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.
Claims21
A high throughput method for generating and screening of probes comprising the steps of:
- (a) providing a reference intracellular Lipid Binding Protein (iLBP) having a binding specificity for a set of unbound molecules of interest;
- (b) generating polynucleotides encoding an iLBP library comprising an assortment of iLBP muteins of the reference iLBP from a polynucleotide template to generate a library of iLBP muteins;
- (c) expressing the iLBP muteins;
- (d) purifying the iLBP muteins by binding to a solid matrix;
- (e) labeling the matrix-bound iLBP muteins with a single fluorophore at a specific site on each iLBP mutein to produce multiple probes with altered specificity for the set of unbound molecules of interest relative to the reference labeled iLBP, labeled at the specific site;
- (f) retrieving the probes with altered specificity for the set of unbound molecules of interest relative to the reference labeled iLBP from the solid matrix;
- (g) screening the probes by fluorescence in the presence and absence of each unbound molecule in the set of unbound molecules of interest to determine binding to the set of unbound molecules of interest;
- and
- (h) characterizing each of the probes according to a set of fluorescence responses of each of the probes to the set of unbound molecules of interest, wherein the response of the probes differs from the response of the reference labeled iLBP,
- wherein the iLBP encoded by the template is a Fatty Acid Binding Protein (FABP), or variant thereof that retains the capability of binding fatty acid.
A high throughput method for generating and screening of probes comprising the steps of:
- (a) providing a reference intracellular Lipid Binding Protein (iLBP) having a binding specificity for a set of unbound molecules of interest;
- (b) generating polynucleotides encoding an iLBP library comprising an assortment of iLBP muteins of the reference iLBP from a polynucleotide template to generate a library of iLBP muteins;
- (c) expressing the iLBP muteins;
- (d) purifying the iLBP muteins by binding to a solid matrix;
- (e) washing the bound iLBP muteins;
- (f) removing the iLBP muteins from the solid matrix;
- (g) labeling the unbound iLBP muteins with a single fluorophore at a specific site on each iLBP mutein to produce multiple probes having altered specificity for the set of unbound molecules of interest relative to the reference iLBP, labeled at the specific site;
- (h) screening the probes by fluorescence in the presence and absence of each unbound molecule in the set of unbound molecules of interest to determine binding to the set of unbound molecules of interest;
- and
- (i) characterizing each of the probes according to a set of fluorescence responses of each of the probes to the set of unbound molecules of interest, wherein the response of the probes differs from the response of the reference labeled iLBP,
- wherein the iLBP encoded by the template is a FABP, or variant thereof that retains the capability of binding fatty acid.
Dependencies are inferred from the claim text.