# US 5,739,042 A · Method of assay

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- Number: US 5,739,042 A
- Document ID: US5739042A
- Status: Granted
- Kind: A
- Application number: 08/663,121
- Filed: 1996-09-06
- Granted: 1998-04-14
- Earliest claimed: 1993-12-23
- Assignees: Sinvent AS
- Inventors: Jomar Frengen
- Web page: https://patentgrep.com/patent/US5739042A

## CPC

- G01N 33/54313: {the carrier being characterised by its particulate form}
- Y10S 435/962: Prevention or removal of interfering materials or reactants or other treatment to enhance results, e.g. determining or preventing nonspecific binding
- Y10S 436/824: Immunological separation techniques
- 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)
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

## Abstract

The present invention relates to a binary assay method capable of providing wide dynamic range, a high degree of precision and rapid processing times in which analyte is reacted successively with two independently determinable forms of solid-supported binding partner and also with a labelled ligand having affinity for the analyte or binding partner. The analyte concentration is determined from signals deriving from the resulting two forms of analyte-binding partner-labelled ligand systems by reference to a double standard calibration curve. A kit for use in the method is also disclosed.

## Claims

### Claim 1

In a method for assaying an analyte in a sample comprising reacting the sample with two independently determinable forms of solid-supported binding partner having affinity for the analyte and with a labelled ligand having affinity for the analyte or the binding partner, thereby yielding two forms of labelled ligand-carrying solid-supported binding partner, said two independently determinable forms of solid-supported binding partner being such that signals in respect of said two forms of labelled ligand-carrying solid-supported binding partner may be independently determined whereby the analyte concentration may be obtained by reference to a double standard calibration curve, wherein the improvement comprises adding to the sample the first form of solid-supported binding partner and then after an interval adding the second form of solid-supported binding partner.

### Claim 2 (depends on 1)

A method as claimed in claim 1 wherein the solid supports for said two forms of solid-supported binding partner comprise two distinguishable types of monodisperse particles.

### Claim 3 (depends on 2)

A method as claimed in claim 2 wherein said two types of monodisperse particles are distinguishable by size.

### Claim 4 (depends on 2)

A method as claimed in claim 2 wherein said two types of monodisperse particles are detected by flow cytometry.

### Claim 5 (depends on 1)

A method as claimed in claim 1 wherein the first form of solid-supported binding partner is used in a low amount relative to the second form of solid-supported binding partner.

### Claim 6 (depends on 1)

A method as claimed in claim 1 wherein the second form of solid-supported binding partner is used in excess relative to the labelled ligand.

### Claim 7 (depends on 2)

A method as claimed in claim 2 wherein one or more further distinguishable types of monodisperse particles coated with a binding partner having zero affinity for the analyte are also added and signals deriving therefrom are used to give a measure of non-specific binding of analyte, labelled ligand or a combination thereof to said zero affinity partner.

### Claim 8 (depends on 2)

A method as claimed in claim 2 wherein one or more further distinguishable types of monodisperse particles coated with material having affinity for the labelled ligand are also added and signals deriving therefrom are used to give a measure of residual unbound labelled ligand or unbound labelled ligand-analyte complex.

### Claim 9 (depends on 2)

A method as claimed in claim 2 wherein a plurality of analytes is simultaneously assayed using an appropriate number of labelled ligands and sets of distinguishable particle types.

### Claim 10 (depends on 1)

A method as claimed in claim 1 wherein the label component of the labelled ligand is a fluorescent substance.

### Claim 11 (depends on 1)

A method as claimed in claim 1 wherein the analyte is an antigen and the binding partner therefor is a monoclonal antibody.

### Claim 12 (depends on 1)

A method as claimed in claim 1 wherein the same binding partner is employed in the two independently determinable forms of solid-supported binding partner.