Compositions and methods for high-sensitivity immunoassays

Patent metadata

Field	Value
Publication number	US-12326448-B2
Application number	US-201716322679-A
Country	US
Kind code	B2
Filing date	Aug 7, 2017
Priority date	Aug 5, 2016
Publication date	Jun 10, 2025
Grant date	Jun 10, 2025

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

The claimed invention is:

1. A lipid nanoparticle consisting of:
a) a plurality of phospholipids with aliphatic chains with chain lengths ranging from C19 to C50, wherein each phospholipid is attached to linker moiety, wherein the linker moiety is attached to the phospholipid via a polyethylene glycol linker, wherein the polyethylene glycol linker is (—CH 2 —CH 2 —O) n , wherein n is an integer from 3-2000; and
b) a plurality of phospholipids with aliphatic chains with chain lengths ranging from C19 to C50, wherein each phospholipid is attached to a lanthanide chelate, wherein the lanthanide chelate consists of:
(i) a lanthanide ion selected from the group consisting of Eu 3+ , Tb 3+ , Sm 3+ , and Dy 3+ ; and
(ii) a chelating agent.

2. The lipid nanoparticle of claim 1 , wherein the phospholipids of components a) and b) are independently selected from phosphatidylethanolamine, phosphatidylcholine, and mixtures thereof.

3. The lipid nanoparticle of claim 1 , wherein the chain lengths of the aliphatic chains of the phospholipids range from C22 to C38.

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

5. The lipid nanoparticle of claim 4 , wherein the linker moiety is a covalent linker moiety selected from the group consisting of maleimide, vinylsulfone, thiopyridine, isothiocyanate, aldehyde, trans-cyclooctene, dibenzoyclooctyl, azide, alkyne, imidazolylcarbonyl, tetrazine, thiol, iodoacetamido, bromoacetamido, succinimidyl, pentachlorophenyl, tetrafluorophenyl, and sulfosuccinimdyl.

6. The lipid nanoparticle of claim 1 , wherein the chelating agent is selected from the group consisting of diethylenetriamine pentaacetic acid (DTPA), ethylenediamine tetraacetic acid (EDTA), triethylenetetraminehexaacetic acid (TTHA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-N,N′,N″-triacetic acid (NOTA), deferoxamine, diethylenetriamine penta(methylene phosphonic acid) (DTPMP), 1,4,7,10-tetraaza-1,4,7,10-tetra(2-carbamoylmethyl)cyclododecane (TCMC), 1,4,7,10-tetraazacyclododecane-1,4,7-tri (carbamoylmethyl)-10-acetic acid (DOTAM), 2-[1,4,7,10-tetraazacyclododecane-4,7,10-tris(t-butyl acetate)]-pentanedioic acid (DOTAGA), 1,4,8,11-tetraazacyclotetradecane-1,4,8-triacetic acid (DO3A), tetraazabicyclopentadecatrienetriacetic acid (PCTA), and 1,4,7-triazacyclononane-1,4-bis(acetic acid) (NO2A).

7. The lipid nanoparticle of claim 4 wherein the linker moiety is biotin, and the chelating agent is DTPA.

8. The lipid nanoparticle of claim 1 , wherein the lanthanide is Eu 3+ or Tb 3+ .

9. A labeled antibody, wherein the antibody is polyclonal or monoclonal, and is linked to the lipid nanoparticle of claim 1 .

10. The labeled antibody of claim 9 , wherein the antibody is an antigen binding fragment selected from Fab, Fab′, (Fab′) 2 , or Fv fragment, or is a single chain antibody.

11. The labeled antibody of claim 9 , wherein the antibody is covalently linked to the lipid nanoparticle.

12. The labeled antibody of claim 9 , 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.

13. The labeled antibody of claim 9 , wherein the antibody is an anti-HRP antibody.

14. A labeled streptavidin, wherein streptavidin is covalently or noncovalently linked to the nanoparticle of claim 1 .

15. The labeled streptavidin of claim 14 , wherein the linker moiety on the lipid nanoparticle is biotin, and the streptavidin is non-covalently bound to the lipid nanoparticle.

16. 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.

17. 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.

18. 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 13 ;
forming an ordered quaternary complex comprising the capture antibody, analyte, HRP-conjugated reporter antibody, and the labeled antibody of claim 13 ; and
detecting the amount of lanthanide retained on the surface.

19. The lipid nanoparticle of claim 1 , wherein:
a) is biotin-(PEG) 3 -di-C22-phosphatidylethanolamine and b) is Eu-DTPA-di-C22-phosphatidylethanolamine; or
a) is biotin-(PEG) 12 -di-C22-phosphatidylethanolamine or TCO-(PEG) 12 -di-C22-phosphatidylethanolamine and b) is Eu-DTPA-di-C22-phosphatidylethanolamine.

20. A time-resolved fluorescence bioaffinity assay wherein 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 .

21. 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, and a biotinylated reporter antibody;
forming an ordered ternary complex comprising the capture antibody, the analyte, and the biotinylated reporter antibody;
adding a streptavidin;
forming an ordered quaternary complex comprising the capture antibody, the analyte, the biotinylated reporter antibody, and the streptavidin;
adding a lipid nanoparticle of claim 7 ;
forming an ordered quinary complex comprising the capture antibody, the analyte, the biotinylated reporter antibody, the streptavidin, and the lipid nanoparticle; and
detecting the amount of lanthanide retained on the surface.

Assignees

• Univ Virginia Patent Foundation

Inventors

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

Classifications

• G01N2333/908

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

• G01N2333/36

  from Actinomyces; from Streptomyces (G) · CPC title

• G01N2021/6439

  with indicators, stains, dyes, tags, labels, marks · CPC title

• G01N33/6857

  Antibody fragments · CPC title

• G01N33/54346

  Nanoparticles · CPC title