# US 2013/0157288 A1 · Immobilised-bead immunomultiplex assay

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- Number: US 2013/0157288 A1
- Document ID: US20130157288A1
- Status: Published application
- Kind: A1
- Application number: 13/580,083
- Filed: 2011-03-04
- Published: 2013-06-20
- Earliest claimed: 2010-02-18
- Assignees: Bima Limited
- Inventors: Stephen Kilfeather; Alberto Taurozzi; Paul Lehmann; Elaine Linglee
- Web page: https://patentgrep.com/patent/US20130157288A1

## CPC

- G01N 21/75: Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated (systems in which material is burnt in a flame or plasma G01N21/72, G01N21/73)
- G01N 21/6428: {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)}
- G01N 33/54306: {Solid-phase reaction mechanisms}
- G01N 33/54313: {the carrier being characterised by its particulate form}
- G01N 33/54326: {Magnetic particles}
- G01N 33/582: {with fluorescent label}
- B01L 3/5085: {for multiple samples, e.g. microtitration plates}
- G01N 2021/6441: {with two or more labels}
- G01N 2500/00: Screening for compounds of potential therapeutic value
- 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)
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE

## Abstract

Embodiments of this invention include image-based systems and methods for detection of one or more analytes. A surface has identifiable analyte-specific capture particle(s) immobilised thereto at any point of an assay, to which different analytes attach due to the affinity of analyte-specific capture molecule(s) linked to the surface of the capture particle(s) for the analyte. Analyte-specific detector molecules with conjugated detection moieties are then attached to the analyte, and a computer assisted, image-based detection system captures images of the capture particles with or without attached analytes and detector molecules. By using different subsets of analyte-specific capture molecules, each subset having a characteristic identifiable feature; it is now possible to perform capture particle-based, rapid multiplex assays of biological and non-biological analytes without flow. These image-based systems can be used to aid in diagnosis of disease, evaluation of therapy for disease, or laboratory investigation.

## Claims

### Claim 1

A non-flow based multi-analyte detection system not requiring remobilisation of capture particles, comprising:
(1) a first type of analyte-specific capture particle permanently immobilised on a detection surface, said capture particle having:
(i) at least one capture particle identification parameter that distinguishes said first type of said analyte-specific capture particle from another type of capture particle,
(ii) said first type of analyte-specific capture molecule having a binding moiety specific for a first analyte, said binding moiety attached to said capture particle;
(2) a first type of analyte-specific detector molecules, each of said detector molecules having:
(i) an analyte-specific binding moiety; and
(ii) a luminescent moiety having an identification parameter different from that of said first type of capture particle;
(3) a second type of analyte-specific capture particle immobilised said detection surface, said second type of capture particle having:
(i) at least one capture particle identification parameter that is different from the capture particle identification parameter of said first type of analyte-specific capture particle;
(ii) said second type of analyte-specific capture particle having a binding moiety specific for a second analyte, said binding moiety bound to said capture particle, where
(4) upon binding of a first analyte to said first type of analyte-specific capture particle, upon binding of said first type of analyte-specific detector molecule to said first analyte bound to said first type of analyte-specific capture molecule, and upon binding of a second analyte to said second type of analyte-specific capture particle, and upon binding of said second type of analyte-specific detector molecule to said second analyte bound to said second type of analyte-specific capture molecule, forming:
a first complex of said first type of analyte-specific detector molecule, said first analyte, and said first type of analyte-specific capture particle immobilised on said detection surface and a second complex of said second type of analye-specific detector molecule, said second analyte, and said second type of analyte-specific capture particle molecule immobilised on said detection surface; and
(5) a light source, and a camera operably linked to a computer.

### Claim 2 (depends on 1)

The system of claim 1, further comprising:
(a) a multi-well plate holder compatible with at least one type of well-plate, and
(b) a multi-well plate having at least one well.

### Claim 3

(canceled)

### Claim 4 (depends on 1)

The system of claim 1, where at least one type of capture particle has a shape selected from the group consisting of cylindrical, conical, spherical, elliptical, ribbon-like, ovoid, spiral, amoeba-like, tube-like, and flat-sided comprising 4 or more flat sides.

### Claim at position 5

5-6. (canceled)

### Claim 7 (depends on 1)

The system of claim 1, where at least one type of said capture particle comprises a material selected from the group consisting of polymers, composites, inorganics, and natural products.

### Claim at position 7

8-12. (canceled)

### Claim 13 (depends on 1)

The system of claim 1 wherein said capture particles have a magnetically sensitive core or coating.

### Claim 14 (depends on 1)

The system of claim 1 where said capture particle identification parameter of at least one of said types of analyte-specific capture particles is selected from the group consisting of size, electromagnetic emission profile, and intensity of electromagnetic emission profile.

### Claim 15 (depends on 1)

The system of claim 1 where at least one type of said capture particle is immobilised on said detection surface by a tethering molecule, a physical force, or incorporation of said capture particle within a matrix.

### Claim 16 (depends on 15)

The system of claim 15 where said immobilisation is accomplished using covalent attachment, ionic interaction, hydrogen bonding, electrostatic forces, positive or negative pressure, or Van der Waals interaction.

### Claim 17

(canceled)

### Claim 18 (depends on 15)

The system of claim 15 where said matrix is selected from the group consisting of liquid-derived solid matrices, resins, glues, adhesives, and gels.

### Claim 19

(canceled)

### Claim 20 (depends on 1)

The system of claim 1, wherein said capture particle comprises a capture molecule selected from the group consisting of an antibody, antibody fragment, Fab region, receptor or receptor fragment, lectin, substrate of the target analyte, vitamin, an inorganic molecule, and derivatives combinations thereof.

### Claim at position 16

21-23. (canceled)

### Claim 24 (depends on 1)

The system of claim 1 where said analyte is a soluble inorganic, biological or synthetic molecule.

### Claim 25 (depends on 1)

The system of claim 1, wherein said computer has instructions stored therein to perform one of more of the following steps:
(1) a Capture Particle Distinguishing Step, comprising:
(i) moving said multi-well plate holder,
(ii) focusing said lens,
(iii) capturing an image of a capture particle using at least a first filter,
(iv) identifying and classifying said capture particle;
(2) a Recording Step, comprising:
(i) re-imaging said capture particle using a second filter different from said first filter, thereby recording of an image of said fluorescent or luminescent complex;
(ii) measuring the intensity of fluorescence or luminescence characteristic of said detector molecule;
(3) an Analysis Step, comprising:
(i) using of said measurement described in said step (2) to determine the presence and/or quantity of at least one analyte.

### Claim 26 (depends on 25)

The system of claim 25 where said computer has
(a) instructions stored therein to image an additional field of view within a well of a multi-well plate,
(b) instructions to determine a number of capture particles of each subset to be imaged to achieve a degree of precision of 85% prior imaging of other wells,
(c) instructions to move said well plate holder and re-focusing said lens followed by repeating said Capture Particle Distinguishing Step and said Recording Step on an additional field of view.

### Claim 27 (depends on 25)

The system of claim 25, where said computer has instructions stored therein to perform the steps:
(a) predict fluorescence or luminescence values under conditions where said detector molecule fluorescence or luminescence exceeds the limit of detection (‘white-out’), comprising:
(b) repeating said Recording Step at a lower sensitivity of fluorescence or luminescence detection; and
(c) calculating the predicted fluorescence or luminescence that would have been obtained at the higher sensitivity of fluorescence or luminescence detection at which ‘white-out’ was observed; wherein said predicted value then being used in said Analysis Step.

### Claim 28

(canceled)

### Claim 29 (depends on 25)

The system of claim 25 where the computer has instructions stored therein to recognise debris, being objects that are not said capture particles, but have been imaged in said Capture Particle Distinguishing Step and Recording Step and said instructions include steps for exclusion of debris from incorporation into said Analysis Step.

### Claim 30 (depends on 1)

A method for detecting and quantifying an analyte, comprising:
providing a system of claim 1;
providing a sample containing the analyte to be measured;
incubating said sample with said capture particle before, during or after incubation of said first capture particle with a solution containing a known concentration of said first analyte, said capture particle having a fluorophore, luminescent moiety or chromophore conjugated thereto;
permitting said labelled analyte to compete with the unlabelled analyte for binding to said capture particle;
immobilising said capture particle on said detection surface in a well of a multiwell plate;
adding a first type of detector molecule to said well thereby producing a complex of said capture particle, analyte and detector molecule immobilised on said surface;
using an image-based detector to determine the amount of fluorophore, luminescent moiety or chromophore present on said complex; and
calculating the amount of said analyte in said sample.

### Claim 31 (depends on 30)

The method of claim 30, where said computer contains instructions to distinguish said capture particle from debris, where said debris has a surface selected from the group excluding cylindrical, conical, spherical, elliptical, ribbon-like, ovoid, spiral, amoeba-like, tube-like and flat sided comprising 4 or more flat sides.

### Claim at position 25

32-37. (canceled)

### Claim 38 (depends on 30)

The method of claim 30, where said analyte is an enzyme and said capture particle has a substrate attached thereto and said enzyme catalyzes an addition reaction to add an additional moiety to or a removal reaction that removes an already existing moiety from said substrate, said detection molecule having a chromophore, fluorophore, or luminescent moiety conjugated thereto binds to said additional moiety or to said existing moiety, further comprising;
(a) incubating said capture particle and substrate with said enzyme and said additional moiety, thereby producing a complex of said capture particle, substrate and said additional moiety; or
(b) incubating said capture particle with said pre-existing moiety with said enzyme; and
(c) incubating said complex obtained in step (a) or step (b) with said detection moiety, and
(d) determining the presence and/or activity of said enzyme by detecting the fluorescence or luminescence on said detection moiety.

### Claim 39 (depends on 30)

The method of claim 30, where said analyte is an enzyme and said capture particle having a substrate having a fluorescent tag attached thereto and said fluorescent tag has a fluorescent-conjugated detector molecule comprising a flurophore, chromophore or luminescent moiety, thereby forming a labeled complex, further comprising;
(a) incubating said labeled complex with a solution containing said enzyme; and
(b) determining the presence and/or activity of said enzyme by detecting and quantifying the loss of intensity of said fluorescence.

### Claim at position 28

40-41. (canceled)

### Claim 42 (depends on 40)

The method of claim 40, where the analyte is selected from the group consisting of small molecule therapeutic agents, peptide therapeutic agents, cell-derived receptors, DNA binding molecules, and antibodies.

### Claim 43

A kit, comprising:
(1) a multi-well plate;
(2) a subset of capture particles with or without capture molecules pre-conjugated to said capture particles;
(3) a substance used to immobilise said capture particles,
(4) a subset of detector molecules with or without pre-conjugation to a fluorophore, luminescent moiety, or chromophore;
(5) a fluorophore, luminescent moiety, or chromophore with a moiety facilitating conjugation or binding to said detector molecule;
(6) reagents for use in the preparation of said multi-well plate, said capture particles, said analytes, said samples, said detector molecules, or said fluorophore, luminescent moiety, or chromophore, for performing the assay;
(7) a covering for the top of said multi-well plate.
(8) instructions for use.

### Claim 44

A system for image-based analysis of a plurality of analytes, comprising:
a. a detection surface having a plurality of types of capture particles thereon, each of said types of capture particles having a uniquely identifiable spectral feature, and each type of said capture particles having at least one analyte-specific capture molecule attached thereto;
b. a plurality of types of analyte-specific detector molecules, each of said plurality of types corresponding to one of said types of analyte-specific capture molecules;
c. a light source;
d. an imaging sensor; and
e. a computer having a program stored in a computer memory device having instructions thereon to determine the type and amount of each of said plurality of analytes.

### Claim at position 32

45-46. (canceled)

### Claim 47

A method for image-based analysis of a plurality of analytes, comprising:
a. providing a detection surface having a plurality of types of capture particles immobilized thereon, each of said types of capture particles having a uniquely identifiable spectral feature, and each type of said capture particles having at least one analyte-specific capture molecule attached thereto;
b. applying a sample containing two or more analytes to said detection surface thereby forming a plurality of analyte-specific capture molecule-analyte complexes (“ASCMAC”);
c. providing a plurality of types of analyte-specific detection molecules, each type of which comprises a luminescent moiety different from luminescent moieties of other types of detection molecules.
d. applying to said ASCMAC, a said plurality of analyte-specific detector molecules with conjugated luminescence moieties attached thereto thereby forming a plurality of ACSMAC-analyte-specific detector molecule complexes (“ASCMASDC”); and
e. capturing an image of said ASCMASDC.

### Claim at position 34

48-50. (canceled)