Stabilized standards for busulfan immunoassay
Inventors
Salamone, Salvatore J. • Courtney, Jodi Blake • Li, Yunying
Assignees
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Publication Number
US-8329416-B2
Publication Date
2012-12-11
Expiration Date
Abstract
Use of busulfan amide as stabilized standards in immunoassays for quantifying the amount of busulfan in samples of human biological fluids, methods for carrying out said immunoassay and kits for use in said immunoassay.
Core Innovation
The invention provides stabilized standards for a busulfan immunoassay by using an amide of formula II as a stable substitute for unstable busulfan standards. Because busulfan standards decompose, the disclosed amide standards are used to enable calibration and quantification of busulfan in a patient's sample. The busulfan amide standards are correlated with busulfan in the immunoassay using a predetermined busulfan equivalence factor that is constant across the dose-response range.
The disclosure states that an immunoassay is performed using multiple standards in separate containers and reagents packaged in containers separate from the standards. The reagents include an antibody selectively reactive with busulfan, and not substantially cross-reactive with tetramethylene and tetrahydrothiophene-3-ol-1,1-dioxide. The immunoassay reagents further include a conjugate of a polyamine polymer with a compound of a specified formula, or a compound of an alternative specified formula.
The invention includes rationale and evidence for stability and correlation. FIG. 1 is referenced for busulfan decomposition, and FIG. 2 is referenced for amide stability, with the amide of formula II described as stable for weeks to greater than one year while busulfan decomposes. FIG. 3 is referenced for a constant calibration factor and correlation, with calibration equivalence verified by experimental context including ELISA and GC-MS-verified calibration equivalence.
Claims Coverage
The document provides one independent claim (kit) defining the immunoassay kit configuration and the chemistry of the reagents and standards, plus dependent claims that refine how calibration standards are used and the stated concentration range in an inert liquid medium. The independent claim contains inventive features focused on using amide-of-formula-II standards with a busulfan equivalence factor and employing selective, low-cross-reactivity antibody chemistry within a polyamine polymer conjugate reagent format.
Amide-of-formula-II standards correlated to busulfan
A kit having a plurality of standards in separate containers, each standard containing a different predetermined concentration or amount of an amide of the formula, wherein the immunoassay uses a predetermined busulfan equivalence factor to correlate the amide standards to busulfan.
Separate packaging of standards and immunoassay reagents
Standards are packaged in separate containers, and reagents for carrying out the busulfan immunoassay are packaged in containers separate from those of the standards.
Selective antibody reagent with limited cross-reactivity
Reagents for carrying out the immunoassay comprise one reagent container containing an antibody selectively reactive with busulfan and not substantially cross-reactive with tetramethylene and tetrahydrothiophene-3-ol-1,1-dioxide.
Polyamine polymer conjugate reagent
The other reagent container comprises a conjugate of a polyamine polymer with a compound of a specified formula, wherein A is lower alkylene, X′ is a functional linking group, Y is an organic spacing group, X is a terminal functional group capable of binding to the polyamine polymer, and p and z are independent integers from 0 to 1; or a compound of an alternative specified formula wherein A′ is lower alkyl or lower alkenylene.
Calibration with at least three standards
A kit wherein the plurality of standards comprises at least three standards such that measured results from immunoassay of the standards can be plotted versus the concentration of the amide to generate a curve or a straight line.
Inert liquid medium and stated concentration range for standards
A kit wherein each standard contains the amide of formula II in an inert liquid medium, with the amide concentration ranging from 0 to 16,000 ng/ml.
Overall claim coverage centers on a busulfan immunoassay kit that uses amide-of-formula-II standards correlated to busulfan by a predetermined equivalence factor, combined with an antibody selectively reactive with busulfan and not substantially cross-reactive with specified compounds, and a polyamine polymer conjugate reagent. Dependent claims further require at least three calibration standards and specify an inert liquid medium with an amide concentration range of 0 to 16,000 ng/ml.
Stated Advantages
Stabilized standards for a busulfan immunoassay that substitute for unstable busulfan standards.
Amide standards remain stable for weeks to greater than one year while busulfan decomposes.
Busulfan quantification using calibration based on a predetermined busulfan equivalence factor constant across the dose-response range.
Antibody selectivity with busulfan and lack of substantial cross-reactivity with tetramethylene and tetrahydrothiophene-3-ol-1,1-dioxide.
Documented Applications
Determining the amount of busulfan in a patient's sample using a busulfan immunoassay with the disclosed kit format.
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