# US 2024/0201183 A1 · Assay for rapid detection of cerebrospinal fluid leaks

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- Number: US 2024/0201183 A1
- Document ID: US20240201183A1
- Status: Published application
- Kind: A1
- Application number: 18/404,228
- Filed: 2024-01-04
- Published: 2024-06-20
- Earliest claimed: 2021-06-30
- Assignees: NSPC Technologies, LLC; Gwangju Institute of Science and Technology
- Applicants: NSPC Technologies, LLC; Gwangju Institute of Science and Technology
- Inventors: Min-Gon Kim
- Web page: https://patentgrep.com/patent/US20240201183A1

## CPC

- G01N 33/54388: {based on lateral flow}
- G01N 33/561: Immunoelectrophoresis
- G01N 33/6803: {General methods of protein analysis not limited to specific proteins or families of proteins}
- G01N 2333/42: Lectins, e.g. concanavalin, phytohaemagglutinin
- G01N 2333/79: Transferrins, e.g. lactoferrins, ovotransferrins
- 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)

## Abstract

The invention encompasses methods and test strips for detecting the presence of cerebrospinal fluid (CSF) in a biological sample with a lateral flow device which uses lectin conjugates, anti-antigen conjugates, an immobilized serum line, and an immobilized anti-antigen line.

## Claims

### Claim 1

A method of identifying a subject as having a cerebrospinal fluid leak comprising:
detecting if cerebrospinal fluid (CSF) is present in a liquid sample from the subject which is a blood sample, a serum sample, an otorrhea sample, a rhinorrhea sample, or comprises drainage from a spinal suture area by
a) distributing the liquid sample on to a sample pad of a lateral flow device which comprises, in lateral flow sequence: a sample pad, a conjugate pad, a binding portion, and an absorbent pad, so as to permit the sample to flow along the lateral flow device; and wherein:
(i) the conjugate pad thereof, downstream in lateral flow from the sample pad, comprises both (A) and (B) thereon, wherein:
(A) is a first plurality of transferrin-binding antibodies conjugated to a first plurality of nanoparticles, and
(B) is a plurality of lectin molecules conjugated to a second plurality of nanoparticles;
(ii) the binding portion thereof, downstream in lateral flow from the conjugate pad, comprises the following separate domains:
a. a domain which comprises a second plurality of transferrin-binding antibodies which are adhered to a surface of the lateral flow device;
b. a dried blood serum domain reagent adhered to a surface of the lateral flow device;
b) determining an intensity of an optical signal present at domain (ii)(a) and also at domain (ii)(b);
c) calculating an optical value, wherein the optical value is determined as =(T1 intensity)2×(T2 intensity), wherein T1 intensity is the optical signal intensity at domain (ii)(a), and T2 intensity is the optical signal intensity at domain (ii)(b), and determining if the optical value is above or below a pre-determined cut-off value for the lateral flow device,
wherein if the optical value is above the predetermined cut-off value for the lateral flow device then the liquid sample is identified as containing cerebrospinal fluid, and wherein if the optical value is below the pre-determined cut-off value for the lateral flow device then the sample is identified as not containing cerebrospinal fluid, and
where CSF has been detected as present in the sample, identifying the subject as having a cerebrospinal fluid leak.

### Claim 2 (depends on 1)

The method of claim 1, further comprising obtaining the sample from the subject.

### Claim 3 (depends on 1)

The method of claim 1, wherein the sample comprises drainage from a spinal suture area.

### Claim 4 (depends on 1)

The method of claim 1, wherein, in the sample pad portion, the first plurality of transferrin-binding antibodies have been oxidized previously to remove sialic acid residues therefrom.

### Claim 5 (depends on 1)

The method of claim 1, wherein, in the binding portion, the second plurality of transferrin-binding antibodies have been oxidized previously to remove sialic acid residues therefrom.

### Claim 6 (depends on 1)

The method of claim 1, wherein the plurality of lectin molecules comprises a Sambucus nigra lectin.

### Claim 7 (depends on 1)

The method of claim 1, wherein the optical signals are determined using an optical reader device.

### Claim 8 (depends on 7)

The method of claim 7, wherein the optical reader device further provides the optical value calculated in step c).

### Claim 9 (depends on 1)

The method of claim 1, wherein the dried blood serum reagent domain adhered to the surface of the lateral flow device comprises human serum.

### Claim 10 (depends on 1)

The method of claim 1, wherein the liquid biological sample distributed on the sample pad portion has been diluted 90× to 110× with a buffer solution from a raw sample obtained from a human prior to sample being distributed on the sample pad portion and/or wherein a dried blood serum reagent has been diluted 90× to 100× prior to being adhered to the surface of the lateral flow device.

### Claim 11 (depends on 10)

The method of claim 10, wherein the buffer solution comprises phosphate buffered saline comprising polyvinylpyrrolidone, a surfactant and is adjusted to pH 7.4.

### Claim 12 (depends on 1)

The method of claim 1, wherein the sample comprise a blood, serum, otorrhea or rhinorrhea sample obtained from a human.

### Claim 13 (depends on 1)

The method of claim 1, wherein predetermined cutoff value has been determined beforehand to account for Hook effect on binding of human serum glycoproteins to the dried blood serum reagent domain at concentrations of human serum glycoproteins in a non-diluted human serum sample and provide a positive signal.

### Claim 14 (depends on 13)

The method of claim 13, wherein the predetermined cutoff value has been determined beforehand such that human serum diluted 90× to 110× with a buffer solution from a previously-undiluted serum sample obtained from a human, wherein the sample is not diluted with cerebrospinal fluid and does not contain cerebrospinal fluid, gives an optical value below the predetermined cutoff value, or wherein the predetermined cutoff value has been determined beforehand such that human serum diluted 90× to 110× with a buffer solution from a previously-undiluted serum sample obtained from a human, wherein the sample is diluted with cerebrospinal fluid or does contain cerebrospinal fluid, gives an optical value below the predetermined cutoff value.

### Claim 15 (depends on 1)

The method of claim 1, further comprising empirically determining the predetermined cutoff value beforehand by running a plurality of samples on the lateral flow device of human serum diluted 90× to 110× with a buffer solution from a previously-undiluted serum sample previously obtained from one or more humans, wherein the serum samples do not contain CSF, and calculating the optical value for each of the plurality of samples from (T1 intensity)2×(T2 intensity), and selecting an optical value which is in excess of every one of the calculated optical values for each of the plurality of samples as the predetermined cutoff value.

### Claim 16 (depends on 1)

The method of claim 1, further comprising empirically determining the predetermined cutoff value beforehand by (a) running a plurality of samples on the lateral flow device of human serum diluted 90× to 110× with a buffer solution from a previously-undiluted serum sample previously obtained from one or more humans, wherein the serum samples do not contain CSF, and calculating the optical value for each of those plurality of samples from (T1 intensity)2×(T2 intensity), and (b) running a plurality of samples on the lateral flow device of human serum diluted 90× to 110× with a buffer solution from a previously-undiluted serum sample previously obtained from one or more humans, wherein the serum samples do contain CSF, and calculating the optical value for each of those plurality from (T1 intensity)2×(T2 intensity), wherein steps (a) and (b) can be performed in any order, and (c) selecting an optical value, as the predetermined cut off, which optical value is (i) in excess of every one of the calculated optical values for each of the plurality of samples in step (a) which do not contain CSF, and (ii) is below every one of the calculated optical values for each of the plurality of samples in step (b) which do contain CSF, so as to determine the predetermined cutoff value.