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US 11,029,320 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.
Claims9
A method of calibrating bilirubin probes to determine binding affinity of the bilirubin probes to bilirubin, the method comprising:
- mixing a bilirubin probe with an aqueous sample of a known concentration of bilirubin (Bt), wherein the bilirubin probe comprises a first fluorophore and a second fluorophore;
- measuring a change in fluorescence of the bilirubin probe in response to binding to bilirubin probe to bilirubin;
- and
- determining binding affinity of the bilirubin probes to bilirubin based on the measured change in fluorescence by fitting the measured change in fluorescence with the following equations 2 or 3:
- I
- λ
-
-
- em
- I
- 0
- =
- 1
- -
- (
- K
- d
- +
- B
- t
- +
- P
- T
- )
- -
- (
- K
- d
- +
- B
- t
- +
- P
- T
- )
- 2
- -
- 4
-
- B
- T
-
- P
- T
- 2
-
- P
- T
- (
- 2
- )
- R
- =
- -
- (
- P
- T
- 2
- +
- (
- 2
-
- K
- d
- -
- 2
-
- B
- T
- )
-
- P
- T
- +
- K
- d
- 2
- +
- 2
-
- B
- T
-
- K
- d
- +
- B
- T
- 2
- )
-
- (
- rR
- 0
- 2
- -
- R
- 0
- )
- +
- (
- P
- T
- +
- K
- d
- -
- B
- T
- )
-
- rR
- 0
- 2
- +
- (
- -
- P
- T
-
- K
- d
- +
- B
- T
- )
-
- R
- 0
- 2
-
- (
- B
- T
-
- r
- 2
-
- R
- 0
- 2
- -
- (
- P
- T
- +
- K
- d
- +
- B
- T
- )
-
- rR
- 0
- +
- P
- T
- )
- (
- 3
- )
- wherein Iλem is the fluorescence intensity of the probe in the sample with blank subtracted, Io is the intensity of the probe in the absence of bilirubin, PT is the total bilirubin probe concentration, BT is the total bilirubin concentration, R is the measured fluorescence ratio (Iλ1/Iλ2), wherein Iλ1 is a fluorescence intensity from the first fluorophore at wavelength λ1 and Iλ2 is a fluorescence intensity from the second fluorophore at wavelength λ2, Ro is the ratio in the absence of bilirubin, r is the Iλ2/Iλ1 ratio of the probe in the absence of the second fluorophore, and Kd is the dissociation constant.
160 more elements
Dependencies are inferred from the claim text.