|Title of Invention||
AN ANALYTICAL INSTRUMENT TO MEASURE THE REAL TIME KINETICS OF LIGAND-LIGATE INTERACTION
|Abstract||A Disclosed herein is 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.|
This invention relates to an analytical instrument 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 construction of instrument required studying of the real time kinetics of ligand-ligate interaction radiolabeled ligand. The user of the instruments will be essentially from the academic community interested in the understanding of 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 ligate 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 ligand on to radio iodine.
Our co-pending application no. 496/mas/1999, describes and claims an analytical instrument to measure the dissolution of the ligand-Agate 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.
Another 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 colimm carrying an immobiUsed ligand-ligate complex on a nitrocellulose disc, said micro-column being placed in a tube containing radio active AURA.
DESCRIPTION OF PRIOR ART:
Presently, the hand-ligate interaction study method utilises an equipment known as "BIAcore". The said equipment utilises the Biosensors effectively to understand ligand-ligate 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.
OBJECT OF THE INVENTION:
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.
It is the main object of the present invention to provide an analytical instrument to measure the real time kinetics of ligand-ligate 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 immobilised ligate (on nitrocellulose) is allowed to react with radiolabeled ligand and the binding of the radioactivity on the Nitrocellulose (NC) disc is misfired as a function of time. Using this information it is possible to analyzer the real time kinetics of association. Similarly a ligand-ligate complex immobilised on NC-disc is obtained by incubating immobilised ligate (on NC disc) with radiolabeled ligand taken in a solution phase. The unbound radio ligand is washed off and the NC bound radiolabeled 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 real time kinetics of ligand-ligate interaction comprising a flow cell, placed in a radioactive comers, 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.
Now the invention will be described in detail with reference to drawings accompanying this complete specification.
Fig. 1 of the drams shows in elevation the block diagram of the components incorporated in the instrument; and
Fig, 2 shows binding profile of the ligand to the immobilised ligate.
In order to do this measurement with minimal experimental manipulation a flow cell has been designed. The design of this has been shown m Fig. 1. Various components are clearly illustrated. The NC disc with immobilised ligate is introduced into the flow cell and placed m a radioactive counter (shown in Fig. 1). Radiolabeled ligand of known strength is allowed to pass through the flow cell. On contacting the NC disc the radiolabel starts binding to the NC. This binding is continuously monitored, as the flow cell is always kj)t in the well of the coater. At the end of the binding period the radiolabeled ligand is replaced with buffer and continuously washed out, where the wash liquid is drained rather than re-circulated. The decrease in the radioactivity of the micro cell can be determined with time. Thus this micro cell can be used for measuring the total dynamics of ligand-ligate interaction with minimal experimental manipulation. The software required for this has also been made and forms a part of the invention. Compared to the lead shield, this has the advantage that this can measure both the association and the dissociation, and has a greater efficiency of radioactive detection. A typical binding profile of the iodinated ligand to the immobilised ligate is shown in Fig. 2.
The radioactive counter is a scintillation counter, which is a solid-state radiation detector. Such scintillation comer have been in use since the beginning of the century, making use of the property of certain chemical compounds to emit short light pulses after excitation by the passage of charged particles or by photons of high energy.
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 ligand is tagged on to radio iodine and used as a radioligand. The ligate (antibody to chug) is adsorbed on Nitrocellulose (adsorbed by keeping fixes 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 radiolabeled ligand-ligate complex. This complex is taken in a microceli and placed in the counting well of the equipment. The microceli is then flushed with buffer to allow the radioactivity (by the washed out ligand) to be washed out. The radioactivity remaining in the microceli 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 bound radioligand. As the washing starts, radiogram 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 microceli. 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.
1. 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
2. The analytical instrument as claimed in claim 1, wherein the nitrocellulose disc
carrying the immobilised ligate is obtained by incubating inraiobilised ligate on a
nitrocellulose disc with a radiolabeled ligand taken in a solution phase.
3. The analytical instrument as claimed in claim 1, wherein the means for receiving
the radiolabeled ligand is a peristaltic pump.
4. The analytical instrument as claimed in any one of claims 1 to 3, wherein the flow
cell is placed in a Gamma-counting well.
5. An analytical instrument to measure the real time kinetics of ligand-ligate
interaction substantially as herein before described and illustrated in Fig. 1 of the
|Indian Patent Application Number||495/MAS/1999|
|PG Journal Number||38/2007|
|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|