Indian Patents. 210137:A METHOD FOR THE PREPARATION OF AN ANALYTICAL PROBE TO MEASURE THE REAL TIME KINETICS OF LIGAND-LIGATE INTERACTION

Title of Invention	A METHOD FOR THE PREPARATION OF AN ANALYTICAL PROBE TO MEASURE THE REAL TIME KINETICS OF LIGAND-LIGATE INTERACTION
Abstract	A method for the preparation of a an analytical probe to measure the real time kinetics of ligand ligate interaction comprising the steps of immersing a nitrocellulose disc in a I I ligate solution to form the disc in a ligate solution carrying the ligate and forming a radio ligand that binds to the ligate in a time dependent manner by tagging a ligand on to radio iodine.

Full Text	This invention relates to a method for the preparation of an analytical probe to measure the real time kinetics of ligand-ligate interaction. FIELD OF TECHNOLOGY: This invention relates to industrial and laboratory instrument technology. More particularly this invention relates to design and preparation of a probe required to study the real time kinetics of ligand-ligate interaction using radiolabeled ligand. In our co-pending application No. 495/Mas/99 we have described and claimed an analytical instrument to measure the real time kinetics of ligand-ligate interaction comprising a flow cell, placed in a radioactive counter, a nitrocellulose disc carrying an immobilised ligate being introduced into the flow cell and means for receiving a radiolabeled ligand of known strength from a container of said radiolabeled ligand. In our co-pending application no. 496/mas/1999 describes and claims an analytical instrument to measure the dissolution of the ligand-ligate interaction which comprises of a nitrocellulose disc carrying an immobilised ligand-ligate complex, said disc being placed in a test tube having a lead shield and a gamma counting well to measure the dissolution of ligand-ligate interaction. In our co-pending application no. 497/mas/1999 describes and claims an analytical instrument to measure the real time kinetics of ligand-ligate interaction comprising a micro column carrying an immobilised ligand-ligate complex on a nitrocellulose disc, said micro-colimm being placed in a tube containing radio active AURA. DESCRIPTION OF PRIOR ART: The existent state of the art utilises a method, known in the field as plasmon resonance technology. This measure the amoimt of a ligand boxed on immobilised ligate using a light probe. This is the most popular approach in use, where ligand-ligate interactions have been and is being studied extensively. The method utilises a high technology adsorption surface (CHIP), on which binding of the ligand is monitored as the solution of the ligand is passed through this chip. LIMITATION: One of the primary need in the use of this technique is a high technology item, called the CHIP, This is a small gold plated surface which is used for the adsorption of the ligand in three dimension, measuring 1X2X5 micron dimension, and extremely expensive. The attachment of the ligate to the chip is also very expensive requiring costly reagents. The chip has limited life span in most of the cases, and is rebuked in large numbers for the study of the reaction. OBJECTS OF THE PRESENT INVENTION: It is the primary object of the invention to overcome the problems associated with prior art. The probe prepared by the method of the present invention to study and measure the real time kinetics of ligand-ligate interaction uses a radio labeled ligand for absorption of the ligate and utilises it for the purpose of understanding the real time kinetics of ligand-ligate interaction as the ligate adsorb on the nitrocellulose disc binds to the ligand in a time dependent manner. This invention thus provides a method for the preparation of an analytical probe to measure the real time kinetics of ligand-ligate interaction comprising the steps of immersing a nitrocellulose disc in a ligate solution to form the disc carrying the ligate and forming a radioligand that binds to the ligate in a time maimed by tagging a ligand on to radio iodine. Now the invention will be described in the accompanying description and with reference to the accompanying drawing. Fig. 1 of the drawing is a graph illustrating the binding of ligand (Hick) to immobilised ligate (NC-VM8). It is to be noted that the binding the ligand to the immobilised ligate increases with time. PROPOSED SOLUTION: It is possible to adsorb the ligate on nitrocellulose and this can be used as an alternate to the CHIP in the study of the real time kinetics of the interaction between the ligand and the ligate. The adsorption of proteins on nitrocellulose is very well documented. However its use is limited to qualitative techniques used routinely in the laboratory. As of now, there has not been any instances known to our best of knowledge where it has been used as a major tool in understanding reactions on quantitative basis. We propose the use of nitrocellulose for adsorption of the ligate and utilise it for the purpose of understanding the real time kinetics of ligand-ligate interaction, much like a chip of the Biscoe. The Table 1 shows that the ligate can indeed be adsorbed on nitrocellulose effectively. A ligate adsorbed on the nitrocellulose disc indeed binds to the ligand in a time dependent maimed is shown in Fig. 1, i.e., the binding of the ligand to the immobilised ligate is increasing with time. Table 1 Adsorption of antibody to nitrocellulose disc (6 mm dial) Binding ability of the adsorbed ligate (Monoclonal antibody) to the radiolabel led ligand (chg.) As an example of the system the following is described. The ligand is human chronic gonadotropin and the ligate is the antibody to chg. The Uganda is tagged on to radioiodine and used as a radioligand. The ligate (antibody to chg.) is adsorbed on Nitrocellulose (adsorbed by keeping fresh nitrocellulose disc with the ligate solution). On adding the radioligand to this ligate coated nitrocellulose disc the radioligand binds to the ligate. After completion of the binding (few hours) the nitrocellulose disc will have radiolabel led ligand-ligate complex. This complex is taken in a microcell and placed in the counting well of the equipment. The microcell is then flushed with buffer to allow the radioactivity (by the washed out Ugand) to be washed out. The radioactivity remaining in the microcell is continuously monitored. The radioactivity represents the qxiantity of the Ugand still remaining on the nitrocellulose disc. The radioactivity (radiograms) absorbed on the nitrocellulose disc represents the quantity of the bound radiograms. As the washing starts, radiograms is washed out of the nitrocellulose disc and hence lost in the wash and (the ligate is irreversibly adsorbed on the Nitrocellulose and hence is not lost), results in reduction of radioactivity in the microcell. The rate is an index of the kinetics of the interaction between the Ugand and the ligate. It is to be noted that the object of the description is to explain the important aspects of the invention. It is to be noted further that within the scope of invention various modifications are permissible. I CLAIM: 1. A method for the preparation of an analytical probe to measure the real time kinetics of ligand-ligate interaction comprising the steps of: (a) immersing a nitrocellulose disc in a ligate solution to form the disc carrying the ligate; (b) forming a radioligand that binds to the Agate in a time dependent manner by tagging a ligand on to radio iodine. 2. A method for the preparation of a an analytical probe to measure the real time kinetics of ligand-ligate interaction substantially as herein before described and illustrated m the accompanying drawing.

Full Text

This invention relates to a method for the preparation of an analytical probe to measure the real time kinetics of ligand-ligate interaction.
FIELD OF TECHNOLOGY:
This invention relates to industrial and laboratory instrument technology. More particularly this invention relates to design and preparation of a probe required to study the real time kinetics of ligand-ligate interaction using radiolabeled ligand.
In our co-pending application No. 495/Mas/99 we have described and claimed an analytical instrument to measure the real time kinetics of ligand-ligate interaction comprising a flow cell, placed in a radioactive counter, a nitrocellulose disc carrying an immobilised ligate being introduced into the flow cell and means for receiving a radiolabeled ligand of known strength from a container of said radiolabeled ligand.
In our co-pending application no. 496/mas/1999 describes and claims an analytical instrument to measure the dissolution of the ligand-ligate interaction which comprises of a nitrocellulose disc carrying an immobilised ligand-ligate complex, said disc being placed in a test tube having a lead shield and a gamma counting well to measure the dissolution of ligand-ligate interaction.
In our co-pending application no. 497/mas/1999 describes and claims an analytical instrument to measure the real time kinetics of ligand-ligate interaction comprising a micro column carrying an immobilised ligand-ligate complex on a nitrocellulose disc, said micro-colimm being placed in a tube containing radio active AURA.
DESCRIPTION OF PRIOR ART:
The existent state of the art utilises a method, known in the field as plasmon resonance technology. This measure the amoimt of a ligand boxed on immobilised ligate using a light probe. This is the most popular approach in use, where ligand-ligate interactions have been and is being studied extensively. The method utilises a high technology adsorption surface (CHIP), on which binding of the ligand is monitored as the solution of the ligand is passed through this chip.

LIMITATION:
One of the primary need in the use of this technique is a high technology item, called the CHIP, This is a small gold plated surface which is used for the adsorption of the ligand in three dimension, measuring 1X2X5 micron dimension, and extremely expensive. The attachment of the ligate to the chip is also very expensive requiring costly reagents. The chip has limited life span in most of the cases, and is rebuked in large numbers for the study of the reaction.
OBJECTS OF THE PRESENT INVENTION:
It is the primary object of the invention to overcome the problems associated with prior art.
The probe prepared by the method of the present invention to study and measure the real time kinetics of ligand-ligate interaction uses a radio labeled ligand for absorption of the ligate and utilises it for the purpose of understanding the real time kinetics of ligand-ligate interaction as the ligate adsorb on the nitrocellulose disc binds to the ligand in a time dependent manner.
This invention thus provides a method for the preparation of an analytical probe to measure the real time kinetics of ligand-ligate interaction comprising the steps of immersing a nitrocellulose disc in a ligate solution to form the disc carrying the ligate and forming a radioligand that binds to the ligate in a time maimed by tagging a ligand on to radio iodine.
Now the invention will be described in the accompanying description and with reference to the accompanying drawing.
Fig. 1 of the drawing is a graph illustrating the binding of ligand (Hick) to immobilised ligate (NC-VM8). It is to be noted that the binding the ligand to the immobilised ligate increases with time.

PROPOSED SOLUTION:
It is possible to adsorb the ligate on nitrocellulose and this can be used as an alternate to the CHIP in the study of the real time kinetics of the interaction between the ligand and the ligate.
The adsorption of proteins on nitrocellulose is very well documented. However its use is limited to qualitative techniques used routinely in the laboratory. As of now, there has not been any instances known to our best of knowledge where it has been used as a major tool in understanding reactions on quantitative basis. We propose the use of nitrocellulose for adsorption of the ligate and utilise it for the purpose of understanding the real time kinetics of ligand-ligate interaction, much like a chip of the Biscoe. The Table 1 shows that the ligate can indeed be adsorbed on nitrocellulose effectively. A ligate adsorbed on the nitrocellulose disc indeed binds to the ligand in a time dependent maimed is shown in Fig. 1, i.e., the binding of the ligand to the immobilised ligate is increasing with time.
Table 1
Adsorption of antibody to nitrocellulose disc (6 mm dial) Binding ability of the adsorbed ligate (Monoclonal antibody) to the radiolabel led ligand (chg.)

As an example of the system the following is described. The ligand is human chronic gonadotropin and the ligate is the antibody to chg.
The Uganda is tagged on to radioiodine and used as a radioligand. The ligate (antibody to chg.) is adsorbed on Nitrocellulose (adsorbed by keeping fresh nitrocellulose disc with the ligate solution). On adding the radioligand to this ligate coated nitrocellulose disc the radioligand binds to the ligate. After completion of the binding (few hours) the nitrocellulose disc will have radiolabel led ligand-ligate complex. This complex is taken

in a microcell and placed in the counting well of the equipment. The microcell is then flushed with buffer to allow the radioactivity (by the washed out Ugand) to be washed out. The radioactivity remaining in the microcell is continuously monitored. The radioactivity represents the qxiantity of the Ugand still remaining on the nitrocellulose disc.
The radioactivity (radiograms) absorbed on the nitrocellulose disc represents the quantity of the bound radiograms. As the washing starts, radiograms is washed out of the nitrocellulose disc and hence lost in the wash and (the ligate is irreversibly adsorbed on the Nitrocellulose and hence is not lost), results in reduction of radioactivity in the microcell. The rate is an index of the kinetics of the interaction between the Ugand and the ligate.
It is to be noted that the object of the description is to explain the important aspects of the invention. It is to be noted further that within the scope of invention various modifications are permissible.

I CLAIM:
1. A method for the preparation of an analytical probe to measure the real time
kinetics of ligand-ligate interaction comprising the steps of:
(a) immersing a nitrocellulose disc in a ligate solution to form the disc carrying the ligate;
(b) forming a radioligand that binds to the Agate in a time dependent manner by tagging a ligand on to radio iodine.
2. A method for the preparation of a an analytical probe to measure the real time
kinetics of ligand-ligate interaction substantially as herein before described and
illustrated m the accompanying drawing.

Documents:

494-mas-1999-abstract.pdf

494-mas-1999-claims filed.pdf

494-mas-1999-claims granted.pdf

494-mas-1999-correspondnece-others.pdf

494-mas-1999-correspondnece-po.pdf

494-mas-1999-description(complete) filed.pdf

494-mas-1999-description(complete) granted.pdf

494-mas-1999-description(provisional).pdf

494-mas-1999-drawings.pdf

494-mas-1999-form 1.pdf

494-mas-1999-form 26.pdf

494-mas-1999-form 3.pdf

494-mas-1999-form 5.pdf

494-mas-1999-other documents.pdf

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Patent Number

210137

Indian Patent Application Number

494/MAS/1999

PG Journal Number

38/2007

Publication Date

21-Sep-2007

Grant Date

21-Sep-2007

Date of Filing

29-Apr-1999

Name of Patentee

INDIAN INSTITUTE OF SCIENCE

Applicant Address

BANGALORE -560 012.

Inventors:

#	Inventor's Name	Inventor's Address
1	G.S. MURTHY	BANGALORE -560 012.

PCT International Classification Number

G 01 N 33/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA