Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals – Carrier is inorganic
Reexamination Certificate
1998-01-20
2001-01-30
Brusca, John S. (Department: 1631)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
Carrier is inorganic
C435S006120, C436S526000
Reexamination Certificate
active
06180418
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to assays and more specifically to binding assays, such as antibody/hapten or DNA interactions, taking advantage of the differing strengths of these interactions from each other and from non-specific binding interactions, and using labels that respond to a magnetic field.
2. Description of the Related Art
Binding assays, for example immunoassays, are widely used in the food, medical, and pharmaceutical industries as diagnostic tests for a wide range of target molecules. Many binding assays have been produced and marketed since the principle was first developed.
Immunoassays typically exploit the binding capabilities of antibodies. Antibodies are protein molecules which are frequently considered fighters of infections. They fight infections by binding to the infectious material in a specific manner, forming a complex. This is a signal to the organism to reject that complex. Antibodies may also be produced to specifically bind to a wide range of compounds, as a key fits a lock. However other molecules (e.g., chelators, strands of polynucleic acids, receptors including cellular receptors) that are capable of recognizing and selectively binding other molecules may be employed to detect a wide range of species, such as polynucleic acids (DNA or RNA), polypeptides, glycolipids, hormones, polymers, metal ions, and certain low molecular weight organic species including a number of illegal drugs. To be useful in an assay, this recognition event must generate a signal that is macroscopically observable. The method employed to generate such a signal is one way of distinguishing the various types of immunoassays.
In the initial embodiment of an immunoassay, radioactivity was employed. This radioimmunoassay (RIA) is quite sensitive and widely used, but the hazards, expense, and restrictions associated with handling radioactive material makes alternative immunoassays desirable. Recently, enzyme and fluorescence assays have replaced radioassays.
A few immunoassays use magnetically active labels to detect chemical compounds, and associate the force applied by these magnetic labels in a magnetic field with the amount of the analyte present. See U.S. Pat. Nos. 5,445,970 and 5,445,971, both issued on Aug. 29, 1995 to Rohr.
The Rohr patents use magnetically active beads, an externally applied magnetic field, and a balance for monitoring the force applied by these beads to a chemically modified substrate. In a typical embodiment (a sandwich assay), both the magnetically active beads and the substrate are modified to have antibodies for the analyte bound to their surfaces. The antibody—antibody interactions between the beads and the substrate will not produce specific binding interactions, although some non-specific adsorption may occur. If an external magnetic field is applied, the field will separate the beads from the substrate. If the analyte is introduced into the system, it will bind to the beads and the substrate in a sandwich configuration, thus linking the beads to the substrate through a specific binding interaction. When an external magnetic field is applied to this system, the field will, depending on its orientation, push or pull on the beads, changing the force measured by the balance, thereby indicating the presence and (in principle) amount of analyte present.
One of the drawbacks of Rohr's work is that it only measures an integrated signal related to the behavior of a multitude of beads experiencing a range of magnetic conditions. This is particularly problematic, because the work of the present inventors has shown that the beads in this system interact in complex ways.
Referring to
FIG. 1
, a significant fraction of beads in Rohr's system will tend to clump together, and the behavior of these clumped beads is not easily modeled.
FIG. 1
depicts typical paramagnetic beads on a substrate, in an applied magnetic field, as they might be seen through a typical optical microscope. One sees from
FIG. 1
that some of the beads aggregate into strings and clusters. In some cases, the bead dipoles may align, creating a larger magnetic moment than anticipated. Rohr's invention takes into account none of this behavior, and thus is plagued by low sensitivity. A more reliable qualitative and quantitative system would monitor the behavior of individual beads, and correct for or cancel the effect of complex interactions that would be a source of errors in Rohr's system. Additionally, such a system would inherently be more precise, since each bead could be monitored.
A second drawback of Rohr's work is the unstated assumption that time is not a variable in this system. Each of the assays disclosed in Rohr's patents show the signal to be associated with only the concentration of the analyte. The work of the present inventors has shown that the magnetic beads in an applied field will dissociate from a substrate as a function of time and temperature, following a Boltzmann distribution curve.
An improved binding assay sensor would allow for the discrimination between complex binding interactions at the single bead level. An improved binding assay sensor would also provide additional information beyond the presence or absence of a binding interaction, such as information about the strength of that specific interaction. A part of developing such additional information, it would be valuable to have a method for developing force-binding curves for target analytes.
SUMMARY OF THE INVENTION
Accordingly, it is an object of this invention to selectively detect a wide range of target species, with a high degree of sensitivity.
It is a further object of this invention to detect target species using a transduction mechanism that is independent of the mass of the species.
It is a further object of this invention to obtain additional information beyond the presence or absence of a binding interaction, such as information about the strength of that specific interaction.
It is a further object of the invention to characterize the strength of specific binding interactions.
It is a further object of the invention to develop force-binding curves for a wide range of analyte species.
It is a further object of the invention to use magnetically active beads in an assay that can discriminate the activity of single beads.
It is a further object of the invention to use magnetically active beads in an assay that can isolate the effects of complex magnetic interactions between clusters of beads.
It is a further object of the invention to use magnetically active beads in an assay that can account for the effects of time and temperature on populations of beads bound to a substrate, i.e., the Boltzmann effect.
These and additional objects of the invention are accomplished by the structures and processes hereinafter described.
An aspect of the present invention is a sensor for a selected target species having (a) a substrate which has been chemically modified by attachment of substrate modifiers; (b) one or more magnetically active beads which have been chemically modified by attachment of bead modifiers, where these bead modifiers will have a binding affinity for the substrate modifiers in the presence of the target species, and a measurably different binding affinity for the substrate modifiers in the absence of the target species; (c) an adjustable source of a magnetic field for exerting a force on the beads; and (d) an imaging system, for observing and counting beads bound to said substrate.
In a preferred embodiment, the invention further has a system for identifying clusters of beads, and for removing the effect of such clusters from measurements of the target analyte.
Another aspect of the present invention is a method for sensing the presence of selected target species, having the steps of (a) modifying a substrate by attaching a substrate modifier; (b) modifying one or more magnetically active beads by attaching bead modifiers, where these bead modifiers have a binding affinity for the substrate mo
Brusca John S.
Karasek John J.
Lundgren Jeffrey S.
The United States of America as represented by the Secretary of
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