Direct detection of single molecules on microparticles

Patent metadata

Field	Value
Publication number	US-12061200-B2
Application number	US-201916729067-A
Country	US
Kind code	B2
Filing date	Dec 27, 2019
Priority date	Dec 28, 2018
Publication date	Aug 13, 2024
Grant date	Aug 13, 2024

Abstract

The disclosure provides methods of analyzing an analyte of interest in a biological sample using fluorescent agents and macroconjugates which comprise a core containing a cross-linked polymer or protein, tags, specific binding members or fragments thereof, and optionally carrier proteins. Also provided are methods of analyzing two or more analytes of interest in a biological sample in a single assay using microparticles and detection conjugates comprising different fluorophore labels, acquiring transmitted light and fluorescent images of the microparticles, and using a customized image analysis process to analyze the acquired images.

First claim

The invention claimed is:

1. A method of analyzing an analyte of interest in a biological sample, the method comprising the steps of:
a. capturing an analyte of interest on a binding surface, which binding surface comprises a plurality of specific binding members immobilized thereto that bind to the analyte;
b. reacting a plurality of macroconjugates with the captured analyte; wherein each macroconjugate comprises:
i. a core which comprises a cross-linked protein or polymer comprising a polysaccharide, a dendrimer, a polyether compound, or nanoparticle;
ii. a plurality of specific binding members, fragments thereof, or combinations thereof;
iii. a plurality of tags, wherein the method comprises reacting a plurality of fluorescent agents with the tags, wherein each fluorescent agent comprises a detectable label and a molecule which is capable of binding to the tag;
iv. a plurality of carrier proteins, wherein: (a) the plurality of carrier proteins are covalently attached to the core; (b) the plurality of tags are covalently attached to the plurality of carrier proteins and (c) the plurality of specific binding members are covalently attached to the plurality of carrier proteins; and

c. imaging the binding surface and analyzing the images.

2. The method of claim 1 , wherein the polysaccharide is a dextran, amino dextran or combinations thereof.

3. The method of claim 1 , wherein the dendrimer is a PAMAM dendrimer or a DNA dendrimer.

4. The method of claim 1 , wherein the polyether compound is polyethylene glycol.

5. The method of claim 1 , wherein the carrier proteins are serum albumin proteins.

6. The method of claim 5 , wherein the serum albumin proteins are bovine serum albumin.

7. The method of claim 1 , wherein the tags comprise biotin.

8. The method of claim 1 , wherein the specific binding members comprise antibodies.

9. The method of claim 1 , wherein the fragments of specific binding members comprise antibody fragments.

10. The method of claim 1 , wherein the molecule which is capable of binding to the tag is streptavidin.

11. The method of claim 1 , wherein the detectable label in the fluorescent agent is phycoerythrin or allophycocyanin.

12. The method of claim 11 , wherein the fluorescent agents comprise streptavidin-phycoerythrin conjugates (SAPE) or streptavidin-allophycocyanin (SAAPC) conjugates.

13. The method of claim 1 , wherein the binding surface is imaged on a flat smooth surface of a solid support.

14. The method of claim 1 , wherein imaging the binding surface involves acquiring a transmitted light image of the binding surface and one or more fluorescent images corresponding to the fluorescent agents.

15. The method of claim 14 , wherein analyzing the images further comprises calculating average pixel intensity on the binding surface in the one or more fluorescent images.

16. The method of claim 15 , wherein, if the average pixel intensity in a region of interest is less than a predetermined value, analyzing the images comprises:
(i) counting high fluorescence intensity peaks in the region of interest; and
(ii) determining the ratio of peaks to the region of interest area in the one or more fluorescent images.

17. The method of claim 16 , further comprising calculating a composite signal using a conversion factor to combine the data points obtained for different analyte concentrations after step (ii).

18. The method of claim 1 , wherein the binding surface comprises one or more microparticles.

19. The method of claim 18 , wherein each of the one or more microparticles has a size greater than 1 micron.

Assignees

• Abbott Lab

Inventors

• Ruan Qiaoqiao
• Macdonald Patrick J
• Swift Kerry M
• Tetin Sergey Y
• Calfin Brenda
• Lin Zhen
• Haack Richard
• Pope Mark R
• Prostko John
• Qiu Xiaoxing
• Bogdan M Felicia

Classifications

• G01N15/1429

  Signal processing · CPC title

• G01N33/54346

  Nanoparticles · CPC title

• G01N33/582 Primary

  with fluorescent label · CPC title

• G01N33/587

  Nanoparticles · CPC title

• G01N33/566

  using specific carrier or receptor proteins as ligand binding reagents {where possible specific carrier or receptor proteins are classified with their target compounds} · CPC title