# US 12,111,324 B2 · Quantification of biomarkers present in dried physiological samples

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- Number: US 12,111,324 B2
- Document ID: US12111324B2
- Status: Granted
- Kind: B2
- Application number: 17/046,116
- Filed: 2019-04-09
- Published: 2021-04-01
- Granted: 2024-10-08
- Earliest claimed: 2018-04-10
- Assignees: Quanterix Corporation
- Applicants: Quanterix Corporation
- Inventors: Edward Kevin Hrusovsky; David Wilson; Dandan Shan; Lei Chang; Linan Song; Andreas Jeromin; Carmen Ioana Tobos; Purvish Prahlad Patel
- Web page: https://patentgrep.com/patent/US12111324B2

## CPC

- G01N 33/6896: {Neurological disorders, e.g. Alzheimer's disease}
- A61B 5/0071: {by measuring fluorescence emission}
- A61B 5/0075: {by spectroscopy, i.e. measuring spectra, e.g. Raman spectroscopy, infrared absorption spectroscopy (A61B5/0071 takes precedence)}
- A61B 5/0082: {adapted for particular medical purposes}
- A61B 5/4064: {Evaluating the brain (for intracranial pressure A61B5/031; for cerebral blood gases A61B5/14553; using EEG A61B5/369)}
- G01N 1/38: Diluting, dispersing or mixing samples
- G01N 33/582: {with fluorescent label}
- G01N 2001/386: {Other diluting or mixing processes}
- G01N 2800/28: Neurological disorders
- G01N 2800/2821: Alzheimer
- G01N 2800/285: Demyelinating diseases; Multipel sclerosis
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- A61B: DIAGNOSIS; SURGERY; IDENTIFICATION (usefulness limited to only animals A61D)

## 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.

## Claims

### Claim 1

A method, comprising:
i) eluting a dried sample of physiological fluid in a diluent to form a liquid sample, wherein the physiological fluid obtained is from a human subject, and wherein the diluent comprises one or more immunoglobulins, a detergent, urea, and one or more blocking agents;
ii) contacting at least a portion of the liquid sample with a first capture antibody capable of binding tau protein (Tau);
iii) contacting the mixture of step ii) with a plurality of detectable agents, wherein the plurality of detectable agents comprises a first detectable agent comprising a biotinylated first detection antibody capable of binding Tau, and wherein the contacting comprises the formation of an immunocomplex of first capture antibody-Tau-first detection antibody;
iv) washing the mixture of step iii);
v) contacting the combination of step iv) with a streptavidin-bound label, wherein the streptavidin-bound label interacts with a biotin of the biotinylated first detection antibody of the immunocomplex;
vi) washing the mixture of step v);
vii) generating a detectable signal from the streptavidin-bound label of step vi);
viii) detecting the detectable signal of step vii), 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; and
ix) obtaining a concentration of the Tau in the liquid sample based upon the detectable signal.

### Claim 2 (depends on 1)

The method of claim 1, wherein the dried sample of physiological fluid comprises one or more dried spots of capillary blood.

### Claim 3 (depends on 1)

The method of claim 1, wherein the one or more immunoglobulins comprises human IgG at a concentration of between 1 mg/mL and 10 mg/mL.

### Claim 4 (depends on 1)

The method of claim 1, wherein the diluent further comprises phosphate, one or more ionic salts, bovine serum albumin (BSA), a sugar, Triton X-100, and ProClin 300.

### Claim 5 (depends on 1)

The method of claim 1, wherein the detergent comprises Triton X-100.

### Claim 6 (depends on 1)

The method of claim 1, wherein the one or more blocking agents comprises TRUblock and/or Superchemiblock.

### Claim 7 (depends on 1)

The method of claim 1, wherein the one or more immunoglobulins comprises bovine gamma globulin (BgG), human immunoglobulin (IgG), or a combination thereof.

### Claim 8 (depends on 1)

The method of claim 1, wherein the dried sample is derived from venous blood, capillary blood, serum, plasma, whole blood, cerebrospinal fluid, urine, or saliva.

### Claim 9 (depends on 1)

The method of claim 1, wherein the first capture antibody is associated with a bead.

### Claim 10 (depends on 9)

The method of claim 9, wherein the bead is a paramagnetic bead.

### Claim 11 (depends on 1)

The method of claim 1, wherein:
a) the step ii) further comprises contacting the at least portion of the liquid sample with a second capture antibody capable of binding a second analyte, wherein the second analyte is neurofilament light chain (NF-L), glial fibrillary acidic protein (GFAP), ubiquitin carboxyl-terminal hydrolase L1 (UCH L1), amyloid beta 40 (A beta 40), amyloid beta 42 (A beta 42), S100 calcium-binding protein B (S100B), or neuron-specific enolase (NSE); and
b) the plurality of detectable agents contacted in the step iii) further comprises a second detectable agent comprising a second antibody capable of binding the second analyte, and wherein said contacting of step iii) further comprises the formation of a second immunocomplex of second capture antibody-second analyte-second detection antibody.

### Claim 12 (depends on 11)

The method of claim 11, wherein the second analyte is neurofilament light chain (NF-L), glial fibrillary acidic protein (GFAP), or ubiquitin carboxyl-terminal hydrolase L1 (UCH LI).

### Claim 13 (depends on 1)

The method of claim 1, wherein the subject is a subject suspected of a neural injury.

### Claim 14 (depends on 13)

The method of claim 13, wherein the neural injury is a traumatic brain injury.

### Claim 15 (depends on 14)

The method of claim 14, wherein the traumatic brain injury is collateral to childbirth in the subject.