5-fluoro-uracil immunoassay
Inventors
Salamone, Salvatore J. • Courtney, Jodi Blake • Stocker, Dennis
Assignees
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Publication Number
US-9551724-B2
Publication Date
2017-01-24
Expiration Date
Abstract
Novel conjugates of 5-fluoro-uracil and novel 5-fluoro-uracil immunogens and monoclonal antibodies generated by these immunogens which are useful in immunoassays for the quantification and monitoring of 5-fluoro-uracil in biological fluids.
Core Innovation
The document discloses an immunoassay system for quantifying 5-fluorouracil (5-FU) in human biological fluids for therapeutic drug management. It emphasizes generation of antibodies with high selectivity for 5-FU and low or insubstantial cross-reactivity to tegafur and other pyrimidines to avoid false positives.
A key aspect is the preparation of immunogens and assay conjugates built from 5-FU derivatives. The immunogen uses a 3-substituted derivative, while the conjugate reagent uses a 1-substituted derivative, with the derivatives conjugated to immunogenic carriers or polyamine polymers via linker or spacer groups.
The document also describes competitive binding immunoassay formats using labeled detection, including fluorescence polarization and nanoparticle aggregation or absorbance, with microtiter solid support and ELISA approaches referenced. It further includes evaluation of cross-reactivity and potency using indirect competitive ELISA, and it reports markedly lower cross-reactivity to interfering bases including uracil, thymine, cytosine, and tegafur.
Claims Coverage
The provided claim coverage centers on one independent claim and dependent refinements defining an immunogen architecture. In total, the inventive scope is anchored on an immunogenic carrier containing a polyamine polymer linked to a compound specified by Y, X, and p.
Immunogen with polyamine polymer-linked compound
An immunogen containing an immunogenic carrier containing a polyamine polymer linked to a compound defined by Y as an organic spacing group, X as a terminal functional group involved in binding to the polyamine polymer, and p as an integer from 0 to 1.
Terminal functional group pattern for binding via defined X
X includes either a carbonyl ester-forming group where R3 is hydrogen or where the attached oxygen atom forms a reactive ester, or a cyano-like group N=C= with R4 being oxygen or sulfur.
Spacer group Y defined by alkylene chain length limits
Y is an alkylene group with 1 to 10 carbon atoms, and integer parameters n (0 to 6), o (0 to 6), and m (1 to 6) define alkylene chain lengths in the represented structure.
Integer p fixed to 0 or 1
p is fixed to either 0 or 1.
Ester product class defined by formed ester type
The ester formed is a lower alkyl ester, imidoester, or amidoester.
The inventive features are directed to an immunogen linked through a spacer group Y and terminal functional group X, with p constrained to 0 or 1 and further narrowing to specific spacer-chain limits, terminal functional group patterns, and ester classes.
Stated Advantages
High selectivity for 5-fluorouracil (5-FU).
Low or insubstantial cross-reactivity to tegafur and other pyrimidines to avoid false positives.
Markedly lower cross-reactivity to interfering bases including uracil, thymine, cytosine, and tegafur.
Using the specific immunogen/reagent combination improves assay specificity versus tegafur.
Documented Applications
Therapeutic drug management using an immunoassay system to quantify 5-fluorouracil (5-FU) in human biological fluids.
Competitive binding immunoassays, including fluorescence polarization, nanoparticle absorbance or aggregation detection, microtiter solid support, and indirect competitive ELISA, for 5-FU measurement with cross-reactivity evaluation.
Indirect competitive ELISA-based immunoassays to determine IC50 and evaluate cross-reactivity of antibodies against uracil, thymine, cytosine, and tegafur.
Immunogen and plate-coating conjugate preparation using immunogenic carriers KLH, BTG, and BSA.
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