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US 10,295,551 B2Granted
Development and use of fluorescent probes of unbound bilirubin
AssigneeAlan Marc Kleinfeld
InventorsAlan Marc Kleinfeld, Andrew Henry Huber, James Patrick Kampf, Thomas Kwan, Baolong Zhu
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Abstract
Identification and use of proteins fluorescently labeled and that undergo a change in fluorescence index upon binding bilirubin are described. Probes are disclosed which are labeled at a cysteine or lysine residue and also probes labeled at both cysteine and lysine with two different fluorophores. These probes are useful for determination of unbound bilirubin levels in a fluid sample.
Claims14
A solid substrate comprising:
- a polystyrene or latex bead, or Ni-agarose bead;
- and
- a probe attached to the solid substrate, wherein the probe comprises an iLBP comprising SEQ ID NO:
- 3, wherein SEQ ID NO:
- 3 comprises one or more amino acid substitutions at positions selected from the group consisting of positions 14, 18, 23, 25, 27, 31, 36, 38, 55, 60, 72, 73, 74, 78, 102, 104, 106, 115, and 117 and a first fluorophore wherein the first fluorophore is attached to a lysine residue, the N-terminus amino group of the iLBP, or to a cysteine substitution in SEQ ID NO:
- 3, and wherein C-terminus His-tag of SEQ ID NO:
- 3 is replaced with one or more linkers in combination with one or two 6-His tags wherein the linkers are selected from GGSGSGSG (SEQ ID NO:
- 26), GGGSGGGSGGGTGGGSGGGRRADAA(SEQ ID NO:
- 27), SRAWRHPQFGG (SEQ ID NO:
- 28), RAFIASRRIRRP (SEQ ID NO:
- 29), RLLLRRLRR (SEQ ID NO:
- 30), RIIIRRIRR (SEQ ID NO:
- 41), AAS, NDN, PSNTNHNSNSN (SEQ ID NO:
- 31), SHRATPNTSPH (SEQ ID NO:
- 32) and combinations thereof, wherein the probe binds to bilirubin but does not significantly bind to fatty acid.
12 more elements
Dependencies are inferred from the claim text.