Measurement of binding rate of a binding substance and an analyte
Inventors
Assignees
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Publication Number
US-8133693-B2
Publication Date
2012-03-13
Expiration Date
Abstract
A method of measuring the rate of binding of a binding substance and an analyte, for example in an assay such as an immunoassay, uses an initial step of performing ultrasonication sufficient to disrupt binding between the binding substance and the analyte. After cessation of the ultrasonication, measurements are taken to determine the rate of binding at cessation of said ultrasonication or at a predetermined time thereafter. The ultrasonication results in knowledge of the precise time of the start of the binding reaction which provides a better rate measurement.
Core Innovation
The invention relates to a method of measuring the rate of binding of a binding substance and an analyte. The method disposes the binding substance and the analyte in a medium, performs sonication of the medium sufficient to disrupt binding between the binding substance and the analyte, and then ceases the sonication.
After ceasing the sonication, the method determines the rate of binding at a predetermined time at or after cessation by extrapolation from measurements made after cessation of the sonication. The binding disruption provides a reset of the binding state so that the subsequent measurements are used to determine the binding rate at a defined time.
In the described implementations, binding is measured using optical measurements such as turbidometry, nephelometry, fluorometry, or photometrically. The document further describes immunological binding in a homogeneous immunoassay context and, in an example, monitors absorbance at a specified wavelength for standards and calibration of an analyte such as CRP.
Claims Coverage
The independent claim covers measuring the rate of binding between a binding substance and an analyte by interrupting binding with sonication and determining the binding rate at a predetermined time by extrapolation from post-cessation measurements. The dependent claims refine the measurement modality, binding type, binding substance type, sonication frequency, and extrapolation approach.
Measuring binding-rate at a predetermined time by post-cessation extrapolation
Disposing the binding substance and the analyte in a medium, performing sonication sufficient to disrupt binding between the binding substance and the analyte, ceasing said sonication, and determining the rate of binding at a predetermined time at or after cessation by extrapolation from measurements made after cessation of said sonication.
Determining the rate photometrically
Determining the rate of binding by photometric measurement.
Measuring immunological binding rate
Performing the method such that the binding is immunological binding.
Using antibody, antigen, or hapten as the binding substance
Using a binding substance comprising an antibody, an antigen, or a hapten.
Sonication at least 1 kHz
Performing sonication at a frequency of at least 1 kHz.
Two-stage curve fitting to extrapolate the rate at a predetermined time
Deriving the rate of binding at a predetermined time by extrapolation through fitting a first curve to measurements, using an initial estimate of the rate at said predetermined time to determine a curve fitting algorithm, fitting a second curve to the measurements, and calculating the rate of binding at said predetermined time from the second fitted curve.
Overall, the claims cover a binding-rate measurement method that disrupts binding by sonication, then stops sonication and determines binding rate at a predetermined time using extrapolation from measurements after cessation. Dependent refinements specify measurement modality, binding type, binding substance types, minimum sonication frequency, and curve fitting approaches.
Stated Advantages
Improved timing accuracy versus prior mixing-uncertain methods.
Faster and more sensitive quantification of analyte concentration or presence.
Optional homogeneous immunoassay operation without separating bound/unbound.
Faster start.
Reduced non-specific aggregation disruption.
Documented Applications
Measuring presence or amount of C-reactive protein (CRP) using an example with CRP antibody-coated latex particles and a binding-rate determination with calibration.
Immunological binding measurement in a homogeneous immunoassay context.
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