Compositions and methods for high-sensitivity immunoassays

Patent metadata

Field	Value
Publication number	US-2021373011-A1
Application number	US-201716322679-A
Country	US
Kind code	A1
Filing date	Aug 7, 2017
Priority date	Aug 5, 2016
Publication date	Dec 2, 2021
Grant date	—

Abstract

Lanthanide chelate lipid nanoparticles, and methods of their synthesis and use are described. Biological molecules labeled with the lipid nanoparticles, useful in bioaffinity assays with improved sensitivities are also described.

First claim

1 . A lipid nanoparticle comprising:
a) a lipid with an aliphatic chain, wherein the chain length ranges from C19 to C50, attached to a linker moiety; b) a lipid with an aliphatic chain, wherein the chain length ranges from C19 to C50, attached to a lanthanide chelate, wherein the lanthanide is selected from the group consisting of Eu3+, Tb3+, Sm3+ and Dy3+; and c) optionally, a lipid with an aliphatic chain, wherein the chain length ranges from C19 to C50.

2 . The lipid nanoparticle of claim 1 , wherein the lipids of components a), b) and c), if present, are independently selected from phospholipids, sphingolipids, sulfolipids, glycerolipids, aminolipids, peptide-lipids, fatty acid derivatives, fatty aldehydes, fatty alcohol derivatives, polymer-lipids, carbohydrate-lipids, or polyol-lipids.

3 . The lipid nanoparticle of claim 2 , wherein the lipids are phospholipids.

4 . The lipid nanoparticle of claim 3 , wherein the phospholipids are independently selected from phosphatidylethanolamine, phosphatidylcholine and mixtures thereof.

5 . The lipid nanoparticle of claim 1 , wherein the chain length of the aliphatic chain of the lipid ranges from C22 to C38.

6 . The lipid nanoparticle of claim 5 , wherein the chain length is C22.

7 . The lipid nanoparticle of claim 1 , wherein the linker moiety is attached to the lipid via a polyethylene glycol based linker, wherein the polyethylene glycol based linker is (—CH2-CH2-O)n, wherein n is an integer from 3-2000.

8 . The lipid nanoparticle of claim 7 , wherein n is from 3-100.

9 . The lipid nanoparticle of claim 7 , wherein n is 3-12.

10 . The lipid nanoparticle of claim 1 , wherein the linker moiety is either a covalent linker moiety or biotin.

11 . The lipid nanoparticle of claim 10 , wherein the covalent linker moiety is selected from the group consisting of maleimide, vinylsulfone, thiopyridine, isothiocyanate, aldehyde, trans-cyclooctene, dibenzoyclooctyl, azide, alkyne, imidazolylcarbonyl, tetrazine, thiol, iodoacetamido, bromoacetamido, succinimidyl, pentachlorophenyl, tetrafluorophenyl, sulfosuccinimdyl

12 . The lipid nanoparticle of claim 11 , wherein the covalent linker moiety is maleimide.

13 . The lipid nanoparticle of claim 10 , wherein the linker moiety is biotin.

14 . The lipid nanoparticle of claim 1 , wherein in the lanthanide chelate the chelating agent is selected from the group consisting of DTPA, EDTA, TTHA, DOTA, NOTA, deferoxamine, DTPMP, TCMC, DOTAM, DOTAGA, DO3A, PCTA, NO2A

15 . The lipid nanoparticle of claim 13 wherein the chelating agent is DTPA.

16 . The lipid nanoparticle of claim 1 , wherein the lanthanide is Eu3+ or Tb3+.

17 . The lipid nanoparticle of claim 16 , wherein the lanthanide is Eu3+.

18 . The lipid nanoparticle of claim 1 , wherein the lipid comprises 1-4 aliphatic chains.

19 . The lipid nanoparticle of claim 18 , wherein the lipid comprises 2 aliphatic chains.

20 . A labeled antibody, wherein the antibody is linked to the lipid nanoparticle of claim 1 .

21 . The labeled antibody of claim 20 , wherein the antibody is a polyclonal antibody.

22 . The labeled antibody of claim 20 , wherein the antibody is a monoclonal antibody.

23 . The labeled antibody of claim 20 , wherein the antibody is an antigen binding fragment selected from Fab, Fab′, (Fab′)2, or Fv fragment, or is a single chain antibody.

24 . The labeled antibody of claim 20 , wherein the antibody is covalently linked to the lipid nanoparticle.

25 . The labeled antibody of claim 20 , wherein the antibody is biotinylated and the linker moiety of the nanoparticle is biotin, and wherein said antibody and said nanoparticle are crosslinked with streptavidin.

26 . The labeled antibody of claim 20 , wherein the antibody is an anti-HRP antibody.

27 . A labeled streptavidin, wherein streptavidin is linked to the nanoparticle of claim 1 .

28 . The labeled streptavidin of claim 27 , wherein the streptavidin is covalently linked to the nanoparticle.

29 . The labeled streptavidin of claim 27 , wherein the linker moiety on the lipid nanoparticle is biotin, and the streptavidin is non-covalently bound to the lipid nanoparticle.

30 . A time-resolved fluorescence bioaffinity assay to determine the presence or concentration of an analyte in a sample comprising the steps of:
a) mixing the sample with a labeled reagent, wherein the labeled reagent is a reagent linked to a lipid nanoparticle of claim 1 ; b) reacting the analyte with the labeled reagent, wherein a bioaffinity reaction between the analyte and the labeled reagent takes place resulting in a reaction product, wherein the analyte is bound to the labeled reagent; c) separating said reaction product from unbound labeled reagents, and d) measuring the amount of lanthanide from the reaction product by time-resolved fluorescence.

31 . A time-resolved fluorescence bioaffinity assay to simultaneously determine the presence or concentration of two to four analytes within a sample comprising the steps of:
a) mixing the sample with two to four analyte-specific labeled reagents, wherein each labeled reagent is linked to a lipid nanoparticle of claim 1 , each with a different lanthanide; b) reacting the analytes with the analyte-specific labeled reagents, wherein specific bioaffinity reactions between the analytes and their corresponding labeled reagents takes place, resulting in two to four reaction products, wherein each analyte is its specific labeled reagent, c) separating said reaction products from unbound labeled reagents, and d) measuring the amount of lanthanide from the reaction products by time-resolved fluorescence.

32 . A time-resolved fluorescence sandwich immunoassay to determine the presence or concentration of an analyte in a sample comprising the steps of:
mixing the sample with a capture antibody immobilized on a surface, an HRP-conjugated reporter antibody and a labeled antibody of claim 26 ; forming an ordered quaternary complex comprising the capture antibody, analyte, HRP-conjugated reporter antibody, and a labeled antibody of claim 26 ; and detecting the amount of lanthanide retained on the surface.

33 . A lipid nanoparticle of claim 1 , wherein the lipid attached to a linker moiety is biotin-(PEG)3-di-C22-phosphatidylethanolamine, the lipid attached to a lanthanide chelate is Eu-DTPA-di-C22-phosphatidylethanolamine, and wherein the lipid is di-C22-phosphatidylcholine.

34 . A lipid nanoparticle of claim 1 , wherein the lipid attached to a linker moiety is biotin-(PEG)12-di-C22-phosphatidylethanolamine, and the lipid attached to a lanthanide chelate is Eu-DTPA-di-C22-phosphatidylethanolamine.

35 . A lipid nanoparticle of claim 1 , wherein the lipid attached to a linker moiety is TCO-(PEG)12-di-C22-phosphatidylethanolamine, and the lipid attached to a lanthanide chelate is Eu-DTPA-di-C22-phosphatidylethanolamine.

36 . In a time-resolved fluorescence bioaffinity assay where a labeled reagent is used to determine the presence or concentration of an analyte in a sample, the improvement wherein the label in the labeled reagent is a lipid nanoparticle of claim 1 .

37 . A time-resolved fluorescence sandwich immunoassay to

Assignees

• Univ Virginia Patent Foundation

Inventors

• Farhi Leon S
• Klibanov Alexander L
• Gaylinn Bruce D

Classifications

• G01N33/54346

  Nanoparticles · CPC title

• G01N2333/908

  acting on hydrogen peroxide as acceptor (1.11) · CPC title

• G01N33/54393 Primary

  Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding · CPC title

• G01N21/6428

  Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53) · CPC title

• G01N21/6408

  with measurement of decay time, time resolved fluorescence · CPC title