Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
  • G01N33/545—Synthetic resin
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57407—Specifically defined cancers
  • G01N33/57434—Specifically defined cancers of prostate
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/914—Hydrolases (3)
  • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
  • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
  • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
  • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
  • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
  • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
  • G01N2333/96433—Serine endopeptidases (3.4.21)

Definitions

• the invention relates to an antigen assay and a reagent that make it possible to measure a free antigen, and a complex of a free antigen and a coexisting substance in a sample, by immune agglutination so that an equimolar response is obtained.
• the invention relates to an assay of prostate-specific antigen, and a reagent therefor.
• Prostate cancer is a malignant disease that is observed in men. A large number of patients suffer from prostate cancer in the United States and Europe. In recent years, the number of patients who suffer from prostate cancer has rapidly increased in Japan. Since prostate cancer grows slowly, and may effectively be treated by radiotherapy or anti-androgenic therapy, it is important to find prostate cancer in an early stage.
• PSA Prostate-specific antigen
• fPSA free PSA
• PSA-ACT complex-type PSA
• PSA-ACT complex of PSA and ⁇ 1-antichymotrypsin
• fPSA and PSA-ACT can be measured by an immunoassay.
• an assay based on agglutination that utilizes a latex or the like is used as the immunoassay.
• an anti-PSA monoclonal antibody has different reactivity with fPSA and PSA-ACT, it may be difficult to accurately measure the total PSA level.
• PTL 1 proposes an immunoagglutination assay reagent and an assay that utilizes the same, wherein the immunoagglutination assay reagent including (1) a first particle suspension that includes first insoluble carrier particles immobilizing thereon a first monoclonal antibody that can bind to a free measurement target substance and a complex of the free measurement target substance and the corresponding binding molecule, (2) a second particle suspension that includes second insoluble carrier particles immobilizing thereon a second monoclonal antibody that can bind to the free measurement target substance and a complex of the free measurement target substance and the corresponding binding molecule, and does not compete with the first monoclonal antibody, and (3) a third particle suspension that includes third insoluble carrier particles immobilizing thereon a third monoclonal antibody that does not recognize the free measurement target substance, but recognizes a complex of the free measurement target substance and the corresponding binding molecule.
• the immunoagglutination assay reagent including (1) a first particle suspension that includes first insoluble carrier particles immobilizing thereon
• PTL 2 proposes an assay reagent and an assay that utilizes the same, wherein the assay reagent adjusting reactivity with a free antigen and a complex-type antigen by using carriers having a smaller particle size among two or more types of carriers that differ in particle size and immobilizing thereon at least one monoclonal antibody among three monoclonal antibodies that have reactivity with a free antigen and a complex-type antigen and differ in recognition site, and using carriers having a larger particle size among the two or more types of carriers and immobilizing thereon the remaining monoclonal antibodies.
• PTL 3 proposes a two-step reaction immunoassay that includes (1) reacting a sample that includes a free measurement target substance and a complex-type measurement target substance with a latex 1 on which a monoclonal antibody 1 to the measurement target substance is immobilized to obtain a reaction product 1, and (2) reacting the reaction product 1 with a latex 2 on which a monoclonal antibody 2 that differs in recognition site from the monoclonal antibody 1 is immobilized to obtain a reaction product 2.
• PTL 4 proposes a prostate-specific antigen immunoassay reagent and an assay that utilizes the same, wherein the immunoassay reagent including a latex 1 on which a monoclonal antibody 1 that has reactivity with free PSA and PSA complex is immobilized, and a latex 2 on which a monoclonal antibody 2 that has reactivity with free PSA and PSA complex is immobilized, the monoclonal antibody 2 differing in recognition site from the monoclonal antibody 1, and the latex 2 differing in average particle size from the latex 1.
• the immunoassay reagent including a latex 1 on which a monoclonal antibody 1 that has reactivity with free PSA and PSA complex is immobilized, and a latex 2 on which a monoclonal antibody 2 that has reactivity with free PSA and PSA complex is immobilized, the monoclonal antibody 2 differing in recognition site from the monoclonal antibody 1, and the latex 2 differing in average particle size from the
• the method disclosed in PTL 2 has a problem in that it is necessary to combine latexes that differ in particle size
• the method disclosed in PTL 3 has a problem in that it is necessary to employ a two-step reaction in which the second monoclonal antibody is reacted after reacting the first monoclonal antibody.
• the method disclosed in PTL 4 also has a problem in that it is necessary to combine latexes that differ in particle size.
• PTL 4 discloses a comparative example in which latexes having an identical average particle size (0.22 ⁇ m) are used. However, an agglutination accelerator is not used, and an equimolar response is not obtained.
• an object of the invention is to provide an assay that easily and accurately measures PSA by a one-step reaction without using carriers that differ in average particle size, and a reagent used therefor.
• the expression “measurement of PSA” or “assay of PSA” used herein refers to measurement or assay of the total PSA level unless otherwise specified.
• a PSA assay comprising using insoluble carriers immobilizing thereon two kinds of anti-PSA monoclonal antibodies, and bringing the insoluble carriers into contact with a sample in the presence of an agglutination accelerator, wherein the two kinds of anti-PSA monoclonal antibodies can react with both free PSA and complex of free PSA and ⁇ 1-antichymotrypsin (PSA-ACT) and recognize different epitopes, and the insoluble carriers have an identical average particle size that is more than 0.20 ⁇ m and equal to or less than 0.40 ⁇ m.
• PSA-ACT ⁇ 1-antichymotrypsin
• agglutination accelerator is one or more agglutination accelerators selected from polyethylene glycol, a polysaccharide, polyvinylpyrrolidone, polyvinyl chloride, a poly- ⁇ -glutamate, and poly(2-methacryloyloxyethylphosphorylcholine).
• 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.
• the PSA assay according to (1) wherein the concentration of the agglutination accelerator is adjusted so that an equimolar response to free PSA and complex of free PSA and ⁇ 1-antichymotrypsin (PSA-ACT) is obtained.
• the polysaccharide is one or more polysaccharides selected from dextran, pullulan, and alkylated polysaccharides including methyl cellulose and ethyl cellulose.
• 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.
• a PSA assay reagent comprising at least 1) and 2): 1) antibody-immobilized carriers prepared by using insoluble carriers immobilizing thereon two kinds of anti-PSA monoclonal antibodies, wherein the two kinds of anti-PSA monoclonal antibodies can react with both free PSA and complex of free PSA and ⁇ 1-antichymotrypsin (PSA-ACT) and recognize different epitopes, and the insoluble carriers have an identical average particle size that is more than 0.20 ⁇ m and equal to or less than 0.40 ⁇ m; and/or 2) one or more agglutination accelerators selected from polyethylene glycol, a polysaccharide, polyvinylpyrrolidone, polyvinyl chloride, a poly- ⁇ -glutamate, and poly(2-methacryloyloxyethylphosphorylcholine).
• the agglutination accelerator is one or more agglutination accelerators selected from polyethylene glycol, a polysaccharide, polyvinylpyrrolidone, polyvinyl chloride, a poly- ⁇ -glutamate, and poly(2-methacryloyloxyethylphosphorylcholine), and the amount of the agglutination accelerator is adjusted so that an equimolar response to free PSA and complex of free PSA and ⁇ 1-antichymotrypsin
• the assay according to the aspect of the invention can easily and accurately measure PSA by a one-step reaction without using carriers that differ in average particle size. Therefore, it is possible to implement high-accuracy measurement using a general-purpose automatic analyzer without requiring a special device.
• PSA prostate-specific antigen
• PSA in plasma that can be measured by immunoassay include free PSA (fPSA) and complex of free PSA (fPSA) and ⁇ 1-antichymotrypsin (PSA-ACT).
• fPSA free PSA
• fPSA complex of free PSA
• PSA-ACT ⁇ 1-antichymotrypsin
• the target sample is not particularly limited as long as the sample includes PSA, but is preferably blood, serum, plasma, or the like.
• At least two kinds of anti-PSA monoclonal antibodies that can react with both fPSA and PSA-ACT and recognize different epitopes are used as monoclonal antibodies to PSA.
• the difference in epitope between the two kinds of anti-PSA monoclonal antibodies can be determined by confirming whether normal sandwich immunoassay using PSA (antigen) and these antibodies is possible or not.
• the antibodies are selected as described below.
• an antibody that has high reactivity i.e., high titer
• carriers that ensure sufficient measurement sensitivity.
• the carriers immobilize thereon two kinds of antibodies arbitrarily selected from the antibodies selected to have a high titer, and a combination of antibodies are selected so that the reactivity with PSA-ACT and fPSA shows small difference and sufficient measurement sensitivity is obtained.
• the anti-PSA monoclonal antibodies have a ratio (fKd/cKd) of the dissociation constant (fKd) for free PSA to the dissociation constant (cKd) for complex of free PSA and ⁇ 1-antichymotrypsin (PSA-ACT) of more than 0.1, and more preferably more than 0.2.
• the upper limit of the ratio (fKd/cKd) is preferably 2.0 or less, and more preferably 1.5 or less.
• the dissociation constant (fKd) for free PSA is preferably 10 nM or less, and more preferably 6 nM or less.
• the monoclonal antibodies may include, for example, an Fab fragment obtained by partial hydrolysis by papain or the like, an F(ab′) 2 fragment obtained by partial hydrolysis by pepsin or the like, and/or an Fab′ fragment obtained by reduction of an F(ab′) 2 fragment.
• the insoluble carriers immobilizing antibodies thereon are not particularly limited, but are preferably selected from particles comprising materials derived from latex particles of synthetic polymers, silica, alumina, carbon blacks, metal compounds, metals, ceramics, and/or magnetic substances.
• the synthetic polymer is preferably 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.
• the polystyrene latex may be produced, for example, by the method disclosed in WO2003/005031. Specifically, a reaction vessel containing water as a solvent is charged with specific amounts of a styrene monomer, an initiator (e.g., potassium persulfate), and a polymerization stabilizer (e.g., sodium styrene sulfonate). After optionally adding emulsifier (e.g., sodium lauryl sulfate), the mixture is heated with stirring in a nitrogen atmosphere to effect polymerization.
• an initiator e.g., potassium persulfate
• a polymerization stabilizer e.g., sodium styrene sulfonate
• emulsifier e.g., sodium lauryl sulfate
• the average particle size of the insoluble carriers is more than 0.20 ⁇ m and equal to or less than 0.40 ⁇ m, preferably more than 0.22 ⁇ m and equal to or less than 0.40 ⁇ m, more preferably equal to or more than 0.23 and equal to or less than 0.40 ⁇ m, and still more preferably equal to or more than 0.23 and equal to or less than 0.34 ⁇ m. If the average particle size of the insoluble carriers is 0.20 ⁇ m or less, or exceeds 0.40 ⁇ m, it may be difficult to obtain an equimolar response.
• average particle size ( ⁇ m) used herein in connection with the insoluble carriers refers to a value that is obtained by image analysis using a transmission electron microscope (see the examples) and rounded off to two decimal places. Note that the average particle size can be calculated to four decimal places by image analysis.
• the average particle size is more than 0.20 ⁇ m and equal to or less than 0.40 ⁇ m
• two types of particles e.g. prepared in different lots
• the average particle size ( ⁇ SD) of particles B is P ⁇ Q, M>P
• N and Q are 0.02 ⁇ m or less.
• the average particle size of particles A is 0.22 ⁇ 0.02 ⁇ m and the average particle size of particles B is 0.21 ⁇ 0.02 ⁇ m
• M ⁇ N is 0.20
• P+Q is 0.23 (i.e., the above relationship is satisfied). Therefore, it is considered that the particles A and the particles B have an identical average particle size.
• the average particle size of particles A is 0.40 ⁇ 0.02 ⁇ m and the average particle size of particles B is 0.37 ⁇ 0.02 ⁇ m, M ⁇ N is 0.38 and P+Q is 0.39 (i.e., the above relationship is satisfied). Therefore, it is considered that the particles A and the particles B have an identical average particle size.
• a mixture of particles wherein the particles are considered to have an identical average particle size as mentioned above and are mixed in an arbitrary ratio is within the scope of the invention.
• two or more carriers have an identical average size, but a combination of carriers made from different materials is not excluded.
• a combination of carriers having different average particle sizes is also not excluded, as long as the carrier therefrom has an average particle size within the above range and is used in the main reaction to obtain an equimolar response.
• the insoluble carriers may immobilize the antibodies thereon by a generally-used physical adsorption method, and a chemical binding method, an immunobinding method, or the like is used as well.
• the carriers which have respectively immobilize thereon two kinds of monoclonal antibodies that recognize different epitopes are normally mixed in an appropriate ratio (see the examples), but the two kinds of monoclonal antibodies may be mixed beforehand in an appropriate ratio to be immobilized on the carriers.
• the agglutination accelerator is not particularly limited as long as the agglutination accelerator promotes agglutination of the insoluble carriers that immobilize the antibodies thereon via an antigen-antibody reaction.
• the agglutination accelerator include polyethylene glycol, a polysaccharide, polyvinylpyrrolidone, polyvinyl chloride, a poly- ⁇ -glutamate, poly(2-methacryloyloxyethylphosphorylcholine) (hereinafter may be referred to as “MPC polymer”), and the like.
• the polysaccharide is preferably one or more polysaccharides selected from dextran, pullulan, and alkylated polysaccharides including methyl cellulose and ethyl cellulose.
• the poly- ⁇ -glutamate may be an alkali metal salt (e.g., sodium, potassium, or lithium salt), an alkaline-earth metal salt (e.g., magnesium, calcium, or barium salt), or an ammonium salt.
• polyethylene glycol and polyvinylpyrrolidone are preferable, and polyethylene glycol is more preferable.
• a plurality of agglutination accelerators may be used in combination.
• the number average molecular weight of polyethylene glycol is preferably 3,000 to 1,000,000, for example, more preferably 5,000 to 100,000, and particularly preferably 5,000 to 50,000. When the number average molecular weight of polyethylene glycol is 3,000 to 1,000,000, excellent measurement sensitivity can be achieved.
• Polyvinylpyrrolidone may have a weight average molecular weight of 25,000 to 1,200,000, for example, and preferably 40,000 to 360,000.
• the poly- ⁇ -glutamate may have a weight average molecular weight of 200,000 to 6,000,000, for example.
• the MPC polymer may have a molecular weight of 5,000 to 5,000,000, for example, and preferably 500,000 to 2,000,000.
• the concentration of the agglutination accelerator is preferably adjusted so that the final concentration of the agglutination accelerator is 0.1 to 5 wt %, more preferably 0.2 to 2 wt %, and particularly preferably 0.2 to 0.6 wt %, when the agglutination accelerator is brought into contact with the sample.
• the final concentration of the agglutination accelerator is 0.1 to 5 wt %, excellent measurement sensitivity can be achieved.
• a saccharide e.g., glucose and sucrose
• an inorganic salt e.g., sodium chloride
• a surfactant e.g., polyoxyethylene sorbitan monostearate
• a preservative e.g., sodium azide
• a non-specific reaction inhibitor e.g., IgG antibody derived from a normal animal
• the content of the saccharide in the reagent is preferably about 0.1 to about 10 wt %, the content of the inorganic salt in the reagent is preferably about 0.01 to about 5 wt %, the content of the surfactant in the reagent is preferably about 0.02 to about 5 wt %, the content of the preservative in the reagent is preferably about 0.001 to about 0.1 wt %, and the content of the non-specific reaction inhibitor in the reagent is preferably about 0.001 to about 5 wt %.
• the antigen-antibody reaction according to the embodiments of the invention is effected in a buffer.
• the type, the concentration, and the pH of the buffer are not particularly limited as long as the antigen-antibody reaction occurs.
• Example of the buffer includes a phosphate buffer, a Tris-HCl buffer, a carbonate buffer, a glycine buffer, a Good's buffer, and the like.
• the concentration of the buffer is about 3 to about 500 mM, preferably 5 to 100 mM, and more preferably 5 to 50 mM. It is preferable that the buffer have a pH in a neutral to basic region (normally 6.5 to 9.5).
• the agglutination signal may be measured using an arbitrary method used to measure agglutination.
• the agglutination signal may be measured by measuring the absorbance, the particle count, the particle size, scattered light, or the like.
• PSA is measured as described below.
• agglutination occurs when at least two kinds of antibodies that can react with both fPSA and PSA-ACT are immobilized on carriers (e.g., latex) and thereafter react with a sample that includes fPSA and/or PSA-ACT.
• the total PSA level in the sample can be determined by measuring the degree of agglutination, and comparing the measured degree of agglutination with the degree of agglutination when using a standard solution having a known PSA level.
• the degree of agglutination is preferably detected as a change in absorbance by utilizing a general-purpose biochemical automatic analyzer. In this case, it is preferable to use a change in absorbance at a wavelength of 500 to 900 nm for determination of the degree of agglutination.
• PSA can thereby be quantitatively determined with high accuracy.
• the expression “equimolar response to fPSA and PSA-ACT” used herein means that the ratio of a signal obtained by measuring a sample that includes only fPSA to a signal obtained by measuring a sample that includes only PSA-ACT (equal mol with fPSA) is approximately 1:1.
• the ratio (PSA-ACT/fPSA) (hereinafter may be referred to as “c/f ratio”) of agglutination signals per equal mol of fPSA and PSA-ACT is calculated, and it is considered that an equimolar response is obtained when the c/f ratio is 85 to 115% (most preferably 90 to 110%).
• the PSA assay reagent according to the embodiments of the invention is produced as described below.
• the PSA assay reagent may be produced by providing carriers having immobilized thereon two kinds of antibodies that can react with both PSA-ACT and fPSA and differ in recognition site, setting the mixing ratio of the carriers having immobilized thereon two kinds of antibodies and have an identical average particle size (more than 0.20 ⁇ m and equal to or less than 0.40 ⁇ m) within a specific range, and adjusting the concentration of the agglutination accelerator so that an equimolar response to PSA-ACT and fPSA is obtained.
• the mixing ratio is 1:10 to 10:1, preferably 1:5 to 5:1, and more preferably 1:2 to 2:1.
• Human semen-derived purified PSA (SCIPAC Ltd., Code No. P117-7, degree of purification: 96%) was used as an immunogen.
• the purified PSA was used after dialysis using 20 mM PBS (pH: 7.2).
• the above PSA solution and a complete Freund's adjuvant (CFA) were mixed and emulsified in a ratio of 1:1, and administered subcutaneously to the back of 6-week-old female Balb/C mice in an amount of 25 ⁇ g PSA/mouse. Additional immunization was performed three times at intervals of 2 weeks, and the PSA solution (25 ⁇ g PSA/mouse) was administered intraperitoneally 3 days before cell fusion.
• CFA complete Freund's adjuvant
• the spleen was removed from each mouse immunized with PSA to collect spleen cells.
• the spleen cells and mouse myeloma cells SP2/O—Ag14 were mixed in a ratio of 6:1, and fused in the presence of 50% polyethylene glycol 1540 (Wako Pure Chemical Industries, Ltd.).
• the fused cells were suspended in an HAT medium so that the number of spleen cells was 2.5 ⁇ 10 6 , and dispensed onto a 96-well culture plate (CORNING Inc.) in an amount of 0.2 ml/well.
• the fused cells were cultured at 37° C. for 2 weeks in a 5% CO 2 incubator.
• PSA SCIPAC, Code No. P117-7
• PSA-ACT SCIPAC, Code No. P192-3
• An ELISA plate (Nunc) was coated (50 ⁇ l/well) with a goat anti-mouse IgG (Fc) antibody (Jackson Inc.) (5 ⁇ g/ml), and allowed to stand at 4° C. overnight. 2) After washing the ELISA plate with a washing solution (0.05% Tween 20-PBS) three times (400 ⁇ l/well), a blocking reagent (0.05% Tween 20-PBS) was dispensed into each well in an amount of 200 ⁇ l/well, and the ELISA plate was allowed to stand at room temperature for 1 hour.
• a washing solution 0.05% Tween 20-PBS
• a blocking reagent 0.05% Tween 20-PBS
• an HRP-rabbit anti-human PSA antibody ( ⁇ 500) was dispensed into each well in an amount of 50 ⁇ l/well, and the ELISA plate was allowed to stand at room temperature for 1 hour. Note that the HRP-rabbit anti-human PSA antibody was prepared by a periodic acid method using a rabbit anti-human PSA antibody (DAKO) and peroxidase (Toyobo Co., Ltd.).
• an OPD color reagent was dispensed into each well in an amount of 50 ⁇ l/well, and the ELISA plate was allowed to stand at room temperature for 10 minutes.
• a stop solution (1.5 N sulfuric acid) was dispensed into each well in an amount of 50 ⁇ l/well to terminate the reaction, and the absorbance at a wavelength of 492 nm was measured using a plate leader.
• the cell lines in the wells that reacted with both fPSA and PSA-ACT during the above screening were cloned by a limiting dilution method to establish hybridomas. 28 types of established hybridomas were thus obtained.
• the hybridomas (0.5 ⁇ 10 6 cells) obtained by cloning were administered intraperitoneally to 8-week-old female Balb/C mice to which 0.5 ml of pristane was administered intraperitoneally 2 weeks ago. Abdominal fluid was collected when 2 weeks had elapsed, and an IgG fraction was purified using a protein A column (Amersham plc). Purified fractions of the 28 types of monoclonal antibodies were thus obtained.
• Each of the 28 types of monoclonal antibodies is immobilized on a latex, and agglutination of the latex was observed as described below. Two kinds of antibodies (#91 antibody and #51 antibody) that showed a large signal, and combinations thereof with the latex were selected.
• the hybridomas that produce the antibodies #63251, #63279, and #63291 are deposited at International Patent Organism Depositary (IPOD), National Institute of Advanced Industrial Science and Technology as accession numbers FERM BP-11453, FERM BP-11454, and FERM BP-11455.
• the monoclonal antibodies obtained from these hybridomas may be referred to as “#51 antibody”, “#79 antibody”, and “#91 antibody”, which respectively corresponds to the first- and second-final numbers of the identification numbers.
• a glass reaction vessel (2 l) equipped with a stirrer, a reflux condenser, a thermometer, a nitrogen inlet tube, a heating oil bath, and the like was charged with 1200 g of water, 200 g of a styrene monomer, 1.2 g of potassium persulfate, and 0.2 g of sodium styrene sulfonate, and the atmosphere inside the reaction vessel was sufficiently replaced with nitrogen with stirring (about 200 rpm). After polymerizing the monomer at 70° C. for about 18 hours, the reaction solution was filtered through filter paper (ADVANTEC No. 2) to obtain latex particles.
• ADVANTEC No. 2 filter paper
• the average particle size ( ⁇ SD) of the latex particles was determined by photographing the latex particles using a transmission electron microscope, randomly selecting three fields of view, subjecting 100 or more latex particles within each field of view to image analysis to determine the average particle size ( ⁇ SD) of the latex particles for each field of view, and averaging the average particle size ( ⁇ SD) of the latex particles for each field of view.
• the average particle size thus determined was 0.19 ⁇ m ( ⁇ 0.01 ⁇ m).
• Latex particles were obtained in the same manner as in section 1), except that 1200 g of water, 200 g of a styrene monomer, 2.4 g of potassium persulfate, and 0.1 g of sodium styrene sulfonate were used.
• the average particle size of the latex particles thus obtained was 0.23 ⁇ m ( ⁇ 0.01 ⁇ m).
• Latex particles were obtained in the same manner as in section 1), except that 1200 g of water, 200 g of a styrene monomer, 0.9 g of potassium persulfate, and 0.2 g of sodium styrene sulfonate were used.
• the average particle size of the latex particles thus obtained was 0.25 ⁇ m ( ⁇ 0.01 ⁇ m).
• Latex particles were obtained in the same manner as in section 1), except that 1200 g of water, 200 g of a styrene monomer, 1.3 g of potassium persulfate, and 0.1 g of sodium styrene sulfonate were used.
• the average particle size of the latex particles thus obtained was 0.29 ⁇ m ( ⁇ 0.01 ⁇ m).
• Latex particles were obtained in the same manner as in section 1), except that 1200 g of water, 200 g of a styrene monomer, 1.3 g of potassium persulfate, and 0.08 g of sodium styrene sulfonate were used.
• the average particle size of the latex particles thus obtained was 0.34 ⁇ m ( ⁇ 0.01 ⁇ m).
• Latex particles were obtained in the same manner as in section 1), except that 1200 g of water, 200 g of a styrene monomer, 1.3 g of potassium persulfate, and 0.03 g of sodium styrene sulfonate were used.
• the average particle size of the latex particles thus obtained was 0.40 ⁇ m ( ⁇ 0.01 ⁇ m).
• Latex particles were obtained in the same manner as in section 1), except that 1200 g of water, 200 g of a styrene monomer, 1.2 g of potassium persulfate, and 0.01 g of sodium styrene sulfonate were used.
• the average particle size of the latex particles thus obtained was 0.42 ⁇ m ( ⁇ 0.01 ⁇ m).
• Polystyrene latexes (average particle size: 0.19 to 42 ⁇ m) obtained as described above
• #91 antibody-latex complex (#91Lx)-containing liquid 1)
• the latex and the #91 antibody were respectively diluted with a 20 mM glycine buffer (pH: 9) to prepare a 1% latex liquid and a #91 antibody liquid (0.4 mg/ml).
• the latex liquid and the #91 antibody liquid were mixed (1:1, v/v), and the mixture was stirred for about 1 hour.
• a blocking reagent (10% BSA) was added to the mixture (0.1:2, v/v), and the mixture was stirred for about 1 hour.
• a blocking reagent (10% BSA) was added to the mixture (0.1:2, v/v), and the mixture was stirred for about 1 hour.
• the supernatant liquid was removed by centrifugation, suspended in a 5 mM MOPS buffer (pH: 7.0), and diluted so that the absorbance at a wavelength of 600 nm was 3 Abs/ml to obtain a #51 antibody-immobilized latex (#51Lx) liquid.
• fPSA and PSA-ACT concentration: 5 ng/ml were used as measurement samples, and the reactivity with fPSA and PSA-ACT was determined using the following PSA measurement reagents and measurement conditions.
• the ratio “PSA-ACT/fPSA” (c/f ratio) of the reactivity with PSA-ACT to the reactivity with fPSA was determined. The results are shown in Table 1.
• Each combination of the antibody-containing liquids used in this example is prepared by respectively using latex particles having an identical average particle size (0.19 to 0.42 ⁇ m) and immobilizing thereon the #91 antibody or the #51 antibody.
• Nanopia registered trademark
• PSA calibrator PSA concentration: 0, 4.2, 10, and 29 ng/ml, Sekisui Medical Co., Ltd.
• the measurement samples were prepared by dissolving fPSA and PSA-ACT (Fitzgerald) in PBS (pH: 7.4) including 1% BSA and 0.1% NaN 3 .
• the c/f ratio was less than 80% and an equimolar response was not obtained when the average particle size was 0.20 ⁇ m or less, or exceeded 0.40 ⁇ m.
• fPSA and PSA-ACT were used as measurement samples.
• the reactivity with fPSA and PSA-ACT was determined using the following PSA measurement reagents under the same measurement conditions as those employed in Test Example 1, and the ratio “PSA-ACT/fPSA” (c/f ratio) of the reactivity with PSA-ACT to the reactivity with fPSA was determined.
• the results are shown in Tables 2 to 4.
• agglutination accelerator PEG (polyethylene glycol) (the numerical value is the number average molecular weight)
• PVP polyvinylpyrrolidone
• MPC polymer
• Pullulan PI-20 molecular weight: about 200,000, Hayashibara Co., Ltd.
• PGA-Na poly- ⁇ -sodium glutamate
• the antibody-containing liquid used in this example is prepared by using latex having an identical average particle size (0.29 ⁇ m) and immobilizing thereon the #91 antibody and the #51 antibody.
• the c/f ratio was determined using a combination of the monoclonal antibodies #79 and #91, #14 and #51, #91 and #17, or #14 and #17 instead of the combination of the monoclonal antibodies #51 and #91, and using the following measurement reagent including a 0.6% MPC polymer as the agglutination accelerator. The remaining conditions were the same as those employed in Test Example 1. fPSA and PSA-ACT (concentration: 35 ng/ml) were used as samples.
• Table 6 shows the Kd values of the monoclonal antibodies used in the test examples for fPSA and PSA-ACT. As shown in Table 6, it was confirmed that it is preferable that the anti-PSA monoclonal antibodies have a ratio (fKd/cKd) of the dissociation constant (fKd) for free PSA to the dissociation constant (cKd) for complex of free PSA and ⁇ 1-antichymotrypsin (PSA-ACT) of more than 0.1 and equal to or less than 2.0, and have a dissociation constant (fKd) for free PSA of 10 nM or less.
• fKd dissociation constant
• the Kd value in Table 6 was determined under the following experimental conditions.
• Biacore registered trademark
• GE Healthcare form Biacore
• i) to (viii) indicate the product name and the catalog No. from Biacore (currently available from GE Healthcare))
• Biacore registered trademark
• the PSA antigen RSA or PSA-ACT is diluted to 5.0 ⁇ g/ml with the running buffer (HBS-EP), and added for 120 seconds at a flow rate of 30 ⁇ l/min.
• HBS-EP running buffer
• a free running operation is performed using the running buffer (HBS-EP) to effect dissociation for 120 seconds (flow rate: 30 ⁇ l/min).
• the sensor chip is regenerated using a regenerant (Glycine 1.75 prepared by mixing Glycine 1.5 and Glycine 2.0 in a ratio of 1:1).
• the assay and the reagent according to the embodiments of the invention can easily and accurately measure PSA using a general-purpose automatic analyzer, and are useful for early detection of prostatic diseases (particularly prostate cancer).

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