Quantification of biomarkers present in dried physiological samples
Inventors
HRUSOVSKY, Edward Kevin • Wilson, David • Shan, Dandan • Chang, Lei • Song, Linan • Jeromin, Andreas • TOBOS, Carmen Ioana • PATEL, Purvish Prahlad
Assignees
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Publication Number
US-12111324-B2
Publication Date
2024-10-08
Expiration Date
Abstract
The present disclosure relates to digital immunoassays performed on diluted samples obtained from dried blood spots, for example dried capillary blood samples, to detect the presence or absence of a physiological condition by quantifying one or a combination of biomarkers at concentrations indicative of the condition.
Core Innovation
The invention provides a method in which a dried sample of physiological fluid from a human subject is eluted in a diluent to form a liquid sample. The diluent comprises one or more immunoglobulins, a detergent, urea, and one or more blocking agents, and the method forms an immunocomplex using a first capture antibody capable of binding Tau and a biotinylated first detection antibody capable of binding Tau.
After washing, the immunocomplex is contacted with a streptavidin-bound label that interacts with the biotin of the biotinylated first detection antibody, and a detectable signal is generated from the streptavidin-bound label. The detectable signal is detected and used to obtain a concentration of Tau in the liquid sample, with the limit of detection (LOD) for Tau less than about 250 pg/mL and/or the limit of quantitation (LOQ) for Tau less than about 250 pg/mL.
The disclosure further characterizes use with dried samples, including dried spots of capillary blood, and expands multiplex measurement by contacting the liquid sample with capture antibodies and detection antibodies for additional neurological biomarkers. It also includes multivariate classification and risk assignment based on biomarker concentration inputs, optionally incorporating demographics, to diagnose or exclude conditions such as traumatic brain injury and multiple sclerosis.
Claims Coverage
The document provides one independent method claim and dependent claims. Across the independent claim, the coverage concentrates on eluting dried human physiological-fluid samples in an immunoglobulin/detergent/urea/blocking-agent diluent, performing Tau-specific immunocomplex formation with biotin-streptavidin detection, and requiring a Tau sensitivity performance with LOD and/or LOQ below about 250 pg/mL.
Eluting dried human physiological fluid in specified diluent and forming Tau immunocomplex
Eluting a dried sample of physiological fluid in a diluent to form a liquid sample, wherein the physiological fluid is from a human subject and the diluent comprises one or more immunoglobulins, a detergent, urea, and one or more blocking agents; contacting at least a portion of the liquid sample with a first capture antibody capable of binding Tau; contacting the mixture with a biotinylated first detection antibody capable of binding Tau, wherein immunocomplex formation comprises a first capture antibody-Tau-first detection antibody.
Streptavidin-bound label detection and Tau concentration from signal with LOD/LOQ limits
Washing the mixture; contacting with a streptavidin-bound label that interacts with the biotin of the biotinylated first detection antibody of the immunocomplex; generating a detectable signal from the streptavidin-bound label; detecting the detectable signal and obtaining a concentration of the Tau in the liquid sample based upon the detectable signal, wherein the limit of detection (LOD) for Tau is less than about 250 pg/mL and/or the limit of quantitation (LOQ) for Tau is less than about 250 pg/mL.
Overall, the claim coverage centers on a Tau immunoassay workflow that begins with eluting dried human physiological-fluid samples using a diluent containing immunoglobulins, detergent, urea, and blocking agents. It then relies on biotinylated Tau detection antibodies and a streptavidin-bound label to generate a detectable signal that is converted into a Tau concentration under specified LOD/LOQ constraints, with dependent coverage extending to additional analytes and sample format refinements.
Stated Advantages
Achieves ultrasensitive detection for Tau with LOD and/or LOQ less than about 250 pg/mL.
Enables multiplex immunoassays using biomarker panels including Tau and additional neuroinjury/neurodegeneration analytes (NF-L, GFAP, UCH-L1, Aβ30/42, S100B, NSE).
Improves multiplexing performance by addressing cross-reactivity, non-specific binding, dilution non-linearity, and spike-recovery issues.
Provides quantitative signal-to-concentration determination of Tau based on the detectable signal.
Improved sensitivity and/or quantification for Tau with an LOD and/or LOQ for Tau of less than about 250 pg/mL.
Documented Applications
Diagnosis/assessment of severity in neuroinjury and neurodegeneration by using biomarker panels (including Tau and additional biomarkers such as NF-L, GFAP, UCH-L1, Aβ30/42, S100B, and NSE) measured in blood and CSF.
Human subject use with physiological fluids, including use of dried samples and diagnostic comparison against healthy/control populations and in relation to time-to-sampling/clinical context.
Neural injury context including traumatic brain injury.
Detecting and classifying neurological conditions and/or neural injury in a human subject using Tau detection and optionally additional biomarker analytes (NF-L, GFAP, UCH L1, Tau, Aβ40, Aβ42, S100B, NSE).
Diagnosing or excluding conditions including traumatic brain injury using neurological biomarker quantification that includes Tau (and additional biomarkers in multiplex contexts).
Diagnosing or excluding conditions including multiple sclerosis using neurological biomarker quantification that includes Tau (and additional biomarkers in multiplex contexts).
Risk assignment and multivariate classification based on biomarker concentration inputs derived from the assay, optionally incorporating demographics.
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