Development and use of fluorescent probes of unbound analytes
A method for high throughput screening of probes is described. These probes are useful for characterization and measurement of unbound metabolites in a fluid sample, particularly characterization and measurement of levels of unbound free fatty acids. By practice of the disclosed invention, a profile of unbound metabolites can be determined for an individual which can be used to determine the individual's relative risk for disease such as stroke, cardiac disease and cancer.
1. A method to determine a concentration of unbound bilirubin in body fluids of a mammal which comprises:
providing an unbound bilirubin probe having a ΔR/ΔR ADIFAB2 which is <0.1 for unbound fatty acids by selecting a fluorescently labeled FABP mutein which binds to unbound bilirubin, wherein the fluorescence intensity of the unbound bilirubin probe is quenched by the binding of unbound bilirubin, and wherein ΔI/I 0 for the unbound bilirubin probe is >1.45 ΔI/I 0 for ADIFAB2, wherein ΔI is the difference in fluorescence intensity of the probe in the presence (I) and absence (I 0 ) of unbound bilirubin by performing the following steps:
(i) providing a reference Fatty Acid Binding Protein (FABP) mutein comprising the amino acid sequence of SEQ ID NO: 2 having amino acid substitutions selected from the group consisting of 18G31M72A, 14I72W117W, 14M72I117W, 126K73F72A, 38172W117W, 14L38A72G117F114E, 18Y31V55V72A and 14M72V117W,
(ii) generating polynucleotides encoding a FABP library comprising an assortment of FABP muteins of the reference FABP mutein from a polynucleotide template to generate a library of FABP muteins,
(iii) expressing the FABP muteins,
(iv) muteins by binding to a solid matrix,
(v) labeling the matrix bound FABP muteins with a single fluorophore at a specific site on each FABP mutein to produce multiple probes with altered specificity for unbound bilirubin relative to the reference labeled FABP mutein from the solid matrix,
(vi) retrieving the probes with altered specificity for unbound bilirubin relative to the reference FABP mutein,
(vii) screening the probes by fluorescence in the presence and absence of unbound bilirubin to determine Kd for unbound bilirubin,
withdrawing a body fluid from the mammal;
contacting the body fluid with the unbound bilirubin probe;
measuring the fluorescence intensity of the unbound bilirubin probe in the body fluid to determine I,
measuring the fluorescence intensity of the unbound bilirubin probe in a fluid without bilirubin present to determine I 0 , and
calculating the unbound bilirubin concentration using I, I 0 and Kd according to the equation: unbound bilirubin concentration=Kd(I/I 0 −1).
2. The method of claim 1 , wherein the body fluid is selected from the group consisting of whole blood, blood plasma, blood serum, urine, CSF, saliva, gastric juices, interstitial fluid, synovial fluid and lymph.
3. The method of claim 1 , wherein the bilirubin binding FABP mutein comprises a substitution at position 117.
4. The method of claim 1 , wherein the bilirubin binding FABP mutein comprises a substitution at position 31.
5. The method of claim 1 , wherein the bilirubin binding FABP mutein comprises a substitution at position 18.
6. The method of claim 1 , wherein the bilirubin binding FABP mutein comprises 1-5 additional substitutions.