Method of determining affinity and kinetic properties

Details Method of determining affinity and kinetic properties Method of determining affinity and kinetic properties

1995-11-22

1998-05-19

Chin, Christopher L.

Chemistry: analytical and immunological testing

Involving kinetic measurement of antigen-antibody reaction

204403, 422 57, 422 681, 422 8201, 422 8205, 422 8208, 422 8209, 422 8211, 435 72, 4352871, 4352872, 4352887, 435808, 436518, 436527, 436805, 436806, 310311, 310312, G01N 33557

Patent

active

057535183

DESCRIPTION:

BRIEF SUMMARY
The present invention relates to the determination of the affinity and kinetic properties of particularly low molecular weight compounds with regard to their interactions with a receptor molecule.
Ligand-receptor interactions are usually characterized in the terms of the binding affinity. Determination of rate constants would be helpful to characterize the interaction, but unfortunately only a few methods are available where the kinetic properties of the interaction can be directly visualized. One such method, which has recently been described (Karlsson R. et al., J. Immunol. Meth. 145, 229-240, 1991), uses mass sensitive detection and a flow system for direct kinetic analysis of ligands interacting with their immobilized receptors at a sensor surface. However, this method is restricted to the study of high molecular weight ligands, the increase of mass on the sensor surface in the case of low molecular weight components (M.sub.w <approx. 3000 Da) which interact with an immobilized receptor being too small to be accurately measured.
EP-A-276 142 discloses a method of quantitatively measuring, on a surface of an optical structure capable of exhibiting surface plasmon resonance (SPR), small molecules, such as drugs and hormones, using a competitive assay format. The method is characterized in that the sample antigen will compete with the same antigen (or an antigen analogue) coupled to a comparatively large (optical thickness enhancing) "label particle", such as a latex particle, for the binding to an immobilized receptor. The amount of labelled antigen bound will then be inversely proportional to the concentration of antigen in the sample.
Rogers, K. R., et al., Biosensors & Bioelectronics 6 (1991) 507-516 describes the use of fluorescence-labelled ligands to determine the number of free sites on a receptor bound to a fibre-optical biosensor. To determine the affinity for the reaction of a non-labelled ligand with the receptor, the receptor is incubated with non-labelled ligand, a fluorescence-labelled ligand is added and the fluorescence measured, and the measured fluorescence signal is compared with that obtained when the receptor is reacted directly with the fluorescence-labelled ligand. When the procedure is repeated with a number of concentrations of non-labeled ligand, the affinity constant for the interaction between the non-labeled ligand and the receptor may be calculated.
In accordance with the present invention, it has now been found that the lack of sensitivity of mass-sensing methods to accurately detect low molecular molecules when the size of the receptor molecule is much larger than the molecule to be detected may be advantageously utilized to compare by competitive kinetics the affinity and kinetic properties of different low molecular weight components interacting with the same receptor, using an approach similar to that used for the concentration measurements in EP-A-276 142 above.
According to this novel concept of competitive kinetics, two ligands are allowed to react simultaneously with the binding site of a common immobilised receptor. One of the ligands is of high molecular weight and the other is one of low molecular weight. The progress of the binding interaction with the receptor is studied by recording the binding curves (plotting of the detector response versus time) for, on one hand, each ligand alone and, on the other hand, when the high molecular weight ligand and the low molecular weight ligand compete for the binding site of the receptor. In the latter case, both ligands bind to the receptor but almost the entire signal will be due to the binding of the high molecular weight component. The binding of the low molecular weight component is instead seen as a distortion of the binding curve obtained with the high molecular weight ligand alone. The change in shape and in position of the binding curve obtained under competing conditions thus reflects the kinetic and affinity properties of the low molecular weight ligand-receptor interaction.
By proceeding in this way, for exam

REFERENCES:
Kim R. Rogers et al "Biosensors & Bioelectronics", vol. 6, 1991 pp. 507-516.
Robert Karlsson et al "Journal of Immunological Methods" 145 (1991) pp. 229-240.

Affiliated with

Also associated with

Rating

Method of determining affinity and kinetic properties does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Method of determining affinity and kinetic properties, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method of determining affinity and kinetic properties will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-1852325