Ultrasensitive assay for tau and methods of use thereof for assessing traumatic brain injury in tissues and biofluids
View Patent ↗Compositions and methods for accurate and sensitive quantitation of T-tau and P-tau are disclosed.
1. An ultrasensitive assay for quantifying total tau (T-tau) and/or phosphorylated tau (P-tau) in a biological sample, comprising:
a) obtaining a biological sample from a subject;
b) contacting said sample with capture antibodies that are immunologically specific for P-tau or T-tau, said capture antibodies binding said T-tau or P-tau when present in the sample;
c) contacting said antibody-bound total tau or phosphorylated tau with a biotinylated detection antibody;
d) contacting antibody-tau complexes formed in step c) with streptavidin; said streptavidin binding any biotinylated antibody in said antibody complexes;
e) contacting the streptavidin-biotin-antibody complexes of step d) with a biotinylated primer, a T4 DNA ligase-pretreated DNA template, DNA polymerase reaction buffer, bovine serum albumin, nucleotide triphosphates supplemented with dUTP-sulforhodamine 101 acid chloride and DNA polymerase thereby forming a reaction mixture, under conditions suitable for rolling circle amplification (RCA) to occur, thereby generating a fluorescent signal;
f) neutralizing said reaction mixture; and
g) subjecting the mixture of step f) to fluorescence analysis using surround optical detection, thereby quantifying the levels of total tau and/or phosphorylated tau in said sample.
2. The assay of claim 1 , wherein said biological sample is obtained from a subject having a traumatic brain injury (TBI).
3. The assay of claim 2 , wherein said sample is obtained within one hour of said TBI.
4. The assay of claim 2 , said sample is obtained at one hour, one day, one week, two weeks, three weeks, four weeks or six months after said TBI.
5. The assay of claim 2 , wherein the severity of said traumatic brain injury is determined to be mild traumatic brain injury or moderate traumatic brain injury.
6. The assay of claim 1 , further comprising administering a compound to said subject prior to said obtaining step.
7. The assay of claim 2 , wherein said assay is performed after treatment of said TBI, for assessing efficacy of said treatment.
8. The assay of claim 1 , wherein said tau-binding capture antibodies bind a site on tau selected from amino acids 150-190 on tau, a phosphorylated serine at position 202 on P-tau, or phosphorylated threonine at position 231 on P-tau.
9. The method of claim 1 , wherein said method is performed on a multi-well plate, comprising separate wells coated with antibodies that bind phosphorylated tau or total tau.
10. The assay of claim 1 , further comprising correlating said quantity of P-tau with CT scan normality, or a Glasgow Coma Score (GCS).
11. The assay of claim 1 , further comprising measuring one or more additional biomarkers.
12. The assay of claim 1 , wherein said biological sample is obtained from a subject lacking a traumatic brain injury (TBI).