessible to adsorption. In the state of the art inert proteins, e.g. bovine serum albumin, blood substitute mixtures etc., detergents such as TWEEN.RTM. 20, water-soluble technical polymers such as polyethylene glycol (molecular weight 20,000 D), polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl sulfate, dextran and gelatin are used as stabilizers (cf. e.g. De Mey, Supra; Beesley, Supra; Behnke, Eur. J. Cell Biol. 41 (1986), 326-338; DE 24 20 531 C3; and Meisel et al., J. Phys. Chem. 85 (1981), 179-187). in addition the possibility of stabilizing gold sols by phospane complex ligands has also been described (Schmid et al., Z. Naturforsch. 45b (1994), 989-994). The stabilizers used according to the state of the art bind adsorptively to the free surfaces of the metal particles. The stabilizers can be desorbed or displaced from the surfaces to a greater or lesser extent as a result of long storage or changes in the environmental conditions such as those which for example can occur in a test by contact with the sample (blood, serum, plasma, urine), incorporation of the conjugates in test strip fleeces etc. This leads to a deterioration of the aggregation stability and to an increase in the unspecific reactivity. Moreover a large number of the stabilizers used are poorly defined products some of which have variable quality e.g. bovine serum albumin, gelatin. As a result variations in the stabilizing effect may also occur. Adsorption processes on particle surfaces are very complex and up to now are only partially understood. It is assumed that the adsorption occurs as a result of a combination of electrostatic interactions, Van-der-Waals forces and hydrophobic interactions (Beesely, Supra). In this process one or other type of binding can predominate depending on the type of the adsorbed biomolecule. The biomolecules adsorbed to the surface of the particles are preferably selected from the group comprising proteins, glycoproteins, peptides, nucleic acids, peptidic nucleic acids, saccharides, antigens and haptens. Biomolecules are particularly preferably selected from the group comprising antibodies, antibody fragments, lectins, enzymes, streptavidin, avidin, protein A, antigens such as recombinant polypeptides or multiple antigens (cf. WO96/03652), e.g. polyhaptens (several haptens or peptides coupled to a carrier such as dextran or to a protein), peptides and haptens (low molecular substances preferably with a MW.ltoreq.1500 such as biotin, fluorescein or digoxigenin). Reference is made to the aforementioned review article by De Mey and Beesley with regard to the exact conditions for adsorbing these biomolecules to gold particles. U. S. Patent no. 5972720 describes a composition comprising colloidal particles to the surface of which biomolecules are adsorbed, wherein the composition additionally contains a polyethylene glycol substituted by thiol or/and disulfide groups. U.S. Patent no. 5851776 describes conjugates and competitive and non-competitive assays for simultaneously detecting the presence or amount of at least two target ligands capable of competing with a single conjugate for binding to at least two different ligand receptors. The invention teaches and claims binding domains coupled to a signal development element to form a conjugate where each binding domain comprises at least one ligand analogue or ligand receptor depending on assay design. The binding domains are constructed such that they function independently from one another in assays for their respective target ligands. Each binding domain may bind its respective binding partners in the assay without affecting the binding reactions of other binding domains coupled to the same signal development element. The average diameter of the particles is in the range of 1-1000 nm which is usual in the state of the art and can be varied according to the application purpose. The average diameter of the particles is preferably in the range of 2-200 nm and particularly preferably 2-100 nm. The composition according to the invention can be used as a detection reagent in particular as an immunological detection reagent. In a preferred embodiment the detection reagent is used in an immunoassay i.e. in a method for the determination of an analyte in a sample liquid by means of immunological methods e.g. by a competitive assay in which a labelled analyte analogue or a labelled analyte-specific receptor e.g. an antibody is used, or a sandwich assay in which a labelled analyte-specific receptor or a labelled further receptor capable of binding to the analyte-specific receptor is used. In one of the embodiments the subject invention relates to a colloidal gold conjugate composition comprising colloidal gold solution, buffer, tris buffer, a stabilizer to block the unblocked site, buffer solution for suspension, antibodies and other ingredients Accordingly the subject invention relates to a colloidal gold conjugate composition comprising 01-.5%gold chloride, 1-5% sodium citrate solution buffer solution for suspension, .1-2%stabilizer the balance being other conventional ingredients The present invention also relates to a process for the preparation of the conjugate composition for detection of malarial parasites in the human blood and plasma comprising: Taking the solution of 0.01% - 0.5% (w/v) Gold chloride Adding a boil sodium citrate solution (1-5%) Stirring the solution is rapidly & boiling it until a pink purple colour develops. Mixing anti HRP2 monoclonal & Pan Malarial monoclonal Antibodies in a buffer solution Mixing the antibody solution with gold colloid for 1 hr to 2 hrs. Adding any animal IgG (mouse or goat or rabbit or sheep) to the above solution Centrifuging this solution and discarding the supernatant Redissolving the pallet in a buffer containing stabilizers Filtering the said slution to get the desired conjugate The gold chloride is obtained from reduction of tetra chloro auric acid by using various chemicals The pallet is redissolved in a buffer containing stabilizers like BSA, gelatin. The buffer solution used is preferably HEPES, MOPS, TRIS, Borate or Phosphate may also be used. The molarity of buffer can range from 1 mM to 25 mM and pH 6.5 to 9.0. The antibody concentration preferably is 1 ug / ml to 10 ug / ml of Gold colloid solution. The concentration of IgG could be 1 ug to 10 ug / ml of gold colloid solution. This solution is mixed for 2 hr to overnight. After this the solution is centrifuged and the supernatant is discarded. The subject invention is a mere statement of invention , where various alterations and modifications are possible without deviating from the scope of the invention henoe the same should not be construed to restrict the scope of the inyention. We Claim: 1. A colloidal gold conjugate composition wherein antibodies namely monoclonal anti HRP2 antibody and monoclonal anti Pan malarial antibody are simultaneously conjugated to gold colloid such that it can be used for differential detection of P. falciparum and Pan malaria species in human serum or plasma comprising of: 0.01%-5% Gold chloride; 1% - 5% Sodium citrate solution; Buffer solution; 0.1%-2% stabilizer; 1µg/ml to 10 µg/ml of monoclonal anti HRP2 antibody and monoclonal anti Pan malarial antibodies; and the balance being other conventional ingredients. 2. A colloidal gold conjugate composition as claimed in claim 1 wherein the said buffer solution is selected from HEPES, MOPS, TRIS, Borate or Phosphate. 3. A colloidal gold conjugate composition as claimed in claim 1 wherein the molarity of said buffer ranges from 1mM to 25mM and pH ranges from 6.5 to 9.0. 4. A colloidal gold conjugate composition as claimed in claim 1 wherein the said stabilizers are BSA and/or gelatin. 5. A process for the preparation of the conjugate composition as claimed in claim 1 for the detection of malarial parasites in the human blood and plasma comprising: taking the solution of 0.01%-0.5% (w/v) gold chloride; adding a boil sodium citrate solution(1-5%) stirring the solution is rapidly & boiling it until a pink purple colour develops; mixing anti HRP2 monoclonal and Pan Malarial monoclonal antibodies in a buffer solution; mixing the antibody solution with gold colloid for 1 hr to 2 hrs; adding any animal IgG (mouse or goat or rabbit or sheep) to the above solution; centrifuging this solution and discarding the supernatant; redissolving the pellet in a buffer containing stabilizers; filtering the said conjugate to get the desired conjugate. 6. A process for preparation of the conjugate composition as claimed in claim 5 wherein the antibody concentration preferably is 1 µg/ml to 10 µg/ml of Gold colloid solution. 7. A process for the preparation of the conjugate composition as claimed in claim 5 wherein the percentage of the buffer is 1/10 of the gold solution. 8. A colloidal gold conjugate composition as substantially herein before described. 9. A process for the preparation of the conjugate composition as substantially herein before described.

Full Text

CONJUGATE COMPOSITION AND PROCESS FOR THE PREPARATION OF THE COMPOSITION
The present invention relates to a colloidal gold conjugate composition comprism colloidal particles to the surface of which biomolecules are adsorbed and a process of preparing the same, The composition is used in immunological test methods. It is particularly useful for detection of malarial parasites in the human blood and plasma.
Conjugates which are composed of biomolecules such as proteins or nucleic acids and colloidal particles are widely used in diagnostic and therapeutic procedures e.g. as markers in detection methods such as immunoassays or as microprojectiles for gene transfer. Particles composed'of metals and metal compounds such as metal oxides, metal hydroxides, metal salts and polymer cores which are coated with metals or metal compounds can be used as particles (cf. e.g. U.S. Pat No. 4,313,734; Leuvering et al., J. Immunoassay 1 (1980), 77-91; Leuvering Dissertation (1984), Sol Particle Immunoassay (SPIA): The Use of Antibody Coated Particles as Labelled Antibodies in Various Types of Immunoassay; Uda et al., Anal. Biochem. 218 (1994), 259-264, DE-OS 41 32 133, page 3, lines 16-18, for applications as markers and Tang et al., Nature 356 (1992), 152-154; Eisenbraun et al., DNA and Cell Biology 12 (1993), 791-797; Williams et al., Proc. Natl. Acad. Sci. USA 88 (1991), 2726-2730 for applications for gene transfer). Furthermore it is also known that non-metallic colloidal particles such as carbon particles (van Amerongen, Anabiotic '92 (1993), 193-199) can also be used. At present colloidal gold particles are used most frequently.
In order to produce biomolecule-gold conjugates gold sols are firstly produced according to generally known processes by reducing tetrachloroauric acid. Subsequently the gold sols are loaded with the desired respective biomolecule e.g. proteins such as antibodies, protein A, protein G, streptavidin etc. The
respective loading conditions (pH, buffer, concentration of biomolecules etc.) depend on the isoelectric point of the biomolecule, the MPA (minimal protecting amount) or/and the specific application of the conjugate (cf. e.g. De Mey, The Preparation and Use of Gold Probes, in: Immunocytochemistry, eds: J. M. Polak and S. V. Noorden, pp 115-145, Wright, Bristol 1986 and J E. Beesley, Colloidal Gold: A New Perspective for Cytochemical Marking, Microscopy Handbooks 17, Oxford University Press, 1989, in particular pp 1-14). Explicit reference is made to the disclosure of these documents.
After the colloidal particles have been loaded with the respective desired biomolecule it is necessary to stabilize the conjugates. This stabilization is intended to minimize an aggregation of the particles and to saturate remaining free surfaces accessible to adsorption. In the state of the art inert proteins, e.g. bovine serum albumin, blood substitute mixtures etc., detergents such as TWEEN.RTM. 20, water-soluble technical polymers such as polyethylene glycol (molecular weight 20,000 D), polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl sulfate, dextran and gelatin are used as stabilizers (cf. e.g. De Mey, Supra; Beesley, Supra; Behnke, Eur. J. Cell Biol. 41 (1986), 326-338; DE 24 20 531 C3; and Meisel et al., J. Phys. Chem. 85 (1981), 179-187). in addition the possibility of stabilizing gold sols by phospane complex ligands has also been described (Schmid et al., Z. Naturforsch. 45b (1994), 989-994).
The stabilizers used according to the state of the art bind adsorptively to the free surfaces of the metal particles. The stabilizers can be desorbed or displaced from the surfaces to a greater or lesser extent as a result of long storage or changes in the environmental conditions such as those which for example can occur in a test by contact with the sample (blood, serum, plasma, urine), incorporation of the conjugates in test strip fleeces etc. This leads to a deterioration of the aggregation stability and to an increase in the unspecific reactivity. Moreover a large number of the stabilizers used are poorly defined products some of which have variable quality e.g. bovine serum albumin, gelatin. As a result variations in
the stabilizing effect may also occur.
Adsorption processes on particle surfaces are very complex and up to now are only partially understood. It is assumed that the adsorption occurs as a result of a combination of electrostatic interactions, Van-der-Waals forces and hydrophobic interactions (Beesely, Supra). In this process one or other type of binding can predominate depending on the type of the adsorbed biomolecule.
The biomolecules adsorbed to the surface of the particles are preferably selected from the group comprising proteins, glycoproteins, peptides, nucleic acids, peptidic nucleic acids, saccharides, antigens and haptens. Biomolecules are particularly preferably selected from the group comprising antibodies, antibody fragments, lectins, enzymes, streptavidin, avidin, protein A, antigens such as recombinant polypeptides or multiple antigens (cf. WO96/03652), e.g. polyhaptens (several haptens or peptides coupled to a carrier such as dextran or to a protein), peptides and haptens (low molecular substances preferably with a MW.ltoreq.1500 such as biotin, fluorescein or digoxigenin). Reference is made to the aforementioned review article by De Mey and Beesley with regard to the exact conditions for adsorbing these biomolecules to gold particles.
U. S. Patent no. 5972720 describes a composition comprising colloidal particles to the surface of which biomolecules are adsorbed, wherein the composition additionally contains a polyethylene glycol substituted by thiol or/and disulfide groups.
U.S. Patent no. 5851776 describes conjugates and competitive and non-competitive assays for simultaneously detecting the presence or amount of at least two target ligands capable of competing with a single conjugate for binding to at least two different ligand receptors. The invention teaches and claims binding domains coupled to a signal development element to form a conjugate where each binding domain comprises at least one ligand analogue or ligand
receptor depending on assay design. The binding domains are constructed such that they function independently from one another in assays for their respective target ligands. Each binding domain may bind its respective binding partners in the assay without affecting the binding reactions of other binding domains coupled to the same signal development element.
The average diameter of the particles is in the range of 1-1000 nm which is usual in the state of the art and can be varied according to the application purpose. The average diameter of the particles is preferably in the range of 2-200 nm and particularly preferably 2-100 nm.
The composition according to the invention can be used as a detection reagent in particular as an immunological detection reagent. In a preferred embodiment the detection reagent is used in an immunoassay i.e. in a method for the determination of an analyte in a sample liquid by means of immunological methods e.g. by a competitive assay in which a labelled analyte analogue or a labelled analyte-specific receptor e.g. an antibody is used, or a sandwich assay in which a labelled analyte-specific receptor or a labelled further receptor capable of binding to the analyte-specific receptor is used.
In one of the embodiments the subject invention relates to a colloidal gold conjugate composition comprising colloidal gold solution, buffer, tris buffer, a stabilizer to block the unblocked site, buffer solution for suspension, antibodies and other ingredients
Accordingly the subject invention relates to a colloidal gold conjugate composition comprising 01-.5%gold chloride, 1-5% sodium citrate solution buffer solution for suspension, .1-2%stabilizer the balance being other conventional ingredients
The present invention also relates to a process for the preparation of the conjugate composition for detection of malarial parasites in the human blood and plasma comprising:
Taking the solution of 0.01% - 0.5% (w/v) Gold chloride
Adding a boil sodium citrate solution (1-5%)
Stirring the solution is rapidly & boiling it until a pink purple colour develops.
Mixing anti HRP2 monoclonal & Pan Malarial monoclonal Antibodies in a buffer
solution
Mixing the antibody solution with gold colloid for 1 hr to 2 hrs.
Adding any animal IgG (mouse or goat or rabbit or sheep) to the above solution
Centrifuging this solution and discarding the supernatant
Redissolving the pallet in a buffer containing stabilizers
Filtering the said slution to get the desired conjugate
The gold chloride is obtained from reduction of tetra chloro auric acid by using various chemicals
The pallet is redissolved in a buffer containing stabilizers like BSA, gelatin.
The buffer solution used is preferably HEPES, MOPS, TRIS, Borate or Phosphate may also be used. The molarity of buffer can range from 1 mM to 25 mM and pH 6.5 to 9.0. The antibody concentration preferably is 1 ug / ml to 10 ug / ml of Gold colloid solution.
The concentration of IgG could be 1 ug to 10 ug / ml of gold colloid solution. This solution is mixed for 2 hr to overnight. After this the solution is centrifuged and the supernatant is discarded.
The subject invention is a mere statement of invention , where various alterations and modifications are possible without deviating from the scope of the invention henoe the same should not be construed to restrict the scope of the inyention.

We Claim:
1. A colloidal gold conjugate composition wherein antibodies namely
monoclonal anti HRP2 antibody and monoclonal anti Pan malarial
antibody are simultaneously conjugated to gold colloid such that it
can be used for differential detection of P. falciparum and Pan
malaria species in human serum or plasma comprising of:
0.01%-5% Gold chloride;
1% - 5% Sodium citrate solution;
Buffer solution;
0.1%-2% stabilizer;
1µg/ml to 10 µg/ml of monoclonal anti HRP2 antibody and
monoclonal anti Pan malarial antibodies;
and the balance being other conventional ingredients.
2. A colloidal gold conjugate composition as claimed in claim 1 wherein the said buffer solution is selected from HEPES, MOPS, TRIS, Borate or Phosphate.
3. A colloidal gold conjugate composition as claimed in claim 1 wherein the molarity of said buffer ranges from 1mM to 25mM and pH ranges from 6.5 to 9.0.
4. A colloidal gold conjugate composition as claimed in claim 1 wherein the said stabilizers are BSA and/or gelatin.
5. A process for the preparation of the conjugate composition as claimed in claim 1 for the detection of malarial parasites in the human blood and plasma comprising:
taking the solution of 0.01%-0.5% (w/v) gold chloride; adding a boil sodium citrate solution(1-5%)
stirring the solution is rapidly & boiling it until a pink purple colour
develops;
mixing anti HRP2 monoclonal and Pan Malarial monoclonal antibodies in
a buffer solution;
mixing the antibody solution with gold colloid for 1 hr to 2 hrs;
adding any animal IgG (mouse or goat or rabbit or sheep) to the above
solution;
centrifuging this solution and discarding the supernatant;
redissolving the pellet in a buffer containing stabilizers;
filtering the said conjugate to get the desired conjugate.
6. A process for preparation of the conjugate composition as claimed in claim
5 wherein the antibody concentration preferably is 1 µg/ml to 10 µg/ml of
Gold colloid solution.
7. A process for the preparation of the conjugate composition as claimed in
claim 5 wherein the percentage of the buffer is 1/10 of the gold solution.
8. A colloidal gold conjugate composition as substantially herein before
described.
9. A process for the preparation of the conjugate composition as
substantially herein before described.

Documents:

2380-DEL-2004-Abstract-(26-09-2008).pdf

2380-del-2004-abstract.pdf

2380-DEL-2004-Claims-(01-09-2009).pdf

2380-DEL-2004-Claims-(26-09-2008).pdf

2380-del-2004-claims.pdf

2380-DEL-2004-Correspondence-Others-(01-09-2009).pdf

2380-DEL-2004-Correspondence-Others-(15-12-2010).pdf

2380-DEL-2004-Correspondence-Others-(26-09-2008).pdf

2380-DEL-2004-Correspondence-Others-(29-12-2008).pdf

2380-del-2004-correspondence-others.pdf

2380-del-2004-correspondence-po.pdf

2380-del-2004-description (complete).pdf

2380-del-2004-description (provisional).pdf

2380-DEL-2004-Form-1-(26-09-2008).pdf

2380-del-2004-form-1.pdf

2380-DEL-2004-Form-13-(01-09-2009).pdf

2380-del-2004-form-13.pdf

2380-DEL-2004-Form-2-(26-09-2008).pdf

2380-del-2004-form-2.pdf

2380-DEL-2004-Form-26-(26-09-2008).pdf

2380-del-2004-form-26.pdf

2380-del-2004-form-3.pdf

2380-del-2004-form-5.pdf

2380-DEL-2004-Others-Document-(26-09-2008).pdf

2380-DEL-2004-Petition 138-(15-12-2010).pdf

2380-DEL-2004-Post-Grant Opposition (14-09-2010).pdf

2380-del-2004-Post-Grant-Opposition-(01-11-2010).pdf

2380-DEL-2004-Post-Grant-Opposition-(15-11-2010).pdf

2380-DEL-2004-Post-Grant-Opposition-(20-09-2010).pdf

2380-DELNP-2004-Description (Complete)-(01-09-2009).pdf

2380-delnp-2004-form-13-(01-09-2009).pdf

« Previous Patent

Next Patent »

Patent Number

236000

Indian Patent Application Number

2380/DEL/2004

PG Journal Number

38/2009

Publication Date

18-Sep-2009

Grant Date

11-Sep-2009

Date of Filing

29-Nov-2004

Name of Patentee

J. MITRA & CO. LTD

Applicant Address

A-180, OKHLA INDUSTRIAL AREA, PHASE-1, NEW DELHI-110 020, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	LALIT MAHAJAN	A-180, OKHLA INDUSTRIAL AREA PHASE-1, NEW DELHI-110 020,INDIA

PCT International Classification Number

A61K 39/39

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA