Development and use of fluorescent probes of unbound analytes
AssigneeFFA Sciences LLC
InventorsALAN MARC KLEINFELD, ANDREW HENRY HUBER, JAMES PATRICK KAMPF, THOMAS KWAN, BAOLONG ZHU
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Abstract
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.
Claims43
A high throughput method for generating and screening of probes comprising the steps of:
- generating polynucleotides encoding a protein library comprising an assortment of proteins which are mutations of a template protein capable of binding to a molecule of interest participating in a binding reaction;
- expressing the proteins;
- purifying the proteins by binding to a solid matrix;
- associating the matrix bound proteins with fluorophores to produce probes;
- retrieving the probes from the solid matrix;
- and
- screening the probes in a fluorometer in the presence and absence of the molecule.
A method for high throughput generating and screening of probes comprising the steps of:
- generating a library of polynucleotides encoding affinity-tagged FABP muteins from a template FABP;
- expressing the FABP muteins and tags;
- purifying the FABP muteins by binding the tags to a solid matrix;
- associating the FABP muteins with fluorophores to produce probes;
- reformatting the probes in an array;
- adding a sample comprising an unbound metabolite to be tested;
- scanning the probes in a fluorometer in the presence and absence of the unbound metabolite to be tested;
- and
- comparing fluorescence of the probes in the presence and absence of the unbound metabolite.
A probe selected from the group consisting of probes listed in Tables 3-7.
A method to determine the concentration of unbound bilirubin in body fluids of a mammal which comprises:
- withdrawing a body fluid from the mammal;
- contacting the body fluid with a probe selected from the group consisting of L2P14F7, L5P16H4, L1P1C12, L1P12E8 and L1P14D6;
- and
- determining the level of unbound bilirubin by measuring binding to the probe and comparing to a standard.
A fluorescently labeled protein based upon SEQ ID NO. 2 which comprises at least one additional mutation at a position(s) selected from the group consisting of positions 11, 14, 17, 18, 21, 23, 31, 34, 36, 38, 40, 47, 49, 51, 53, 55, 60, 62, 68, 70, 72, 73, 74, 78, 80, 82, 89, 91, 93, 102, 104, 106, 113,115, 117, 119, and 126.
A fluorescently labeled mutant of a parent FABP shown as SEQ ID NO:
- 4 having an altered spectral property when associated with an unbound free fatty acid when compared to the parent FABP shown as SEQ ID NO:
- 4.
A fluorescently labeled FABP comprising a mutation at position 72 of SEQ ID NO:
- 2 and at least one additional mutation at a position selected from the group consisting of 11, 14, 17, 18, 21, 23, 31, 34, 36, 38, 40, 47, 49, 51, 53, 55, 60, 62, 68, 70, 73, 74, 78, 80, 82, 89, 91, 93, 102, 104, 106, 113,115, 117, 119, and 126 and which has a value of ΔR/ΔRADIFAB2 with an unbound free fatty acid which is more than 0.1.
A functional engineered fluorescent protein whose amino acid sequence comprises an amino acid sequence of rat intestinal FABP with at least one amino acid substitution at a position selected from the group consisting of 11, 14, 17, 18, 21, 23, 31, 34, 36, 38, 40, 47, 49, 51, 53, 55, 60, 62, 68, 70, 72, 73, 74, 78, 80, 82, 89, 91, 93, 102, 104, 106, 113,115, 117, 119, and 126.
A polynucleotide comprising a nucleotide sequence encoding a functional engineered protein which is selected from the group consisting of:
- (a) a protein comprising an amino acid sequence of rat intestinal FABP with at least one amino acid substitution at a position selected from the group consisting of 11, 14, 17, 18, 21, 23, 31, 34, 36, 38, 40, 47, 49, 51, 53, 55, 60, 62, 68, 70, 72, 73, 74, 78, 80, 82, 89, 91, 93, 102, 104, 106, 113,115, 117, 119, and 126 of SEQ ID NO:
- 4;
- and
- (b) a fluorescently labeled FABP with at least one mutation which has a value of ΔR/ΔRADIFAB2 with an unbound free fatty acid which is more than 0.1.
An iLBP capable of binding an unbound metabolite and having a value of ΔR/ΔRADIFAB2 with an unbound metabolite which is more than 0.1.
Dependencies are inferred from the claim text.