Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2000-11-15
2003-07-08
Housel, James (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S005000, C435S006120, C435S007100, C435S007200, C435S007920, C435S030000, C435S034000, C435S040500, C435S040510, C436S536000, C436S537000
Reexamination Certificate
active
06589727
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods for measuring molecular interactions and for separating, sorting and sizing particles. In particular, this invention relates to measurement of the affinity between different binding partners, e.g. in an antibody-antigen interaction.
BACKGROUND TO THE INVENTION
Specific molecular recognition is a fundamental process, being the basis of enzyme-ligand interactions, antibody-antigen interactions and the binding of molecules to receptors. Molecular recognition is achieved through non-covalent interactions such as electrostatic interaction (hydrogen bonds) and hydrophobic interactions. Thermodynamic measurements of binding constants and free energy, enthalpy and entropy changes offer insight into the molecular basis of recognition, particularly when coupled with information from X-ray diffraction and, when possible, site-directed mutagenesis.
Direct measurement of the force of interaction has been made by atomic force microscopy (AFM) as well as surface force apparatus. While AFM is capable of measuring bond rupture forces, the technique has the disadvantage that only one measurement can be made at a time. To date, AFM has been used on avidin-biotin interactions (Florin et al, Science, 1995; 264:415), DNA hybridisation (Boland et al, PNAS, 1995; 92:5291), antibody-antigen interactions (Dammer et al, Biophys. J., 1996; 70:2437) and adhesion glycoproteins (Dammer et al, Science, 1995; 267:1173).
Separating biological molecules on the basis of their relative affinifies for ligands is a well recognised technique. For example, in affinity chromatography, the components to be separated are passed down a column that contains a specific ligand. The component of interest binds preferentially and strongly to the column and is retained on the column while the other components are removed. The bound material may be eluted off the column at a later stage.
Separation technologies are an important part of many research experiments. Increasing the sensitivity or selectivity of these techniques is desirable.
Kolomenskii et al, J. Appl. Phys., 1998; 84(4):2404-10, discloses surface cleaning and adhesion studies conducted using laser-generated surface acoustic pulses. The pulses were at a low repetition rate (20 Hz) and constant energy. The procedure was conducted in vacuum, and therefore is not suitable for commercial exploitation. An optical microscope was used to detect the removal of particles and it was not possible to distinguish between particles of different size.
WO-A-98/45692 discloses the use of a piezoelectric crystal sensor for determining the formation/dissociation of clathrate hydrates. Kurosawa et al, Chem. Pharm. Bull, 1990; 38(5):1117-20, reports using such a sensor for the detection of agglutination of antibody-bearing latex. WOA-98/40739 also discloses such a sensor, including a plate on which specific binding entities are immobilised, for use in indicating the presence of cells in a medium. These sensors are used by measuring a change in resonance frequency at constant voltage.
At present, where possible, most viruses are detected by culture of the specimen in cells, since this method is sensitive although time-consuming. Direct detection of viral DNA or RNA in clinical samples can be achieved using PCR and specific primers tailored for the virus of interest. Since PCR involves an amplification step, cross-contamination is a major problem and it is difficult to establish reliable quantitative methods. Other direct methods include electron microscopy, immune electron microscopy, and methods based on antigen detection with enzyme-linked antibodies. These methods are often relatively insensitive and hence require relatively large quantities of the viral particles.
SUMMARY OF THE INVENTION
The present invention is based on the realisation that the bonds between a target molecule, or a target molecule attached to a particles, and a surface, can be ruptured by mechanically oscillating the surface at increasing amplitude, leading to detachment of the target molecule or particle from the surface. The required acceleration, and hence force, will depend on a variety of factors, including the mass of the molecule or particle, the nature of the bond to the surface and the geometric shape or size of the target molecule or particle. The present invention may therefore be used to separate or to size different target molecules, or to detect their presence.
According to a first aspect of the present invention, a method for separating a target analyte from a composition, comprises the steps of:
(i) contacting the composition with a binding partner for the analyte, the binding partner being immobilised on a surface; and
(ii) oscillating the surface at increasing amplitude, to selectively remove the analyte, or other components of the composition, from the surface.
In addition, the present invention may be used in a method for determining the presence or size of particles, or the affinity between binding partners. According to a second aspect of the invention, such a method comprises the steps of:
(i) contacting the binding partners, one of which is immobilised on a surface;
(ii) oscillating the surface at increasing amplitude; and
(iii) detecting a dissociation event.
In this second aspect, the invention may be applied to a variety of physical and chemical bonds, ranging from relatively weak interactions such as hydrogen bonds through to covalent bonds.
Apparatus suitable for use in the present invention comprises a surface having one binding partner immobilised thereon; means for oscillating the surface at increasing amplitude; and means for detecting a dissociation event.
In particular, the apparatus may comprise an acoustic transducer device (ATD), e.g. a quartz crystal microbalance (QCM) or surface acoustic wave device, or any piezoelectric material which can be made to oscillate, e.g. by applying an alternating voltage or magnetic field. These are cheap devices compared to an AFM and can be multiplexed. Another advantage of using such apparatus is that the majority of bonds are broken simultaneously, giving rise to detectable sound and sharp noise peaks at specific accelerations (applied voltage to the ATD). Another advantage is that the ATD can be used as a sensitive microphone, to detect the acoustic emission when the dissociation event occurs.
In most prior art experiments using an ATD, changes in the resonant frequency or phase have been measured when the ATD is driven at constant voltage. In contrast, the present invention involves increasing the driving voltage and hence the amplitude of oscillation of the ATD.
The present invention has widespread applications for separation, sorting and sizing. The Examples show that, in air, streptavidin-labelled spheres can be separated from normal latex spheres using a QCM with a biotinylated surface and with a driving voltage above 0.1 V but below 6 V. The normal latex spheres are removed from the surface, leaving only the streptavidin-labelled spheres attached to the surface (by the stronger streptavidin-biotin bond). This opens up a new form of separation science based on variable force applied for a certain length of time, with application, for instance, in particle-sizing and sorting, cell-sorting, panning for phage as well as the design of new biosensors. Such a separation method is of low cost and can easily be multiplexed and automated. For instance, it is possible to deposit different targets at different positions on the same microbalance and screen a library of ligands against multiple targets simultaneously. Detection and analysis of viral particles, which are of fixed size, is another area of application. Equally importantly, this invention provides a new, sensitive and potentially quantitative tool, to probe the forces involved in molecular recognition.
REFERENCES:
patent: 5552274 (1996-09-01), Oyama et al.
patent: 9840739 (1998-09-01), None
patent: 9845692 (1998-10-01), None
Kolomenskii, Al. A. et al. (1998) “Interaction of Laser-Generated Surface Acoustic Pulses With Fine Part
Dultsev Fedor Nikolaievich
Klenerman David
Ostanin Victor Petrovich
Akubio Ltd.
Foley Shanon
Housel James
Saliwanchik Lloyd & Saliwanchik
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