|Title of Invention||
AN ANALYTICAL INSTRUMENT TO MEASURE THE DISSOLUTION OF THE LIGAND-LIGATE INTERACTION
|Abstract||This invention relates to 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.|
This invention relates to an analytical instrument to measure the dissolution of the 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 to study of the realtime kinetics of ligand-ligate interaction using radiolabelled ligand. The user of the instruments will be essentially from the academic community interested in the understanding of the ligand-ligate interactions.
Copending application No. 494/Mas/99 describes an analytical instrument using nitrocellulose disc for quantitative study of the realtime kinetics of ligand-ligate interaction and application nos. 495/Mas/99 and 497/Mas/99 describe different types of analytical instruments using the said nitrocellulose disc.
DESCRIPTION OF PRIOR ART:
Presently, the ligand-ligate interaction study method utilises an equipment known as BIAcore. The said equipment utilizes 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 questioned as documented in several antigen-antibody interaction studies, as well as form the data from several laboratories from the institute.
OBJECTS OF THE PRESENT INVENTION:
It is 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 dissolution 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 immobilised ligand-ligate complex on nitrocellulose (NC) disc is obtained by incubating immobiUsed ligate on nitrocellulose (NC) disc with radiolabelled ligand taken in a solution phase. The unbound radio Ugand is washed off and radioactivity of the NC is measured (this measures the total ligand-ligate present as immobilised complex). This NC disc is continuosly washed with buffer. The release of radioactivity is monitored and be used in the determination of the kinetic constants of Ugand-Ugate interaction.
This invention thus provides an analytical instrument to measure dissolution of the ligand-ligate interaction, which comprises of a nitrocellulose disc carrying an immobilised Ugand-ligate complex, said disc being placed in a test tube having a lead shield.
Now the invention will be described in detail with reference to drawings accompany this complete specification wherein;
Fig. 1 shows nitrocellulose disc in a tube which is contained in the radiation Aura,
Fig. 2 shows the entire set of the instrument; and
Fig. 3 shows a relation between the released radioactivity and time.
A continuous measurement of released radioactivity was done using a lead shield (Fig. 1). The figure shows the radiation pattern emerging from the NC disc. Fig. 2 shows
the shielding of the radioactivity from the NC disc. It is also obvious that the radioactivity from the solution phase is exposed to the counting well of the gamma counter and hence the measurement of the radioactivity is only from the liquid phase. From Fig. 2 it can be observed that some part of the radiation m the liquid phase is also masked by the lead shield and hence recovery of the alter the determination of dissolution kinetics. Thus using the lead shield the release rate of radioactivity can be measured without any physical manipulation of the experimental system. Fig. 3 shows the releases of radioactivity. This was utilised in the measurement of the real-time kinetics constants of the interaction. Such a lead shield would also help in the automation of the system.
As an example of the system the following is described. The ligand is human chorionic gonadotropin and the ligate is the antibody to hCG.
The ligand is tagged on to radioiodine 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 Ugate coated nitrocellulose disc the radioUgand binds to the ligate. After completion of the binding (few hours) the nitrocellulose disc will have radiolabelled ligand-Ugate complex. This complex is taken in a microcell and placed in the coxmting 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 bound 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 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 accompany description.
We CLAIM :
1. 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.
2. The analytical instrument as claimed in claim 1, wherein the immobilised ligand-
ligate complex is obtained by incubating immobilised ligate on the nitrocellulose
disc with a radio labelled ligand taken in a solution phase.
3. The analytical instrument as claimed in claim 1 or claim 2, wherein the tube
containing the nitrocellulose disc and lead shield is incorporated in a gamma
counting well to measure the dissolution of ligand-ligate interaction.
4. An analytical instrument to measure the dissolution of the ligand-ligate interaction
substantially as herein before described and illustrated in Figs 1 & 2 of the
|Indian Patent Application Number||496/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|