High-sensitivity assay for serum neurofilament light chains; cross-references to related applications.

Abstract

This disclosure provides methods and kits for identifying and treating individuals at risk of or suffering from conditions such as neurological disorders or diseases. Generally, the detection or measurement of one or more biomarkers, such as neurofilament light chains (NfLs), and combinations thereof, using the assays of this disclosure assists in the identification of conditions such as neurological disorders. This disclosure also provides methods for selecting patients for the treatment of neurological disorders.

Claims

1. A method for detecting or quantifying an analyte in a sample (for example, a biological sample such as blood, saliva, or serum, or a sample derived from a biological sample such as a diluted biological sample), (a) Mixing the sample with a composition comprising a chemiluminescent label conjugated to a first antibody or antibody fragment bound to the analyte, wherein the chemiluminescent label is bound to a carrier protein comprising a linker, and the linker is bound to the antibody or antibody fragment; (b) Adding particles to the mixture that have a second antibody or antibody fragment bound to an analyte attached to the surface of the particles; (c) Preparing a mixture for measuring chemiluminescence (e.g., by separating particles having chemiluminescent acridinium conjugated on the surface); (d) to produce chemiluminescence from the preparation; and (e) Detecting the presence of at least one analyte or calculating its concentration by comparing the amount of chemiluminescence with a standard dose-response curve that correlates the amount of light generated for a known concentration of at least one of several analytes. Methods that include...

2. The method according to claim 1, wherein the analyte is a neurofilament.

3. The method according to any one of claims 1 to 2, wherein the chemiluminescent label is conjugated to a first antibody fragment (e.g., F(ab)).

4. The method according to any one of claims 1 to 3, wherein the first antibody or antibody fragment is a mouse monoclonal antibody or a fragment thereof (e.g., F(ab)).

5. The method according to any one of claims 1 to 4, wherein the linker comprises (or is) polyethylene glycol (PEG).

6. The method according to claim 5, wherein polyethylene glycol is 2 to 20 (e.g., 2 to 10, 2 to 5) ethylene glycol units.

7. The method according to any one of claims 1 to 6, wherein the carrier protein is keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), or cationized BSA.

8. The method according to any one of claims 1 to 7, wherein the ratio of acridinium to carrier protein is 50:1 to 1:1 by weight (e.g., 30:1 to 1:1, 25:1 to 5:1).

9. The chemiluminescent label conjugated to the first antibody or antibody fragment that binds to the analyte is i) Labeling a carrier protein by reacting it with a chemiluminescent acridinium compound containing a reactive functional group; and ii) Reacting a linking compound (e.g., a compound containing a linker with reactive functional groups at each end), a first antibody or antibody fragment, and a labeled carrier protein. The method according to any one of claims 1 to 8, formed by...

10. The method according to claim 9, wherein the chemiluminescent acridinium compound is added in excess of the weight of the carrier protein (for example, by 100 × excess or less, or 50 × excess or less, or 40 × excess or less, or 30 × excess or less, or 5 × excess to 25 × excess weight).

11. A chemiluminescent acridinium compound containing a reactive functional group has the following structure, according to any one of claims 9 to 10: RFGLΨ (I) [In the formula, RFG is a reactive functional group for conjugate to the protein, L is either non-existent (i.e., bonded) or, in some cases, base L Ｃ Or Z Ｌ It is a linker that includes, Ψ is a chemiluminescent acridinium containing the following structure: 【Chemistry 1】 "j" and "k" are independent of 0 (for example, all R ２ The base is hydrogen, and all R ３ The base is hydrogen), and is 1, 2, 3, or 4; R １ is hydrogen, -R, -X ｂ , -R Ｌ -X ｂ , -L Ｃ , -R, -L Ｃ -X ｂ (for example, -L １ -X ｂ ), -Z, -R Ｌ , -Z, -L Ｃ , -Z (for example, -L １ -Z), or -R Ｌ , -L Ｃ , -R Ｌ , -Z (for example, -R Ｌ , -L １ , -R Ｌ , -Z); R ２ and R ３ Independently, in each appearance, hydrogen, -R, electron-donating group, -X ｃ , -R Ｌ -X ｃ , -L Ｃ -X ｃ (For example, -L) １ -X ｃ ), and selected from -Z; where two adjacent R ２ or R ３ The groups can combine to form fused ring groups (for example, 5-7 membered fused aryl or heteroaryl groups, 5-7 membered fused heterocyclic groups), where R ２ or R ３ This may include coupling to an imaging agent such as a fluorophore (e.g., rhodamine); L Ｃ divalent C is sometimes substituted (e.g., by 1 to 20 heteroatoms, by 1 to 20 substituents). １～３５ Alkyl, alkenyl, alkynyl, aryl, or arylalkyl radical; Z Ｌ This is a zwitterionic linker group having the following structure: 【Chemistry 2】 "m" is either 0 (i.e., a combination) or 1; "n" and "p" are independently integers between 0 (i.e., combination) and 10 in each occurrence; Z is an independent zwitterionic group having the following structure in each occurrence: 【Transformation 3】 "q" and "l" are independently either 0 or 1; "r" is independently an integer between 0 and 10 (for example, 1 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); X ａ and X ｂ Independently, in each occurrence, it is an anionic group; X ｃ It is a protonated anionic group; L １ is independently, in each occurrence, -O-, -S-, -NH-, -N(R Ｎ ), -(CH ２ ) １～１０ -, -S(=O) １～２ -, -C=C-, -C=C-(CH ２ ) １～３ -, -C(O)-, -O-C(O)-, -C(O)-(CH ２ ) １～４ -, -(CH ２ ) １～４ -C(O)-, -C(O)-O-, -C(O)-N(R Ｎ ), -C(O)-NH-, -N(R Ｎ )-C(O)-, -NH-C(O)-, -C(O)-N(R Ｎ )-(CH ２ ) １～３ -, -(CH ２ ) １～３ -C(O)-N(R Ｎ ), -(CH ２ ) １～３ -N(R Ｎ )-C(O)-, -NH-S(O) １～２ -, -N(R Ｎ )-S(O) １～２ -, -S(O) １～２ -N(R Ｎ ), -S(O) １～２ -NH-, -(CH ２ ) １～３ -NH-S(O) １～２ -, -(CH ２ ) １～３ -N(R Ｎ )-S(O) １～２ -, -(CH ２ ) １～３ -S(O) １～２ -N(R Ｎ ), -(CH ２ ) １～３ -S(O) １～２ -NH-, -O-(CH ２ ) １～４ -, -(CH ２ ) １～４ -O-, -S-(CH ２ ) １～４ -, -(CH ２ ) １～４ -S-, -NH-(CH ２ ) １～４ -, -N(R Ｎ )-(CH ２ ) １～４ -, -(CH ２ ) １～４ -N(R Ｎ )-, -(OCH ２ ) １～１０ -, -(CH ２ O) １～１０ -, -(OCH ２ CH ２ ) １～１０ -, or -(CH ２ CH ２ O) １～１０ -; R Ｌ Independently, in each occurrence, C has possibly one or more (e.g., 1 to 10, 1 to 5) substitution sites (e.g., by 1 to 10 heteroatoms, by 1 to 10 substituents), １～２０ Divalent hydrocarbon radicals (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, arylalkyl); R independently has, in each appearance, hydrogen, or optionally one or more (e.g., 1 to 20, 1 to 10, 1 to 5) substitution sites (e.g., by 1 to 20 heteroatoms, by 1 to 20 substituents), C １～３５ A hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) radical; R' and R'' are independent, in each occurrence, hydrogen or C １～１０ It is alkyl; R Ｎ Independently, in each appearance, hydrogen or C １～５ Alkyl (e.g., methyl, ethyl, propyl); R' is either hydrogen or C １～１０ It is alkyl; or These are salts (for example, halogenated salts such as chloride salts, halosulfonates, haloalkylsulfonates, fluoroalkylsulfonates, and carboxylates such as haloalkylcarboxylates and fluoroalkylcarboxylates).

12. The method according to any one of claims 10 to 11, wherein the reactive functional group of the chemiluminescent acridinium containing the reactive functional group is an N-succinimidyl ester.

13. R ２ and R ３ Independently, -X ｃ , -R Ｌ -X ｃ , -L Ｃ -X ｃ (For example, -L) １ -X ｃ The method according to any one of claims 10 to 12, wherein the method is as follows:

14. R ２ and R ３ These are independently -C(O)OH and -SO ２ OH), -OSO ２ OH), -OP(O)(OR Ｐ )alkoxys substituted with OH, -OH, or combinations thereof (e.g., C １ ~C ４ The method according to any one of claims 10 to 13, wherein the alkoxy is used.

15. The chemiluminescent acridinium containing a reactive functional group is classified as TSPAE-NHS: 【Chemistry 4】 The method according to any one of claims 9 to 14, wherein the salt thereof.

16. The method according to any one of claims 1 to 15, wherein the second antibody or antibody fragment is a biotinylated antibody or antibody fragment.

17. The method according to any one of claims 1 to 16, wherein the detection step can detect differences in concentrations less than 5 pg / mL (for example, less than 4 pg / mL, 1 pg / mL to 5 pg / mL, 2 to 5 pg / mL, 2 to 4 pg / mL, 3 to 4 pg / mL, 2 to 3 pg / mL).

18. The method according to any one of claims 1 to 17, wherein the preparation step comprises separating particles from the mixture, the chemiluminescence is generated from the particles or the separated mixture, and optionally further comprises incubating the mixture before and / or after the addition of the particles.

19. A chemiluminescent label conjugated to a first antibody fragment bound to a neurofilament, wherein the chemiluminescent label is a polyethylene glycol linker (e.g., PEG). ４ PEG like ２ ~PEG １５ It is bound to a carrier protein containing ) and the linker is bound to the antibody fragment, chemiluminescently labeled; and Carrier or excipient An immunoassay composition containing the following:

20. In some cases, solid particles coated with streptavidin conjugated to a biotinylated mouse anti-neurofilament antibody via a linker (e.g., PEG, iodine-PEG), wherein the anti-neurofilament antibody is bound to the neurofilaments. Solid particles in which the bound neurofilaments are further bound to monoclonal mouse antibody fragments linked to carrier proteins conjugated with one or more chemiluminescent acridinium.

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