Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
2001-03-16
2003-01-07
Chin, Christopher L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C436S524000, C436S528000, C435S007100, C435S007920, C210S656000, C210S691000, C210S511000, C530S413000, C422S082110
Reexamination Certificate
active
06503760
ABSTRACT:
TECHNICAL FIELD
This invention relates generally to methods for capturing analytes associated with surface-bound ligands and, more specifically, to use of a solid capturing material for capturing analytes eluted from surface-bound ligands.
BACKGROUND OF THE INVENTION
A variety of analytical techniques are used to characterize interactions between molecules, particularly in the context of assays directed to the detection of biomolecular interactions. For example, antibody:antigen interactions are of fundamental importance in many fields, including biology, immunology and pharmacology. In this context, many analytical techniques involve binding of a “ligand” (such as an antibody) to a solid support, followed by contacting the ligand with an “analyte” (such as an antigen). Following contact of the ligand and analyte, some characteristic is measured which is indicative of the interaction, such as the ability of the ligand to bind the analyte. After measurement of the interaction, the ligand:analyte pair is typically disrupted with an elution and/or regeneration solution in order to regenerate surface-bound ligand for further analytical measurement.
The freed analyte of the ligand:analyte pair, however, is commonly not reused; rather, the freed analyte is typically disposed of together with the elution and/or regeneration solution. This practice is undesirable because researchers very often have only limited quantities of the analyte for analytical measurement purposes, and because researchers very often desire to perform further analytical measurements directed to the analyte itself. Accordingly, there is a need in the art to effectively consolidate freed analyte from a ligand:analyte pair such that the freed analyte is amenable to subsequent analytical measurement.
The need to effectively consolidate freed analyte for subsequent analytical measurement may be illustrated in the context of biosensors which use surface plasmon resonance (SPR) to monitor the interactions between an analyte and a ligand bound to a solid support. In this regard, a representative class of biosensor instrumentation is sold by Biacore AB (Uppsala, Sweden) under the trade name BIAcore® (hereinafter referred to as “the BIAcore instrument”). The BIAcore instrument includes a light emitting diode, a sensor chip covered with a thin gold film, an integrated microfluidic cartridge and photo detector. Incoming light from the diode is reflected in the gold film and detected by the photo detector. At a certain angle of incidence (“the SPR angle”), a surface plasmon resonance wave is set up in the gold layer, which is detected as an intensity loss or “dip” in the reflected light. The theoretical basis behind the BIAcore instrument has been fully described in the literature (see, e.g., Jönsson, U. et al.,
Biotechniques
11:620-627 (1991)).
In addition to SPR analysis using the BIAcore instrument, researchers are beginning to appreciate the synergistic effects of coupling SPR technology with other analytical techniques. In this context, the real-time interaction analysis offered by the BIAcore instrument complements other known methods for investigating both biomolecular structure and function. For example, SPR has recently been coupled with mass spectroscopy (i.e., SPR-MS) to provide an extremely powerful micropreparative technique for biomolecular investigations (see, e.g, PCT International Publication No. WO 97/09608). In connection with SPR-MS, analyte is freed from the surface-bound ligand by matrix-assisted laser desorption/ionization for subsequent analytical measurement by mass spectrometry.
One of the problems posed by eluting analyte away from surface-bound ligands for subsequent analytical measurements is that substantial amounts of analyte can be lost due to nonspecific binding of analyte to the walls and other components of the microfluidic cartridge as the elution and/or regeneration solution flows through the microfluidic cartridge. Moreover, once eluted away from surface-bound ligands, analyte must still be consolidated so that there will be enough sample for subsequent analysis. Accordingly, there is a need in the art for improved methods and micropreparative techniques for consolidating biomolecules associated with surface-bound ligands. The present invention fulfills these needs, and provides further related advantages.
SUMMARY OF THE INVENTION
In brief, the present invention is directed to methods for capturing an analyte associated with a surface-bound ligand, as well as to methods for consolidating the same. In one embodiment, the method involves eluting the analyte from the surface-bound ligand by contacting the surface-bound ligand with a first liquid flow that dissociates the analyte from the surface-bound ligand to generate a free analyte within the first liquid flow. The free analyte is then captured by a solid capturing material that is carried within the first liquid flow, yielding a first liquid flow containing captured analyte. The surface to which the surface-bound ligand is attached may be either a sensing surface, such as a sensing surface of a biosensor, or a non-sensing surface.
In an alternative embodiment, the method involves eluting the analyte from the surface-bound ligand on a surface of a biosensor by contacting the surface-bound ligand with a first liquid that dissociates the analyte from the surface-bound ligand to generate a free analyte within the first liquid. The free analyte is then captured by a solid capturing material that is within the first liquid, yielding a first liquid containing captured analyte. In this embodiment, the surface to which the surface-bound ligand is attached is a surface of a biosensor, and the first liquid may be a flowing or non-flowing liquid.
In both of the above embodiments, the captured analyte may be further consolidated with similarly captured analytes at a location removed from the surface-bound ligand. Such consolidation may, for example, be accomplished by passing the captured analytes of the first liquid through a separation device that prevents passage of the captured analytes, but allows passage of the first liquid. Once consolidated, the captured analytes may be contacted with a second liquid that elutes the analyte of the captured analyte from the solid capturing material to yield free analyte, which may then be used in subsequent analytical techniques or procedures.
These and other aspects of the present invention will be evident upon reference to the following detailed description. To this end, various references are cited throughout this application to further illustrate specific aspects of this invention. Such documents are each incorporated herein by reference in their entirety.
DETAILED DESCRIPTION OF THE INVENTION
As mentioned above, the present invention is directed to methods for capturing an analyte associated with surface-bound ligand with a solid capturing material. In a first embodiment, the solid capturing material is carried within a first liquid flow and the surface to which the surface-bound ligand is attached is a sensing or non-sensing surface. In a second embodiment, the solid capturing material is within a first liquid (flowing or non-flowing) and the surface to which the surface-bound ligand is attached is the surface of a biosensor.
In the first embodiment, a method is disclosed for capturing an analyte associated with a surface-bound ligand by eluting the analyte from the surface-bound ligand by contacting the surface-bound ligand with a first liquid flow that dissociates the analyte to generate a free analyte within the first liquid flow. For example, a surface that has been utilized for capturing a solubilized biomolecule (e.g., “real-time” monitoring of analyte-ligand biomolecular interactions with a biosensor) will have an analyte associated with its surface-bound ligand. The analyte is typically associated (e.g., bound) to the ligand by non-covalent forces (such as electrostatic and Lewis acid-Lewis base forces). In the context of this invention, the agent bound to the surface is referred to as a “su
Jansson Östen
Malmqvist Magnus
Biacore AB
Chin Christopher L.
Counts Gary
Seed IP Law Group PLLC
LandOfFree
Method for capturing analytes eluted from surface-bound ligands 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 for capturing analytes eluted from surface-bound ligands, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for capturing analytes eluted from surface-bound ligands will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3027507