Chemiluminescent energy transfer assays
AssigneeTropix, Inc.
InventorsIrena Bronstein, Brooks Edwards, John Voyta
Life of this patent
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Abstract
A chemiluminescent assays for the determination of the presence or amount of a biopolymer in bound assays using 1,2-dioxetanes in connection with AttoPhos.TM. as chemiluminescent substrates for enzyme-labeled targets or probes is provided. Further disclosed is a kit for conducting a bioassay for the presence or concentration of a biopolymer comprising a) an enzyme complex; b) a 1,2-dioxetane; and c) AttoPhos.TM..
Claims14
A method for determining the presence or amount of a substance in a biological sample, said method comprising the steps of:
- a) complexing an enzyme with said substance from said biological sample through a specific binding reaction to form an enzyme complex;
- b) adding a hydrophobic fluorometric substrate and a 1,2-dioxetane, wherein the enzyme of said enzyme complex cleaves an enzyme cleavable moiety from both said hydrophobic fluorometric substrate and said 1,2-dioxetane, thereby causing the 1,2-dioxetane to decompose to form an excited state donor, such that an energy transfer occurs from said excited state donor to said fluorometric substrate as acceptor, causing said acceptor to emit, wherein said 1,2-dioxetane is of the formula:
- ##STR7## wherein Z=H, or C1, R.sup.1 is C.sub.1 -C.sub.20 alkyl or C.sub.1-12 aryl or aralkyl;
- Y is phenyl unsubstituted or substituted with an electron donating or electron withdrawing group;
- R.sup.2 is OX and is meta-substituted on Y with respect to the dioxetane ring and X is an enzyme cleavable moiety;
- wherein said 1,2-dioxetane, when X is cleaved, is hydrophobic;
- the emission as a measure of the presence or amount of the substance in the biological sample.
A method of determining the presence or amount of an enzyme in a biological sample, comprising the steps of:
- (a) adding to said biological sample a hydrophobic fluorometric substrate and a 1,2-dioxetane, wherein the enzyme cleaves an enzyme cleavable moiety from both said hydrophobic fluorometric substrate and said 1,2-dioxetane, thereby causing the 1,2-dioxetane to decompose to form an excited state donor, such that an energy transfer occurs from said excited state donor to said fluorometric substrate as acceptor, causing said acceptor to emit, wherein said 1,2-dioxetane is of the formula:
- ##STR8## wherein Z=H or Cl;
- R.sup.1 is C.sub.1 -C.sub.20 alkyl or C.sub.1 -C.sub.12 aryl or aralkyl;
- Y is phenyl unsubstituted or substituted with an electron donating or electron withdrawing group;
- R.sup.2 is OX and is metasubstituted on Y with respect to the dioxetane ring and X is an enzyme cleavable moiety;
- wherein said 1,2-dioxetane, when X is cleaved, is hydrophobic;
- (b) detecting the presence or amount of emission from said hydrophobic fluorescent substrate as a measure of the presence or amount of the enzyme in said biological sample.
Dependencies are inferred from the claim text.