|Title of Invention||
AN ANALYTICAL INSTRUMENT TO MEASURE THE REAL TIME KINETICS OF LIGAND-LIGATE INTERACTION
|Abstract||This invention relates to an instrument to study and measure the realtime kinetics of , ligand-ligate interaction using radiolabelled ligand. The instrument has a ligand-ligate complex immobilised on NC disc incubating immobilised ligate with radiolabelled ligand taken in a solution phase, a micro column placed in the original tube radio active AURA being incorporated in a gamma counting well.|
This invention relates to an analytical instrument to measure the realtime 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 constructions of instrument required to study of the realtime kinetic of Ugand-ligate interaction using radiolabelled ligand. The user of the instruments will be essentially from the academic community interested in the understanding the ligand-ligate interactions.
In our co-pending application No. 494/Mas/99, we have described and claimed 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 Ugate solution to form the disc in a ligate solution carrying the ligate and forming a radioligand that binds to the ligate in a time dependent manner by tagging a Ugand on to radio iodine.
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 coimter, a nitrocellulose disc carrying an immobilized Ugate being introduced into the flow cell and means for receiving a radiolabeled ligand of known strength from a container of said radiolabeled ligand.
Our co-pending application no. 496/mas/1999, describes and claims an analytical instrument to measure the dissolution of the Ugand-ligate interaction which comprises of a nitrocellulose disc carrying an immobiUsed Ugand-Ugate complex, said disc being placed in a test tube having a lead shield and a gamma coating well to measure the dissolution of Ugand-Ugate interaction.
DESCRIPTION OF PMOR ART:
Presently, the Ugand-ligate interaction study method utilises an equipment known as "BIAcore". The said equipment uteruses the Biosensors effectively to understand Ugand-Ugate interaction. It is a commercial equipment and well known to the people in the field. The instrument is extremely expensive, and is generally successful in measuring the
association of the ligand and ligate. However, its ability to measure the dissociation of the complex is poor, and is well documented.
The ability of the above instrument to measure the dissociation of the ligand-ligate complex is poor, and is very well documented for several antigen-antibody interactions. Further, the analysis is done manually.
It is the primary object of the invention to overcome the problem/limitation in the prior art and also provide an automated system using radioactivity measurements.
OBJECTS OF THE PRESENT INVENTION:
It is the main object of the present invention to provide an analytical instrument to measure the real time kinetics of ligand-Ugate interaction, which is more efficient.
It is a further object of the present invention to provide an analytical instrument which is not very expensive.
The immobilized ligate (on nitrocellulose) is allowed to react with radiolabelled ligand and the binding of the radioactivity on the Nitrocellulose (NC) disc is measured as a function of time. Using this information it is possible to analyse the realtime kinetics of association. Similarly a ligand-ligate complex immobilized on NC-disc is obtained by incubating immobilized ligate (on NC disc) with radiolabelled ligand taken in a solution phase. The unbound radio ligand is washed off and the NC bound radiolabelled ligand is continuously washed with buffer containing unlabelled ligand. The reduction of radioactivity on the NC-disc is monitored and can be used in the determination of the kinetic constants of ligand-ligate interaction.
This invention thus provides an analytical instrument to measure the realtime kinetics of ligand-ligate interaction comprising a micro column carrying an immobilized ligand-ligate complex on a nitrocellulose disc, said micro-column being placed in a tube containing radio active AURA.
Radio active AURA is nothing but the immensity of the radioactivity at any given point.
Now the invention will be described in detail with reference to drawings accompanying this complete specification.
Fig. 1 of the drawings shows radiolabelled ligand taken in the glass tube.
Fig. 2 shows the goanna counting well.
Fig. 3 shows another aspect of gamma counting well.
Fig. 4 shows a relation between radioactivity and time.
A glass gadget known as a micro column is used for the continuous monitoring of both association and dissociation. The NC-ligate disc is put at the bottom of the micro column and is allowed to react with radiolabelled ligand taken in a bigger test tube (5 ml) (Fig. 1). The NC keeps on assimilating the radioactivity as time passes, and at varying time periods the microcolumn is taken out of the tube, washed and the accumulated radioactivity is counted (Fig. 3). The micro column, after commuting, is put back into the original tube and reaction allowed to proceed further. The binding of the radioactivity to the NC is determined at several time intervals for the study of association kinetics (Fig. 4).
For dissociation measurements the radiolabel bound NC is put in the microcolumn and placed in a tube containing buffer with unlabelled ligand and its radioactivity is measured, by placing it in a radioactivity measuring equipment. At different time points the microcolunm is taken out, and the radioactivity leached into the buffer is measured in a gamma counter (Fig. 5). Using this data the dissociation profile of ligand-ligate interaction. With the help of such an equipment we will be in a position to measure both association and dissociation in a single experiment. The software required for its analysis has also been designed and forms part of the Invention.
As an example of the system the following is described. The ligand is human chronic gonadotropin and the ligate is the antibody to chug.
The ligand is tagged on to radio iodine and used as a radioligand. The ligate (antibody to hCG) 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 radiolabelled ligand-hated 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 ligand) to be washed out. The radioactivity remaining in the microcell is continuously monitored. The radioactivity represents the quantity of the ligand still remaining on the nitrocellulose disc.
The radioactivity (radioligand) absorbed on the nitrocellulose disc represents the quantity of the boom radioligand. As the washing starts, radioligand 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 ligand 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 the invention various modifications are permissible. Further the scope of the invention is described in the accompanied description.
An instrument to measure the realtime kinetics of ligand-ligate
Interaction comprising a micro column carrying an immobilised ligand-ligate
Complex on a nitrocellulose disc, said micro-colure being placed in a tube
Containing radio activamJRAl
2. The analytical instrument as claimed in claim 1, wherein the immobilised ligand-
ligate complex is obtained by incubating immobilised ligate on a nitrocellulose
Disc with a radio labeled ligand taken in a solution phase.
3. The analytical instrument as claimed in claim 1 or claim 2, wherein the micro column carrying the immobilised ligand-ligate complex on a nitrocellulose disc in the original tube containing radio active AURA is incorporated in a gamma coating well to measure the realtime kinetics of ligand-Agate interaction.
4, An analytical instrument to measure the realtime kinetics of ligand-ligate interaction substantially as hereinbefore described and illustrated in Figs. 1 to 3 of the accompanying drawings.
|Indian Patent Application Number||497/MAS/1999|
|PG Journal Number||50/2007|
|Date of Filing||29-Apr-1999|
|Name of Patentee||INDIAN INSTITUTE OF SCIENCE|
|Applicant Address||BANGALORE 560 012.|
|PCT International Classification Number||G 01 N 31/00|
|PCT International Application Number||N/A|
|PCT International Filing date|