# US 10,119,963 B2 · PSA assay and reagent therefor

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- Number: US 10,119,963 B2
- Document ID: US10119963B2
- Status: Granted
- Kind: B2
- Application number: 14/865,728
- Filed: 2015-09-25
- Published: 2016-01-14
- Granted: 2018-11-06
- Earliest claimed: 2011-03-28
- Assignees: Sekisui Medical Co., Ltd.
- Applicants: Sekisui Medical Co., Ltd.
- Inventors: Yuki Takahashi; Tomo Shimizu; Yasushi Nakamura; Shinya Nakayama; Shinichiro Kitahara
- Web page: https://patentgrep.com/patent/US10119963B2

## CPC

- G01N 33/543: with an insoluble carrier for immobilising immunochemicals
- G01N 33/573: for enzymes or isoenzymes
- G01N 33/57555: of the prostate
- G01N 33/577: involving monoclonal antibodies {binding reaction mechanisms characterised by the use of monoclonal antibodies (G01N33/5302 - G01N33/576 take precedence)}
- G01N 2333/96433: Serine endopeptidases (3.4.21)
- 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)

## Abstract

Provided is a method for the simple and highly accurate assay of PSA by a one-step reaction that does not use carriers having different average grain sizes. Also provided is a reagent therefor. The PSA assay method comprises sensitizing insoluble carriers having the same average grain size within a range of greater than 0.20 μm but 0.40 μm or less using two types of anti-PSA monoclonal antibodies having different epitopes that are anti-PSA monoclonal antibodies that will react with both free PSA and PSA-ACT, which is a complex of free PSA and α1-antichymotrypsin, and bringing the carriers into contact with a sample in the presence of an aggregation promoter.

## Claims

### Claim 1

A prostate specific antigen (“PSA”) assay comprising:
providing a mixture of insoluble carriers with two kinds of anti-PSA monoclonal antibodies respectively immobilized thereon, and
bringing said mixture into contact with a sample in the presence of an agglutination accelerator, and thereby, reacting two kinds of anti-PSA monoclonal antibodies at one time, wherein said agglutination accelerator excludes PEG6000,
wherein
the two kinds of anti-PSA monoclonal antibodies can react with both free PSA and complex of free PSA and α1-antichymotrypsin (PSA-ACT) by recognizing different epitopes,
each of the two kinds of anti-PSA monoclonal antibodies has a ratio (fKd/cKd) of a dissociation constant (fKd) for free PSA to a dissociation constant (cKd) for complex of free PSA and α1-antichymotrypsin (PSA-ACT) of more than 0.1 and equal to or less than 2.0 in the absence of said agglutination accelerator,
each of the two kinds of anti-PSA monoclonal antibodies has a dissociation constant (fKd) for free PSA of 10 nM or less,
the insoluble carriers have an identical average particle size that is more than 0.20 μm and equal to or less than 0.40 μm, and
the concentration of the agglutination accelerator has been set so that an equimolar response to free PSA and complex of free PSA and α1-antichymotrypsin (PSA-ACT) is obtained, said equimolar response obtained when c/f ratio is 90 to 115%, said c/f ratio being the ratio (PSA-ACT/fPSA) of the reactivity with PSA-ACT to the reactivity with fPSA.

### Claim 2 (depends on 1)

The PSA assay according to claim 1, wherein the agglutination accelerator is one or more agglutination accelerators selected from a polysaccharide, polyvinylpyrrolidone, polyvinyl chloride, a poly-γ-glutamate, and poly(2-methacryloyloxyethylphosphorylcholine).

### Claim 3 (depends on 1)

The PSA assay according to claim 1, wherein the insoluble carriers are of one or more type selected from particles comprising materials derived from latex particles of synthetic polymers, silica, alumina, carbon blacks, metal compounds, metals, ceramics, and/or magnetic substances.

### Claim 4 (depends on 2)

The PSA assay according to claim 2, wherein the polysaccharide is one or more polysaccharides selected from dextran, pullulan, and alkylated polysaccharides.

### Claim 5 (depends on 3)

The PSA assay according to claim 3, wherein the synthetic polymer is one or more synthetic polymers selected from polystyrene, a styrene-sulfonic acid copolymer, a styrene-methacrylic acid copolymer, an acrylonitrile-butadiene-styrene copolymer, a vinyl chloride-acrylate copolymer, and a vinyl acetate-acrylate copolymer.

### Claim 6 (depends on 4)

The PSA assay according to claim 4, wherein the polysaccharide is methyl cellulose and/or ethyl cellulose.

### Claim 7

A prostate specific antigen (“PSA”) assay comprising:
contacting a sample with a Reagent A to form a mixture, said Reagent A including a mixture of insoluble carriers with two kinds of anti-PSA monoclonal antibodies respectively immobilized thereon, and thereby, reacting two kinds of anti-PSA monoclonal antibodies at one time, and
wherein
the two kinds of anti-PSA monoclonal antibodies can react with both free PSA and complex of free PSA and α1-antichymotrypsin (PSA-ACT) and recognize different epitopes,
each of the two kinds of anti-PSA monoclonal antibodies has a ratio (fKd/cKd) of a dissociation constant (fKd) for free PSA to a dissociation constant (cKd) for complex of free PSA and α1-antichymotrypsin (PSA-ACT) of more than 0.1 and equal to or less than 2.0 in the absence of said agglutination accelerator,
each of the two kinds of anti-PSA monoclonal antibodies has a dissociation constant (fKd) for free PSA of 10 nM or less, and
the insoluble carriers have an identical average particle size that is more than 0.20 μm and equal to or less than 0.40 μm; and
reacting the mixture of the sample and Reagent A with a Reagent B, said Reagent B including one or more agglutination accelerators selected from polyethylene glycol, a polysaccharide, polyvinylpyrrolidone, polyvinyl chloride, a poly-γ-glutamate, and poly(2-methacryloyloxyethylphosphorylcholine), wherein said agglutination accelerator excludes PEG6000,
wherein the concentration of the agglutination accelerator has been set so that an equimolar response to free PSA and complex of free PSA and α1-antichymotrypsin (PSA-ACT) is obtained, said equimolar response obtained when c/f ratio is 90 to 115%, said c/f ratio being the ratio (PSA-ACT/fPSA) of the reactivity with PSA-ACT to the reactivity with fPSA.

### Claim 8 (depends on 7)

The PSA assay according to claim 7, wherein the insoluble carriers are of one or more type selected from particles comprising materials derived from latex particles of synthetic polymers, silica, alumina, carbon blacks, metal compounds, metals, ceramics, and/or magnetic substances.

### Claim 9 (depends on 8)

The PSA assay according to claim 8, wherein the synthetic polymer is one or more synthetic polymers selected from polystyrene, a styrene-sulfonic acid copolymer, a styrene-methacrylic acid copolymer, an acrylonitrile-butadiene-styrene copolymer, a vinyl chloride-acrylate copolymer, and a vinyl acetate-acrylate copolymer.

### Claim 10 (depends on 7)

The PSA assay according to claim 7, wherein the polysaccharide is one or more polysaccharides selected from dextran, pullulan, and alkylated polysaccharides.

### Claim 11 (depends on 10)

The PSA assay according to claim 10, wherein the alkylated polysaccharides are methyl cellulose and/or ethyl cellulose.