WO2010090264A1 - Psaの分析方法、及び前記分析方法を用いた前立腺癌と前立腺肥大症との鑑別方法 - Google Patents
Psaの分析方法、及び前記分析方法を用いた前立腺癌と前立腺肥大症との鑑別方法 Download PDFInfo
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- WO2010090264A1 WO2010090264A1 PCT/JP2010/051622 JP2010051622W WO2010090264A1 WO 2010090264 A1 WO2010090264 A1 WO 2010090264A1 JP 2010051622 W JP2010051622 W JP 2010051622W WO 2010090264 A1 WO2010090264 A1 WO 2010090264A1
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- 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
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- 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
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4724—Lectins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/34—Genitourinary disorders
- G01N2800/342—Prostate diseases, e.g. BPH, prostatitis
Definitions
- the present invention relates to a method for analyzing PSA, a method for distinguishing between prostate cancer and benign prostatic hyperplasia using the same, and a kit for analyzing PSA.
- prostate cancer and benign prostatic hyperplasia can be reliably distinguished by using a lectin that binds to a sugar chain specifically expressed in PSA secreted from prostate cancer cancer cells.
- Prostate cancer mainly affects men over the age of 60, and in Europe and the United States, it is the cause of death after lung cancer among male cancers.
- the incidence of prostate cancer has increased since 1975, one of which is due to the spread of diagnostic methods using prostate specific antigen (hereinafter referred to as PSA).
- PSA prostate specific antigen
- PSA is a protein secreted from prostate gland cells into the glandular cavity of the prostate, and is a protein that is specifically expressed in the prostate, but is not a protein specifically expressed in prostate cancer. Therefore, it is known that it increases even in diseases such as prostatic hypertrophy and prostatitis other than prostate cancer.
- PSA measurement methods widely used include bound PSA conjugated with ⁇ 1-antichymotrypsin (hereinafter sometimes referred to as PSA-ACT) and free PSA (hereinafter sometimes referred to as free PSA). This is a measurement method for total PSA.
- prostate cancer When the measured value of total PSA is 10 ng / mL or more, prostate cancer is found in 50% or more of humans, but in humans of 4-10 ng / mL, 25%, Even at 4 ng / mL, 15% of humans have prostate cancer. In particular, the region where the total PSA value is 4 to 10 ng / mL is called a gray zone. However, even in patients with benign prostatic hyperplasia, there are many patients who show the total PSA value in this gray zone, and patients with prostate cancer and benign prostatic hyperplasia. It is expected to develop a PSA analysis method that can discriminate between them.
- the ratio of free PSA to total PSA is measured in order to differentiate prostatic hypertrophy from prostate cancer.
- the ratio of free PSA to total PSA is reduced.
- Free PSA is measured by a free PSA measurement method that measures only free PSA, and free PSA relative to total PSA is measured.
- a value hereinafter sometimes referred to as “free PSA / total PSA ratio”
- this value indicates 25% or less, it is said that the possibility of prostate cancer is high.
- the free PSA / total PSA ratio is in the range of 0-10%, the probability of prostate cancer is 56%, but 10-15% is 28%, 15-20% It is 16% for 20% and 20-25%, and it is not easy to differentiate prostate cancer from benign prostatic hyperplasia using the free PSA / total PSA ratio.
- PSA is a glycoprotein having one asparagine-linked sugar chain (hereinafter sometimes referred to as N-glycan chain), but PSA in prostate cancer patients is known to have a highly branched complex type sugar chain. It was thought that it had a sugar chain specific to prostate cancer patients.
- N-glycan chain of PSA secreted from LNCaP cells derived from prostate cancer was analyzed with a mass spectrometer, the content of N-acetylhexosamine (HexNAc) and fucose was higher than that of PSA in normal seminal plasma. It has been reported that sialic acid is lacking (Non-patent Document 1).
- the PSA sugar chain of this LNCaP cell is different from the PSA sugar chain in the sera of prostate cancer patients in vivo because it lacks sialic acid, and is considered to be a special PSA. It was.
- sialic acid ⁇ (2,3) galactose residue (Sialic acid alpha 2,3 Gal-R) in the N-glycan chain of PSA in seminal plasma, compared with patients with benign prostatic hyperplasia.
- this sialic acid ⁇ (2,3) galactose residue was reported to be high (Patent Document 1 and Non-Patent Document 2).
- MAA canine endurectin
- PSA of prostate cancer patient canine endurectin (hereinafter referred to as MAA) that specifically binds to the sialic acid ⁇ (2,3) galactose residue is bound to PSA of prostate cancer patient, and free PSA bound to MAA It has been found that prostate cancer and prostatic hypertrophy can be differentiated by measuring the ratio of these (patent document 1 and non-patent document 2).
- PSA-ACT separates PSA having sialic acid ⁇ (2,3) galactose residue from PSA having no sialic acid ⁇ (2,3) galactose residue. I can't. Therefore, in the method for measuring total PSA, prostate cancer patients and benign prostatic hyperplasia patients cannot be differentiated, and it was necessary to measure free PSA.
- the present inventors used the MAA column, which is a lectin described in Patent Document 1, to separate healthy PSA from prostate cancer patient PSA and tried to measure PSA having affinity for MAA.
- the recovery rate of normal PSA having no sialic acid ⁇ (2,3) galactose residue is 70%, and PSA is bound nonspecifically. it was thought.
- the recovery rate of PSA in prostate cancer patients is 40%.
- PSA having sialic acid ⁇ (2,3) galactose residues may not be eluted from MAA. It was.
- TJA-II Trichosanthes japonica Agglutinin-II
- WFA nodafuji lectin
- UEA-I harriere lectin-I
- the present invention comprises contacting a lectin having an affinity for ⁇ -N-acetylgalactosamine residues with a sample that may contain PSA, and determining the amount of PSA having an affinity for the lectin.
- the present invention relates to a method for analyzing PSA. In a preferred embodiment of the method for analyzing PSA according to the present invention, (a) by contacting the lectin with a sample possibly containing PSA, the PSA having affinity for the lectin and not having affinity for the lectin. Separating the PSA, and (b) determining the amount of PSA having affinity for the lectin in the sample.
- the determination of the amount of PSA having affinity for the lectin is determined by measuring the amount of PSA having affinity for the separated lectin. Determination by measuring the amount of PSA and measuring the amount of PSA having affinity for the fractionated lectin, or by measuring the amount of PSA in the sample before fractionation, and by measuring the amount of PSA having no affinity for the fractionated lectin It is. In a preferred embodiment of the PSA analysis method according to the present invention, the measurement of the amount of PSA is measurement of total PSA or free PSA.
- the lectin is Kikarasuri lectin-II or Nodafuji lectin.
- the lectin is a lectin further having an affinity for a fucose ⁇ (1,2) galactose residue.
- the sample is a sample obtained from a patient suspected of having prostate cancer. Furthermore, in a preferred embodiment of the method for analyzing PSA according to the present invention, it is for the diagnosis of prostate cancer.
- the present invention comprises contacting a lectin having an affinity for a fucose ⁇ (1,2) galactose residue with a sample that may contain PSA, and determining the amount of PSA having an affinity for the lectin.
- the present invention relates to a method for analyzing PSA. In a preferred embodiment of the method for analyzing PSA according to the present invention, by contacting (a) a lectin having an affinity for the fucose ⁇ (1,2) galactose residue and a sample that may contain PSA, Separating PSA having affinity for lectin and PSA having no affinity for lectin, and (b) determining the amount of PSA having affinity for lectin.
- the determination of the amount of PSA having affinity for the lectin is determined by measuring the amount of PSA having affinity for the separated lectin. Determination by measuring the amount of PSA and measuring the amount of PSA having affinity for the fractionated lectin, or by measuring the amount of PSA in the sample before fractionation, and measuring the amount of PSA having no affinity for the fractionated lectin It is. In a preferred embodiment of the PSA analysis method according to the present invention, the measurement of the amount of PSA is measurement of total PSA or free PSA.
- the lectin is Kikarasuri lectin-II or harine Nishida lectin.
- the sample is a sample obtained from a patient suspected of having prostate cancer. Furthermore, in a preferred embodiment of the method for analyzing PSA according to the present invention, it is for diagnosis of prostate cancer.
- the present invention contacts a lectin having an affinity for ⁇ -N-acetylgalactosamine residues and a lectin having an affinity for fucose ⁇ (1,2) galactose residues with a sample that may contain PSA. And determining the amount of PSA having affinity for the lectin.
- a lectin having an affinity for the ⁇ -N-acetylgalactosamine residue and a lectin having an affinity for a fucose ⁇ (1,2) galactose residue Separating a PSA having affinity for the lectin and a PSA having no affinity for the lectin by contacting with a sample that may contain PSA; and (b) a PSA having affinity for the lectin. Determining the amount of.
- the determination of the amount of PSA having affinity for the lectin is determined by measuring the amount of PSA having affinity for the separated lectin.
- the measurement of the amount of PSA is measurement of total PSA or free PSA.
- the lectin having affinity for the ⁇ -N-acetylgalactosamine residue is Kikarasuri lectin-II or nodafuji lectin, and fucose ⁇ (1,2) galactose
- the lectin having an affinity for the residue is Kikarasuri lectin-II or gorse lectin.
- the sample is a sample obtained from a patient suspected of having prostate cancer. Furthermore, in a preferred embodiment of the method for analyzing PSA according to the present invention, it is for the diagnosis of prostate cancer.
- the present invention also relates to a method for differentiating prostate cancer from benign prostatic hyperplasia characterized by analyzing the amount of PSA having affinity for a lectin in a sample by the method for analyzing PSA.
- the present invention also relates to an analysis kit for PSA comprising a lectin having an affinity for ⁇ -N-acetylgalactosamine residues.
- an anti-PSA antibody is further included.
- the lectin is Kikarasuri lectin-II or Nodafuji lectin.
- the lectin is a lectin further having an affinity for a fucose ⁇ (1,2) galactose residue.
- the present invention relates to an analysis kit for PSA containing a lectin having affinity for fucose ⁇ (1,2) galactose residues.
- a preferred embodiment of the analysis kit for PSA according to the present invention includes a lectin having an affinity for ⁇ -N-acetylgalactosamine residues and a lectin having an affinity for fucose ⁇ (1,2) galactose residues.
- the differentiation method of prostate cancer and benign prostatic hyperplasia of the present invention and the analysis kit of PSA, it is possible to reliably distinguish prostate cancer from benign prostatic hyperplasia.
- TJA-II and WFA which are lectins that can be used in the present invention, and PSA bound to UEA-I can be recovered with a recovery rate of almost 100%, and prostate cancer secreted by prostate cancer It is possible to accurately measure the amount of PSA having ⁇ -N-acetylgalactosamine residues derived from it.
- TJA-II and WFA columns and UEA-I columns can be regenerated and reused.
- TJA-II It is the photograph which electrophoresed purified TJA-II. M indicates a molecular weight marker, lane 1 indicates reduced TJA-II, and lane 2 indicates non-reduced TJA-II. It is the graph which showed the PSA amount (A) of the serum sample before a fraction by a TJA-II column of a prostate cancer patient and a prostatic hypertrophy patient, and the PSA amount (B) in a TJA-II binding fraction.
- a black circle ( ⁇ ) indicates a patient with prostate cancer
- a white circle ( ⁇ ) indicates a patient with prostatic hypertrophy.
- the method for analyzing PSA of the present invention comprises contacting a lectin that has an affinity for ⁇ -N-acetylgalactosamine residues with a sample that may contain PSA, and the affinity for the lectin. It is characterized in that the amount of PSA having a certain value is determined.
- Examples of the lectin that can be used in the present invention include a lectin having an affinity for a ⁇ -N-acetylgalactosamine residue (GalNAc ⁇ 1 ⁇ R) bound to the non-reducing end. In this case, it is essential that the ⁇ -N-acetylgalactosamine residue at the non-reducing terminal is not substituted with sialic acid or a sulfate group.
- the lectin having affinity for the ⁇ -N-acetylgalactosamine residue is not particularly limited, and examples thereof include TJA-II and WFA.
- Examples of the lectin that can be used in the present invention include lectins having affinity for fucose ⁇ (1,2) galactose residues (Fuc ⁇ 1 ⁇ 2Gal ⁇ 1 ⁇ R).
- the fucose ⁇ (1,2) galactose residue is a terminal sugar chain having a structure in which ⁇ -fucose is bonded to galactose in 1,2.
- the lectin having an affinity for a fucose ⁇ (1,2) galactose residue is not particularly limited, and examples thereof include UEA-I and TJA-II.
- lectins that can be used in the present invention include lectins having affinity for ⁇ -N-acetylgalactosamine residues (GalNAc ⁇ 1 ⁇ R) and fucose ⁇ (1,2) galactose residues bound to the non-reducing end. Can be mentioned.
- PSA of prostate cancer patients there may be PSA that expresses ⁇ -N-acetylgalactosamine residue or fucose ⁇ (1,2) galactose residue alone.
- it is highly possible that either ⁇ -N-acetylgalactosamine residue or fucose ⁇ (1,2) galactose residue is expressed predominantly in the PSA.
- a lectin having affinity for ⁇ -N-acetylgalactosamine residue (GalNAc ⁇ 1 ⁇ R) and fucose ⁇ (1,2) galactose residue bonded to the non-reducing end is used. It is preferable.
- the lectin having an affinity for ⁇ -N-acetylgalactosamine residue (GalNAc ⁇ 1 ⁇ R) and fucose ⁇ (1,2) galactose residue bound to the non-reducing end is not particularly limited, but TJA -II.
- TJA-II is a lectin extracted and purified from roots of yellow cucumber, and has a molecular weight of 64 kDa by non-reducing electrophoresis, but is an SS-bonded dimer and is obtained by reducing electrophoresis. The molecular weights are 32 kDa and 29 kDa. TJA-II shows a strong affinity for ⁇ -N-acetylgalactosamine residues (GalNAc ⁇ 1 ⁇ R) and fucose ⁇ (1,2) galactose residues (Fuc ⁇ 1 ⁇ 2Gal ⁇ 1 ⁇ R).
- WFA is a lectin extracted and purified from Nodafuji seeds, and has a strong affinity for ⁇ -N-acetylgalactosamine residue (GalNAc ⁇ 1 ⁇ R) itself. It also shows strong affinity for GalNAc ⁇ 1 ⁇ 4Gal residues and GalNAc ⁇ 1 ⁇ 4GlcNAc residues.
- UEA-I Ulex europaeus agglutinin-I
- Ulex opapaeus has a molecular weight of about 26,700.
- ⁇ -L-Fuc has sugar specificity and shows strong affinity for fucose ⁇ (1,2) galactose residues (Fuc ⁇ 1 ⁇ 2Gal ⁇ 1 ⁇ 4GlcNAc ⁇ R).
- the lectin having affinity for ⁇ -N-acetylgalactosamine residue the lectin having affinity for fucose ⁇ (1,2) galactose residue, and ⁇ -N-acetylgalactosamine residue
- lectin having affinity for fucose ⁇ (1,2) galactose residue the lectin having affinity for fucose ⁇ (1,2) galactose residue
- ⁇ -N-acetylgalactosamine residue One or a combination of two or more lectins having affinity for the group and fucose ⁇ (1,2) galactose residue can be used.
- lectin used in the present invention a commercially available lectin can be used, but it can also be purified according to a conventional method. Plants (for example, leaves, stems, flowers, roots, or seeds) are chopped or crushed and dissolved in a buffer solution. The supernatant can be collected by centrifugation and purified using ammonium sulfate salting-out, ion exchange column chromatography, hydrophobic column chromatography, gel filtration column chromatography, affinity column chromatography, dialysis, or lyophilization. . Specifically, the purification of TJA-II can be performed according to a report by Yamashita et al. (Yamashita et al., J. Biol.
- WFA can be purified according to the report of Toyoshima et al. (Toyoshima et al., Biochemistry, 10, 4457-4463, 1971).
- UEA-I can be purified according to a report by Hindsgaul et al. (Hindsgaul et al., Carbohydr. Res., 109, 109-142, 1982).
- the method for analyzing PSA of the present invention is not particularly limited as long as the lectin and a sample that may contain PSA are brought into contact and the amount of PSA having affinity for the lectin is determined.
- An analysis method for determining the amount of PSA having affinity for lectin by separating PSA having affinity for lectin and PSA having no affinity for lectin by lectin (hereinafter referred to as analysis method (A))
- analysis method (B) an analysis method for determining the amount of PSA having affinity for the lectin in a state where the lectin and the PSA having affinity for the lectin are bound (hereinafter, referred to as analysis method (B)). There are).
- the analysis method (A) is: (A) As a lectin of the present invention, for example, by contacting a lectin having affinity for ⁇ -N-acetylgalactosamine residue with a sample that may contain PSA, the PSA having affinity for the lectin and the A step of separating PSA having no affinity for lectins (hereinafter, sometimes referred to as “sorting step (a)”), and (b) a step of determining the amount of PSA having an affinity for lectins in the sample. (Hereinafter, sometimes referred to as “determination stage (b)”) including.
- the method for separating PSA having affinity for lectin and PSA having no affinity for lectin in the sorting step (a) is not particularly limited as long as it uses the affinity between lectin and PSA.
- a lectin is bound to a carrier, a sample that may contain PSA is brought into contact with the lectin bound to the carrier (hereinafter sometimes referred to as “lectin affinity column”), and the PSA and lectin bound to the lectin are contacted. This can be done by separating PSA that does not bind to.
- the carrier is not limited as long as it can bind a lectin.
- the shape of the carrier is not particularly limited, but carriers in the form of particulate beads, plates, gels, and the like can be used.
- the carrier preferably has a gel shape.
- the lectin affinity column can be prepared according to a conventional method. For example, using CNBr-activated Sepharose 4B, coupling can be performed according to the manufacturer's recommended protocol to produce a lectin column.
- the amount of lectin bound to the sepharose gel is preferably 2 mg / mL to 10 mg / mL.
- a method of separating PSA having affinity for the lectin and PSA having no affinity for the lectin using a lectin affinity column can be performed according to a glycoprotein separation method using a normal lectin affinity column.
- the lectin affinity column is equilibrated with buffer before applying a sample that may contain PSA.
- a phosphate buffer containing 0.1% bovine serum albumin (BSA) or a tris-HCl buffer containing 0.1% bovine serum albumin (BSA) can be used.
- a sample that may contain PSA is added and allowed to stand for a period of time to allow lectin and PSA to contact.
- the contact time is not particularly limited and can be appropriately determined depending on the type of lectin and the affinity of PSA. However, considering the binding speed and efficiency, the contact time is usually 15 to 30 minutes. .
- the temperature at which the lectin and PSA are brought into contact is not particularly limited, and can be determined as appropriate depending on the type of lectin and the affinity of PSA, but can be performed at 0 ° C. to 40 ° C., The temperature is preferably 0 ° C to 30 ° C. If the temperature is lower than 0 ° C, the column may be frozen, and if the temperature is higher than 40 ° C, nonspecific binding of a protein having no affinity for lectin may occur.
- the temperature at which TJA-II and PSA are brought into contact with each other is not particularly limited, but it is usually preferred that the contact is made at 4 to 10 ° C.
- the temperature at which WFA and PSA are brought into contact is not particularly limited, but it is usually preferred that the contact is made at 4 to 10 ° C.
- a bound fraction having affinity for the lectin (hereinafter sometimes referred to as “bound fraction”) and a non-bound fraction having no affinity for the lectin (hereinafter referred to as “non-bound fraction”) Be separated).
- the fraction not bound to lectin can be obtained by adding a washing buffer to the column and collecting the flow-through fraction.
- the washing buffer solution is not limited as long as it is a buffer solution that allows the unbound components to flow out without dissociating the binding between the lectin and PSA.
- the buffer solution used for the equilibration is not limited. Can be used.
- the volume of the washing buffer can be appropriately determined according to the kind of lectin and the affinity of PSA, but is preferably 3 to 7 times the column volume, more preferably about 5 times.
- the fraction bound to lectin can be obtained by adding an elution buffer to the column and collecting the eluted fraction.
- the elution buffer contains hapten sugar, whereby PSA bound to the lectin can be separated from the lectin.
- the hapten sugar can be appropriately selected depending on the specificity of the lectin, but in the case of TJA-II, lactose or the like can be used as the hapten sugar, for example, 10 mM lactose, 0.1% bovine serum albumin (BSA)
- the eluted fraction can be collected using the phosphate buffer solution.
- WFA GalNAc or the like can be used as a hapten sugar.
- the eluted fraction can be collected using a phosphate buffer containing 10 mM GalNAc and 0.1% bovine serum albumin (BSA).
- the volume of the elution buffer can be appropriately selected, but is preferably 3 to 7 times the column volume, more preferably about 5 times.
- the elution temperature is not particularly limited, but can be carried out at 0 to 40 ° C., preferably 2 to 25 ° C., more preferably 4 to 20 ° C. If the temperature is lower than 0 ° C, the column may be frozen, and if the temperature is higher than 40 ° C, nonspecific binding of a protein having no affinity for lectin may occur.
- the temperature at which PSA is eluted from TJA-II is not particularly limited, but it is preferably eluted at room temperature.
- the temperature at which PSA is eluted from WFA is not particularly limited, but it is preferably eluted at room temperature.
- Determination of the amount of PSA having affinity for lectin in the determination step (b) (1) Determination by measuring the amount of PSA having affinity for the sorted lectin, (2) Determination of the amount of PSA in the sample before fractionation and determination by measurement of the amount of PSA having affinity for the fractionated lectin, or (3) Measurement of the amount of PSA in the sample before fractionation, and fractionation Examples include determination by measuring the amount of PSA having no affinity for lectins.
- the determination (1) by measuring the amount of PSA having affinity for the sorted lectin can be performed by measuring the amount of PSA in the fraction bound to the lectin quantitatively or semi-quantitatively. That is, the determination is performed by measuring the absolute amount of PSA having affinity for the lectin contained in the blood of the patient.
- the determination (2) by measuring the amount of PSA in the sample before fractionation and measuring the amount of PSA having affinity for the fractionated lectin is based on the amount of PSA in the sample before fractionation (or the binding to lectin). And the amount of PSA in the fraction bound to the lectin) and the amount of PSA in the fraction bound to the lectin. Specifically, the PSA in the fraction bound to the lectin relative to the amount of PSA in the sample before fractionation (or the total amount of PSA in the fraction bound to the lectin and the fraction not bound to the lectin). By calculating the ratio of the amount, it is possible to determine the amount of PSA having affinity for the lectin. For example, it can be determined by any of the following formulas.
- PSA binding rate (PSA amount in bound fraction / total amount of PSA in bound and unbound fraction) ⁇ 100%
- PSA binding rate (PSA amount in bound fraction / PSA amount in sample before fractionation) ⁇ 100%
- the determination (3) by measuring the amount of PSA in the sample before fractionation and measuring the amount of PSA having no affinity for the fractionated lectin is based on the amount of PSA in the sample before fractionation (or binding to the lectin).
- the total amount of PSA in the fraction and the fraction not bound to the lectin) and the amount of PSA in the fraction not bound to the lectin can be compared. Specifically, from the amount of PSA in the sample before fractionation (or the total amount of PSA in the fraction bound to the lectin and the fraction not bound to the lectin) in the fraction not bound to the lectin. By subtracting the amount of PSA, it is possible to determine the amount of PSA having affinity for the lectin.
- Lectin-affinity PSA amount PSA amount in the sample before fractionation ⁇ PSA amount in unbound fraction
- Lectin-affinity PSA amount total amount of PSA in bound and unbound fraction ⁇ PSA in unbound fraction amount
- PSA bound to the lectin is separated from the lectin and the amount thereof is measured.
- the amount of PSA in the sample before fractionation is not bound.
- the determination from the amount of PSA in the fraction does not measure the amount of PSA bound to the lectin, and therefore can be determined without separating the PSA bound to the lectin.
- the fractions containing PSA are examined in advance, and the fractions are obtained. It is also possible to measure the minute and analyze the PSA.
- the measurement method of PSA for determining the amount of PSA having affinity for lectin is not particularly limited as long as it can determine PSA quantitatively or semi-quantitatively. Although not intended, a total PSA measurement method or a free PSA measurement method can be used.
- the method for measuring total PSA or the method for measuring free PSA is an immunological method using an antibody or a fragment thereof according to a conventional method (for example, enzyme immunoassay, latex agglutination immunoassay, chemiluminescence immunoassay, fluorescent antibody method, A radioimmunoassay method, an immunoprecipitation method, an immunohistochemical staining method, a Western blot, etc.), and it is also possible to use what is marketed as a PSA measurement kit.
- a conventional method for example, enzyme immunoassay, latex agglutination immunoassay, chemiluminescence immunoassay, fluorescent antibody method, A radioimmunoassay method, an immunoprecipitation method, an immunohistochemical staining method, a Western blot, etc.
- a monoclonal antibody or a polyclonal antibody that can bind to both PSA-ACT and free PSA is used.
- an immunological analysis method is used as a method for measuring free PSA
- a monoclonal antibody or a polyclonal antibody that can bind only to free PSA is used.
- a monoclonal antibody or a polyclonal antibody can be prepared by a known method except that PSA-ACT or free PSA is used as an immunizing antigen.
- a monoclonal antibody can be produced by the method of Koehler and Milstein (Nature 256: 495 -497, 1975).
- Polyclonal antibodies can be immunized periodically, for example, in the skin of rabbits by mixing PSA-ACT or free PSA alone or with BSA, KLH, etc., as a simple antigen or mixed with an adjuvant such as Freund's complete adjuvant. To do. Blood is collected at the time when the antibody titer in the blood rises, and can be used as it is as antiserum or by purifying the antibody by a known method.
- the lectin affinity column using TJA-II or WFA that can be used in the method for analyzing PSA of the present invention recovers about 100% (at least 97% or more) of PSA in the sample. It turns out that it is possible.
- the recovery rate using lactose-containing phosphate buffer as an eluent is 30 to 70%, and the eluate is changed to 0.1 M acetic acid solution. However, the recovery rate was not improved.
- the analysis method (B) is a method in which PSA having an affinity for lectin is directly detected by lectin, and specific examples include lectin blot by electrophoresis or lectin blot by dot blot. Both lectin blots can be performed according to standard methods, but in the case of lectin blots by electrophoresis, a sample that may contain PSA is electrophoresed, and the PSA is transferred to a nitrocellulose membrane or PVDF membrane as a sample membrane. Use.
- a sample that may contain PSA is adsorbed to a nitrocellulose membrane or PVDF membrane by a dot blotting device and used as a sample membrane.
- the sample membrane is blocked with a blocking buffer and contacted in a solution containing a biotin-labeled lectin such as biotin-labeled WFA or biotin-labeled TJA-II.
- a biotin-labeled lectin such as biotin-labeled WFA or biotin-labeled TJA-II.
- the resultant is brought into contact with a chromogenic enzyme or a luminescent enzyme, for example, avidin labeled with HRP, and brought into contact with a chromogenic solution or a luminescent solution, and the obtained signal is detected.
- immunoblotting and enzyme immunization can be partially modified.
- the monoclonal antibody or polyclonal antibody against PSA is solid-phased on a nitrocellulose membrane or an ELISA plate and blocked with a blocking buffer.
- a sample that may contain PSA is contacted with a nitrocellulose membrane, an ELISA plate, or the like, and then contacted with a biotin-labeled lectin such as biotin-labeled WFA or biotin-labeled TJA-II.
- a coloring enzyme or an avidin labeled with a luminescent enzyme such as HRP is bound, and a signal can be detected with the coloring solution or the luminescent solution.
- test sample used in the analysis method of PSA of the present invention examples include biological samples or biological samples containing PSA, or biological samples or biological samples that may contain PSA, such as urine, Blood, serum, plasma, spinal fluid, saliva, cells, tissues, or organs, or preparations thereof (for example, biopsy specimens, in particular, prostate biopsy specimens), etc.
- Plasma or prostate biopsy specimens are preferred, particularly blood, serum, or plasma.
- Liquid samples such as urine, blood, serum, plasma, spinal fluid, saliva, etc. are diluted with an appropriate buffer according to each analysis method in the analysis method (A) or analysis method (B).
- a solid sample such as a cell, tissue, or organ is homogenized with an appropriate buffer having a volume of about 2 to 10 times the volume of the solid sample, and the suspension or its supernatant is obtained by the analysis method (A ) Or the analysis method (B) can be used as they are or after further dilution.
- the analysis method (A) when a lectin affinity column is used, the liquid sample, a solid sample suspension or its supernatant can be diluted with an appropriate buffer.
- the dilution factor is not particularly limited as long as the binding between lectin and PSA is not inhibited, but it is preferably 2 to 400 times, more preferably 2 to 300 times, and most preferably 4 to 200 times.
- the volume of the sample applied to the lectin affinity column is preferably 40% or less, more preferably 30% or less, and most preferably 20% or less of the bed volume of the column. For example, when a 1 mL bed volume lectin affinity column is used, 400 ⁇ L or less is preferable, 300 ⁇ L or less is more preferable, and 200 ⁇ L or less is most preferable.
- the binding rate to TJA-II that recognizes ⁇ -N-acetylgalactosamine residues and fucose ⁇ (1,2) galactose residues, and only ⁇ -N-acetylgalactosamine residues are determined.
- the binding rate to the recognized WFA is different. Therefore, it is preferable to determine a cut-off value that can distinguish between prostate cancer and benign prostatic hyperplasia according to the measured value of the lectin used.
- the cut-off value is most preferably a value that allows 100% of prostate cancer patients to be judged positive and 100% of patients with benign prostatic hyperplasia to be judged negative.
- the prostate cancer patient is considered 100% positive It is preferable to select the value to be used as the cutoff value, but any value in the overlapping range can be used as the cutoff value.
- the cutoff value for detection of prostate cancer patients is a value that can detect PSA of prostate cancer patients. If it is, it will not be limited, For example, it is possible to set more than 200 pg / mL and less than 240 pg / mL, Preferably it is 220 pg / mL.
- the binding rate of PSA to the lectins in Examples described later can be determined from the percentage that can be obtained by the following formula.
- PSA binding rate (PSA amount in bound fraction / PSA amount in sample before fractionation) ⁇ 100%
- the cut-off value of the binding rate of PSA is most preferably a value that allows 100% of prostate cancer patients to be determined as positive and 100% of patients with benign prostatic hyperplasia to be determined as negative. If the analyzed population of prostate cancer and benign prostatic hyperplasia results in overlapping PSA binding rates to lectins between prostate cancer patients and patients with benign prostatic hyperplasia, 100 patients with prostate cancer A value judged as% positive is preferable, but any value in the overlapping range can be used as the cutoff value.
- the cutoff value of the TJA-II binding rate of PSA in Examples described later is not limited as long as it is a value that can detect prostate cancer. For example, it exceeds 1.8% and is less than 3% It is possible to set to 2.4%, preferably 2.4%.
- the diagnostic kit of the present invention contains a lectin having affinity for ⁇ -N-acetylgalactosamine residues.
- the diagnostic kit of the present invention can also contain a lectin having an affinity for fucose ⁇ (1,2) galactose residues.
- the diagnostic kit of the present invention can also contain a lectin having an affinity for ⁇ -N-acetylgalactosamine residues (GalNAc ⁇ 1 ⁇ R) and fucose ⁇ (1,2) galactose residues.
- the lectin contained in the diagnostic kit of the present invention includes those bound to the carrier.
- the diagnostic kit of the present invention comprises a lectin having an affinity for the ⁇ -N-acetylgalactosamine residue, a lectin having an affinity for fucose ⁇ (1,2) galactose residue, and a ⁇ -N-acetylgalactosamine residue.
- a lectin having an affinity for the ⁇ -N-acetylgalactosamine residue a lectin having an affinity for fucose ⁇ (1,2) galactose residue
- a ⁇ -N-acetylgalactosamine residue a lectin having an affinity for fucose ⁇ (1,2) galactose residue.
- the lectin having an affinity for ⁇ -N-acetylgalactosamine residue is not particularly limited, and examples thereof include TJA-II and WFA. Further, the lectin having affinity for the fucose ⁇ (1,2) galactose residue is not particularly limited, and examples thereof include UEA-I and TJA-II. Furthermore, as a lectin having an affinity for ⁇ -N-acetylgalactosamine residue (GalNAc ⁇ 1 ⁇ R) and fucose ⁇ (1,2) galactose residue, TJA-II can be mentioned.
- the diagnostic kit of the present invention can further contain an anti-PSA antibody (eg, an antibody that specifically binds to PSA-ACT or free PSA) or a fragment thereof.
- an anti-PSA antibody eg, an antibody that specifically binds to PSA-ACT or free PSA
- an antibody either a monoclonal antibody or a polyclonal antibody can be used.
- the antibody fragment is not particularly limited as long as it retains the specific binding ability to PSA-ACT or free PSA.
- Fab, Fab ′, F (ab ′) 2 , or Fv is used. Can do.
- the diagnostic kit of the present invention containing the lectin and the anti-PSA antibody can contain the anti-PSA antibody or a fragment thereof in a desired form depending on the immunological technique used.
- a labeled antibody such as an enzyme immunoassay, a chemiluminescence immunoassay, a fluorescent antibody method, or a radioimmunoassay
- a labeled antibody or label labeled with a labeling substance In the form of an antibody fragment.
- the labeling substance examples include peroxidase, alkaline phosphatase, ⁇ -D-galactosidase, or glucose oxidase as an enzyme, fluorescein isothiocyanate or rare earth metal chelate as a fluorescent substance, and 3 H, 14 C as a radioisotope. Or 125 I and the like, and biotin, avidin, chemiluminescent substance, and the like. In the case of an enzyme, a chemiluminescent substance, or the like, since a signal that can be measured by itself cannot be provided, it is preferable to select and include a corresponding appropriate substrate.
- TJA-II that can be used in the present invention is a lectin that recognizes fucose ⁇ (1,2) galactose residues and ⁇ -N-acetylgalactosamine residues present at the non-reducing ends of sugar chains.
- WFA is a lectin that recognizes a ⁇ -N-acetylgalactosamine residue present at the non-reducing end of a sugar chain.
- UEA-I is a lectin that recognizes a fucose ⁇ (1,2) galactose residue present at the non-reducing end of the sugar chain.
- PSA that binds to TJA-II and WFA exists in the body of prostate cancer patients, that is, these sugar chain structures may appear in the PSA sugar chain with canceration.
- PSA of prostate cancer-derived LNCaP cells has been reported to have a HexNac ⁇ 1-HexNAc residue on the non-reducing terminal side.
- HexNAc includes ⁇ -N-acetylgalactosamine (GalNAc ⁇ ) and ⁇ -N-acetylglucosamine (GlcNAc ⁇ ), but they have the same molecular weight and cannot be distinguished by mass spectrometry. Moreover, it was thought that the sugar chain of PSA secreted from this LNCaP cell does not reflect the PSA in the living body of prostate cancer patients because it lacks sialic acid. Therefore, herein, by using TJA-II column, WFA column and UEA-I column, non-reducing terminal ⁇ -N-acetylgalactosamine and fucose ⁇ (1,2) galactose in serum PSA of prostate cancer patients This is the first identification of a residue.
- Patent Document 1 and Non-Patent Document 2 when a structural change of a sugar chain in prostate cancer is to be detected by MAA that specifically recognizes NeuAc ⁇ 2-3Gal ⁇ 1-4GlcNAc, a certain ratio of blood Medium PSA is present in the form of PSA-ACT bound to serum ⁇ 1-antichymotrypsin. Since MAA also binds to ⁇ 1-antichymotrypsin, even if the sugar chain structure of PSA is different, PSA bound to ⁇ 1-antichymotrypsin cannot be separated. Therefore, when PSA is separated by MAA, it is necessary to measure free PSA.
- TJA-II that can be used in the present invention recognizes a fucose ⁇ (1,2) galactose residue or ⁇ -N-acetylgalactosamine residue present at the non-reducing end of the PSA sugar chain
- WFA is a PSA sugar. It recognizes ⁇ -N-acetylgalactosamine residues present at the non-reducing end of the chain, but since these sugar chain structures do not exist on serum ⁇ 1-antichymotrypsin, they can be quantified using a total PSA measurement kit. Is possible.
- the amount of free PSA is 20% or less of the total amount of PSA, and in the analysis method of the present invention, a total PSA measurement kit can be used, so compared with the inventions described in Non-Patent Document 2 and Patent Document 1. It is advantageous in terms of sensitivity.
- TJA-II was purified from 20 g of root nodules of Trichosanthes japonica according to the previous report (Yamashita et al., J. Biol. Chem., 267, 25441-25422, 1992). Specifically, the root of Kikarasuuri was finely chopped and homogenized with 16 mL of 10 mM phosphate buffer (pH 7.4) containing 0.15 M NaCl using a Waring blender. Centrifugation was performed at 1000 ⁇ g for 30 minutes, and 35-55% saturated ammonium sulfate fraction precipitate of the obtained supernatant was dissolved in water and dialyzed against distilled water.
- TJA-II The purified TJA-II was used for coupling to a Sepharose column to produce a TJA-II column and a WFA column.
- CNJA-Sepharose manufactured by GE healthcare
- TJA-II was bound at a density of 3 mg per mL of gel volume according to the manufacturer's recommended protocol to prepare a TJA-II column.
- a WFA column was similarly prepared using WFA (manufactured by EY Laboratory) and CNBr-Sepharose (manufactured by GE healthcare).
- Example 1 Analysis of PSA using TJA-II
- 15 patients diagnosed with prostate cancer and 9 patients with total PSA diagnosed as prostatic hypertrophy of 4.0 ng / mL or more For these patients, the amount of PSA was measured by the method for analyzing PSA of the present invention.
- a TJA-II column (1 mL volume) was equilibrated with a phosphate buffer containing 0.1% bovine serum albumin (BSA). Serum samples 1 ⁇ L to 50 ⁇ L were diluted with a phosphate buffer to make 200 ⁇ L and applied to the column, and held for 30 minutes.
- BSA bovine serum albumin
- the column was washed with a 5-fold volume of washing buffer solution (phosphate buffer solution containing 0.1% BSA), and 1 mL each was fractionated to obtain a TJA-II non-binding fraction.
- the column was eluted with 5 mL of elution buffer (phosphate buffer solution containing 10 mM lactose and 0.1% BSA) and eluted to obtain a TJA-II binding fraction.
- the total PSA amount of the serum sample before fractionation using the TJA-II column, the TJA-II non-binding fraction, and the TJA-II binding fraction was measured using Access Hybridtec Total PSA (Beckman Coulter, Inc.).
- TJA-II binding rate (PSA amount in the TJA-II binding fraction / PSA amount in the TJA-II non-binding fraction and TJA-II binding fraction) ⁇ 100%
- free PSA of the serum sample before fractionation was measured using Access Hybridtec Free PSA (manufactured by Beckman Coulter). The free PSA / total PSA ratio was calculated from the total PSA amount and the free PSA amount.
- stage (stage) indicates the degree of progression of prostate cancer.
- the Gleason score is obtained by dividing the malignancy of prostate cancer into five levels according to the pathological classification. “1” means the most gentle cancer and “5” means the worst cancer, but since most prostate cancers have multiple components with different malignancy, the most common component and the next most common component
- a Gleason score of “6” or less is considered to be low malignancy, “7” is moderate malignancy, and “8” to “10” are high malignancy cancers.
- the amount of PSA in the TJA-II binding fraction and the TJA-II binding rate are not duplicated between prostate cancer patients and patients with benign prostatic hyperplasia. It shows that patients and patients with benign prostatic hyperplasia can be differentiated. Provisionally, if the cutoff value for the amount of PSA in the TJA-II binding fraction is 250 pg / mL and the cutoff value for the TJA-II binding rate is 2%, patients with prostate hypertrophy and prostate cancer with 100% accuracy It is possible to distinguish patients. There was no significant difference between the TJA-II binding rate and the amount of TJA-II binding PSA even at different clinical stages and Gleason scores.
- the recovery rate of PSA from the WFA column was 90% to 98%.
- the binding rate of WFA is almost correlated with the binding rate of TJA-II.
- WFA binds to PSA having ⁇ -N-acetylgalactosamine residue and can separate PSA of prostate cancer patients. is there.
- the WFA binding rate is slightly lower than the TJA-II binding rate, and is 84.0% to 93.8%. This indicates the possibility that PSA having affinity only for TJA-II exists in the blood of prostate cancer patients.
- Example 4 In this example, for PSA in patients with benign prostatic hyperplasia, PSA in prostate cancer patient serum, and PSA in seminal plasma, each PSA on a TJA-II column, UEA-I column, or WFA column The binding rate of was examined.
- the binding rate to the TJA-II column was measured according to the method described in Example 1.
- the measurement of the binding rate to the WFA column was performed according to the procedure described in Example 2. Further, the binding rate to the UEA-I column was measured as follows.
- the amount of PSA binding to UEA-I was measured using agarose (UEA-I agarose: J Oil Mills) bound to Ulex europaeus agglutinin-I (UEA-I).
- Example 1 Specifically, instead of the TJA-II column and the eluent (phosphate buffer solution containing 10 mM lactose and 0.1% BSA), UEA-I agarose and elution buffer (50 mM fucose, phosphorus containing 0.1% BSA) The procedure of Example 1 was repeated except that the acid buffer solution was used, and the UEA-I binding rate was measured. The results are shown in Table 4.
- PSA in the serum of patients with benign prostatic hyperplasia hardly binds to the TJA-II column, UEA-I column, or WFA column.
- PSA in prostate cancer patient serum 16% PSA binds to the TJA-II column, 5% PSA binds to the UEA-I column, and 11% PSA binds to the WFA column.
- Comparative Example 1 ⁇ Comparative Example 1 >> In this comparative example, using MAA which is a lectin described in Patent Document 1, fractionation and measurement of PSA in prostate cancer patients were performed. The purification of MAA was performed according to the method described previously (Kawaguchi et al., J. Biol. Chem., 249, 2786-2792, 1974). Specifically, 50 g of seeds of Maackia amurensis were homogenized with several hundred mL of PBS until they became fine with a homogenizer, and further stirred overnight, and then the precipitate was removed by centrifugation at 9000 rpm for 30 minutes.
- the 50-80% ammonium sulfate fraction of this extract (210 mL) was dialyzed against PBS, the precipitate was removed by centrifugation, and a portion (30 mL) was added to thyroglobulin sepharose (19 mg / mL, 15 mL). After washing with PBS, elution is performed with 0.15 L glycine hydrochloride buffer (pH 2.5) containing 0.1 M lactose and 0.075 M NaCl. The portion having high lectin activity is pooled and concentrated, and then replaced with 50 mM phosphate buffer (pH 4.5) by dialysis.
- Sepharose column was prepared using purified MAA. Using MABr-Sepharose (manufactured by GE healthcare), MAA was bound at a density of 3 mg per 1 mL of gel volume according to the attached protocol recommended by the manufacturer to prepare an MAA column.
- the column was washed with 5 volumes of a washing buffer solution (phosphate buffer containing 0.1% BSA and 0.02% Tween), and fractionated by 1 mL each to obtain a MAA non-binding fraction.
- a washing buffer solution phosphate buffer containing 0.1% BSA and 0.02% Tween
- elution was carried out by fractionating 1 mL each with 5 volumes of elution buffer (phosphate buffer containing 400 mM lactose, 0.1% BSA, 0.02% Tween). Got the minute.
- the oligosaccharide having 3 residues of sialic acid ⁇ (2,3) galactose did not bind to the column, and 95% or more was recovered in the MAA non-binding fraction.
- elution was performed by fractionating 1 mL each with 5 volumes of elution buffer (phosphate buffer containing 400 mM lactose, 0.1% BSA, 0.02% Tween). A bound fraction was obtained. About each fraction, the amount of total PSA was measured using access hybrid technical total PSA (made by Beckman Coulter). As a control, the same operation was repeated using 5 ng of purified normal free PSA, and the total amount of PSA was measured.
- the results of PSA of prostate cancer patients are shown in FIG. 4 (B), and the results of normal free PSA are shown in FIG. 4 (A).
- Normal free PSA was mostly detected in the second MAA unbound fraction. However, the amount of normal free PSA obtained by adding the amounts of PSA in all fractions was 3.5 ng with respect to 5 ng normal free PSA applied to the MAA column, and the recovery rate was 70%, 30% Was considered to remain bound to the column.
- PSA of prostate cancer patients has unadsorbed PSA detected in the second non-MAA bound fraction, slightly adsorbed PSA detected in the shoulder portion of the third non-MAA bound fraction, and 400 mM. It separated into adsorbed PSA eluted with lactose.
- the total amount of PSA in all fractions was 4 ng / mL, and only about 40% was recovered with respect to 10 ng / mL, which is the PSA amount of the serum sample before fractionation. This recovery rate was not improved even when a 0.1 M acetic acid solution was used as the elution solution.
- the PSA of prostate cancer patients used has a free PSA / total PSA ratio of 3.6, and 96.4% of PSA is present in the state of PSA-ACT combined with ⁇ -antichymotrypsin. Since ⁇ -antichymotrypsin has one sialic acid ⁇ (2,3) galactose residue per molecule, PSA-ACT should bind to the MAA column. Unadsorbed PSA detected in the first MAA non-binding fraction and slightly adsorbed PSA detected in the shoulder portion of the third MAA non-binding fraction were detected.
- the oligosaccharide having 3 residues of sialic acid ⁇ (2,3) galactose does not bind to the MAA column, and the presence of non-bonded PSA-ACT indicates that PSA is bound to the MAA lectin column. It is weak. However, despite the weak binding of PSA to MAA, the recovery rate of PSA was poor, elution with hapten sugar was difficult, and it was considered difficult to measure accurately with MAA with good reproducibility. .
- the PSA analysis method and PSA analysis kit of the present invention can reliably differentiate patients with prostate cancer and benign prostatic hyperplasia. Therefore, prostate cancer can be detected at an early stage in the health examination. In addition, since prostate cancer and benign prostatic hyperplasia can be reliably differentiated, the number of subjects for prostate biopsy performed for a definitive diagnosis can be reduced, and the burden on the patient can be reduced. As mentioned above, although this invention was demonstrated along the specific aspect, the deformation
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Abstract
Description
現在、広く使用されているPSA測定法は、α1-アンチキモトリプシンと結合した結合型PSA(以下、PSA-ACTと称することがある)及び遊離型PSA(以下、フリーPSAと称することがある)を測定するトータルPSAの測定法であり、トータルPSAの測定値が10ng/mL以上の場合、50%以上のヒトに前立腺癌が発見されるが、4~10ng/mLのヒトで25%、2~4ng/mLでも15%のヒトに前立腺癌の発生が見られる。特に、トータルPSA値が4~10ng/mLの領域をグレーゾーンと呼んでいるが、前立腺肥大症の患者でも、このグレーゾーンのトータルPSA値を示す患者が多く、前立腺癌と前立腺肥大症の患者を鑑別することのできるPSAの分析方法の開発が期待されている。
レクチンは、現在市販されているものだけでも100種類以上あるが、本発明者らは、糖結合特異性の異なる複数の植物レクチンを組み合わせて、前立腺癌患者のPSAに発現している糖鎖の同定を進め、前立腺癌患者のPSAと前立腺肥大症のPSAとを鑑別することについて鋭意研究したところ、キカラスウリレクチン-II(Trichosanthes japonica Agglutinin-II;以下、TJA-IIと称することがある)、ノダフジレクチン(Wisteria floribunda;以下、WFAと称することがある)又はハリエニシダレクチン-I(Ulex europaeus agglutinin-I;以下、UEA-Iと称することがある)に親和性のあるPSAが、前立腺癌患者の血液中に存在していることを見出し、そして前記TJA-II、WFA、又はUEA-Iを用いることにより、前立腺癌患者のPSAと前立腺肥大症のPSAとを鑑別することができることを見出した。すなわち、前立腺癌患者のPSAの多くが、β-N-アセチルガラクトサミン残基(GalNAcβ1→R)及び/又はフコースα(1,2)ガラクトース残基(Fucα1→2Galβ1→R)を有していることを見出した。
本発明は、このような知見に基づくものである。
本発明によるPSAの分析方法の好ましい態様においては、(a)前記レクチンとPSAを含む可能性のある試料とを接触させることにより、前記レクチンに親和性のあるPSAと前記レクチンに親和性のないPSAとを分別する段階、及び(b)前記試料中のレクチンに親和性のあるPSAの量を判定する段階を含む。
また、本発明によるPSAの分析方法の好ましい態様においては、前記レクチンに親和性のあるPSA量の判定が、分別されたレクチンに親和性のあるPSA量の測定による判定、分別前の試料中のPSA量の測定、及び分別されたレクチンに親和性のあるPSA量の測定による判定、又は分別前の試料中のPSA量の測定、及び分別されたレクチンに親和性のないPSA量の測定による判定である。
また、本発明によるPSAの分析方法の好ましい態様においては、前記PSA量の測定が、トータルPSA又はフリーPSAの測定である。
また、本発明によるPSAの分析方法の好ましい態様においては、前記レクチンが、キカラスウリレクチン-II又はノダフジレクチンである。
また、本発明によるPSAの分析方法の別の好ましい態様においては、前記レクチンが、更にフコースα(1,2)ガラクトース残基と親和性を有するレクチンである。
更に、本発明によるPSAの分析方法の好ましい態様においては、前記試料が、前立腺癌の疑いのある患者から得られた試料である。
更に、本発明によるPSAの分析方法の好ましい態様においては、前立腺癌の診断用である。
本発明によるPSAの分析方法の好ましい態様においては、(a)前記フコースα(1,2)ガラクトース残基と親和性を有するレクチンとPSAを含む可能性のある試料とを接触させることにより、前記レクチンに親和性のあるPSAと前記レクチンに親和性のないPSAとを分別する段階、及び(b)前記レクチンに親和性のあるPSAの量を判定する段階、を含む。
また、本発明によるPSAの分析方法の好ましい態様においては、前記レクチンに親和性のあるPSA量の判定が、分別されたレクチンに親和性のあるPSA量の測定による判定、分別前の試料中のPSA量の測定、及び分別されたレクチンに親和性のあるPSA量の測定による判定、又は分別前の試料中のPSA量の測定、及び分別されたレクチンに親和性のないPSA量の測定による判定である。
また、本発明によるPSAの分析方法の好ましい態様においては、前記PSA量の測定が、トータルPSA又はフリーPSAの測定である。
また、本発明によるPSAの分析方法の好ましい態様においては、前記レクチンが、キカラスウリレクチン-II又はハリエニシダレクチンである。
更に、本発明によるPSAの分析方法の好ましい態様においては、前記試料が、前立腺癌の疑いのある患者から得られた試料である。
更に、本発明によるPSAの分析方法の好ましい態様においては、前立腺癌の診断用である。
本発明によるPSAの分析方法の好ましい態様においては、(a)前記β-N-アセチルガラクトサミン残基に親和性のあるレクチン及びフコースα(1,2)ガラクトース残基と親和性を有するレクチンと、PSAを含む可能性のある試料とを接触させることにより、前記レクチンに親和性のあるPSAと前記レクチンに親和性のないPSAとを分別する段階、及び(b)前記レクチンに親和性のあるPSAの量を判定する段階、を含む。
また、本発明によるPSAの分析方法の好ましい態様においては、前記レクチンに親和性のあるPSA量の判定が、分別されたレクチンに親和性のあるPSA量の測定による判定、分別前の試料中のPSA量の測定、及び分別されたレクチンに親和性のあるPSA量の測定による判定、又は分別前の試料中のPSA量の測定、及び分別されたレクチンに親和性のないPSA量の測定による判定である。
また、本発明によるPSAの分析方法の好ましい態様においては、前記PSA量の測定が、トータルPSA又はフリーPSAの測定である。
また、本発明によるPSAの分析方法の好ましい態様においては、前記β-N-アセチルガラクトサミン残基に親和性のあるレクチンがキカラスウリレクチン-II又はノダフジレクチンであり、フコースα(1,2)ガラクトース残基と親和性を有するレクチンがキカラスウリレクチン-II又はハリエニシダレクチンである。
更に、本発明によるPSAの分析方法の好ましい態様においては、前記試料が、前立腺癌の疑いのある患者から得られた試料である。
更に、本発明によるPSAの分析方法の好ましい態様においては、前立腺癌の診断用である。
また、本発明は、β-N-アセチルガラクトサミン残基と親和性のあるレクチンを含む、PSAの分析キットにも関する。
本発明によるPSAの分析キットの好ましい態様においては、抗PSA抗体を更に含む。
また、本発明によるPSAの分析キットの好ましい態様においては、前記レクチンが、キカラスウリレクチン-II又はノダフジレクチンである。
更に、本発明によるPSAの分析キットの別の好ましい態様においては、前記レクチンが、更にフコースα(1,2)ガラクトース残基と親和性を有するレクチンである。
本発明によるPSAの分析キットの好ましい態様においては、β-N-アセチルガラクトサミン残基に親和性のあるレクチン及びフコースα(1,2)ガラクトース残基と親和性を有するレクチンを含む。
本発明のPSAの分析方法は、β-N-アセチルガラクトサミン残基と親和性のあるレクチンと、PSAを含む可能性のある試料とを接触させ、前記レクチンに親和性のあるPSAの量を判定することを特徴とする。
更に、本発明に用いることのできるレクチンとしては、非還元末端に結合したβ-N-アセチルガラクトサミン残基(GalNAcβ1→R)及びフコースα(1,2)ガラクトース残基と親和性を有するレクチンを挙げることができる。前立腺癌患者のPSA中には、β-N-アセチルガラクトサミン残基、又はフコースα(1,2)ガラクトース残基を単独で発現したPSAが存在する可能性がある。また、前立腺癌患者によっては、そのPSAにβ-N-アセチルガラクトサミン残基、又はフコースα(1,2)ガラクトース残基のいずれかが、優位に発現している可能性も高い。従って、本発明のPSAの分析方法においては、非還元末端に結合したβ-N-アセチルガラクトサミン残基(GalNAcβ1→R)及びフコースα(1,2)ガラクトース残基と親和性を有するレクチンを用いることが好ましい。前記非還元末端に結合したβ-N-アセチルガラクトサミン残基(GalNAcβ1→R)及びフコースα(1,2)ガラクトース残基と親和性を有するレクチンとしては、特に限定されるものではないが、TJA-IIを挙げることができる。
前記分析方法(A)は、
(a)本発明のレクチンとして例えばβ-N-アセチルガラクトサミン残基と親和性のあるレクチンと、PSAを含む可能性のある試料とを接触させることにより、前記レクチンに親和性のあるPSAと前記レクチンに親和性のないPSAとを分別する段階(以下、「分別段階(a)」と称することがある)、及び
(b)前記試料中のレクチンに親和性のあるPSAの量を判定する段階(以下、「判定段階(b)」と称することがある)
を含む。
レクチンアフィニティーカラムは、PSAを含む可能性のある試料をアプライする前に、緩衝液で平衡化させる。平衡化緩衝液としては、例えば、0.1%牛血清アルブミン(BSA)含有リン酸緩衝液、又は0.1%牛血清アルブミン(BSA)含有トリス塩酸緩衝液を用いることができる。
カラムを平衡化した後に、PSAを含む可能性のある試料を添加し、一定時間静置し、レクチンとPSAとを接触させる。接触時間は、特に限定されるものではなく、レクチンの種類とPSAの親和性によって、適宜決定することができるが、結合の速度と効率を考慮すると、通常15分~30分で行うことが多い。
レクチンとPSAとを接触させる温度も、特に限定されるものではなく、レクチンの種類とPSAとの親和性によって、適宜決定することができるが、0℃~40℃で行うことが可能であり、好ましくは0℃~30℃である。0℃未満では、カラムが凍結してしまうことがあり、40℃を超えるとレクチンに親和性のないタンパク質の非特異結合が起こることがある。例えば、TJA-IIとPSAを接触させる温度も特に限定されるものではないが、通常、4℃~10℃で接触させるのが好ましい。また、WFAとPSAとを接触させる温度も特に限定されるものではないが、通常、4℃~10℃で接触させるのが好ましい。
レクチンへの非結合画分は、洗浄用緩衝液をカラムに添加し、素通り画分を回収することによって得ることができる。洗浄用緩衝液は、レクチンとPSAの結合を解離させずに、非結合成分を流出させることのできる緩衝液であれば、限定されるものではないが、例えば、前記平衡化に用いた緩衝液を使用することができる。洗浄用緩衝液の容量は、レクチンの種類とPSAとの親和性に応じて、適宜決定することができるが、好ましくはカラム容量の3~7倍であり、より好ましくは5倍程度である。
レクチンへの結合画分は、溶出用緩衝液をカラムに添加し、溶出画分を回収することによって得ることができる。溶出用緩衝液は、ハプテン糖を含んでおり、それによりレクチンに結合したPSAをレクチンから分離させることができる。ハプテン糖はレクチンの特異性によって適宜選択することが可能であるが、TJA-IIの場合は、ラクトースなどをハプテン糖として用いることができ、例えば10mMラクトース、0.1%牛血清アルブミン(BSA)含有リン酸緩衝液を用いて、溶出画分を回収することができる。また、WFAの場合は、GalNAcなどをハプテン糖として用いることができ、例えば10mMGalNAc、0.1%牛血清アルブミン(BSA)含有リン酸緩衝液を用いて、溶出画分を回収することができる。溶出用緩衝液の容量は、適宜選択することが可能であるが、好ましくはカラム容量の3~7倍であり、より好ましくは5倍程度である。溶出の温度も特に限定されるものではないが、0℃~40℃、好ましくは2~25℃、より好ましくは4~20℃で行うことができる。0℃未満では、カラムが凍結してしまうことがあり、40℃を超えるとレクチンに親和性のないタンパク質の非特異結合が起こることがある。例えば、TJA-IIからPSAを溶出させる温度は、特に限定されるものではないが、室温で溶出させるのが好ましい。また、WFAからPSAを溶出させる温度も、特に限定されるものではないが、室温で溶出させるのが好ましい。
(1)分別されたレクチンに親和性のあるPSA量の測定による判定、
(2)分別前の試料中のPSA量の測定、及び分別されたレクチンに親和性のあるPSA量の測定による判定、又は
(3)分別前の試料中のPSA量の測定、及び分別されたレクチンに親和性のないPSA量の測定による判定
を挙げることができる。
PSAの結合率=(結合画分中のPSA量/結合画分及び非結合画分のPSAの合計量)×100%
PSAの結合率=(結合画分中のPSA量/分別前の試料中のPSA量)×100%
レクチン親和性PSA量=分別前の試料中のPSA量-非結合画分中のPSA量
レクチン親和性PSA量=結合画分及び非結合画分のPSAの合計量-非結合画分中のPSA量
また、分取した結合画分及び非結合画分のPSA量は、すべての画分についてPSA量の測定を行うことが好ましいが、予めPSAが含まれる分取画分を調べておき、その画分について測定を行い、PSAの分析を行うことも可能である。
前記分析方法(B)は、レクチンにより直接レクチンに親和性のあるPSAを検出する方法であるが、具体的には、電気泳動によるレクチンブロット、又はドットブロットによるレクチンブロットを挙げることができる。どちらのレクチンブロットも、定法に従って行うことができるが、電気泳動によるレクチンブロットの場合、PSAを含む可能性のある試料を電気泳動し、ニトロセルロース膜、PVDF膜にPSAを転写し、サンプルメンブレンとして用いる。ドットブロットによるレクチンブロットの場合、ドットブロッティング装置により、PSAを含む可能性のある試料をニトロセルロース膜、又はPVDF膜などに吸着させ、サンプルメンブレンとして用いる。サンプルメンブレンは、ブロッキングバッファーでブロッキングを行い、ビオチン標識したレクチン、例えばビオチン標識WFA又はビオチン標識TJA-IIを含む溶液中で接触させる。その後、発色酵素、又は発光酵素、例えばHRPで標識されたアビジンと接触させ、発色液又は発光液を接触させ、得られたシグナルを検出する。
例えば、前記分析方法(A)において、レクチンアフィニティーカラムを用いる場合、前記液体試料、又は固形の試料の懸濁液若しくはその上清は、適当な緩衝液により希釈して用いることができる。希釈倍率は、レクチンとPSAとの結合が阻害されなければ、特に限定されないが、好ましくは2~400倍であり、より好ましくは2~300倍であり、最も好ましくは4~200倍である。また、レクチンアフィニティーカラムにアプライする試料の容量は、カラムのベッドボリュームの40%以下が好ましく、30%以下がより好ましく、20%以下が、最も好ましい。例えば1mLのベッドボリュームのレクチンアフィニティーカラムを用いる場合、400μL以下が好ましく、300μL以下がより好ましく、200μL以下が最も好ましい。
前記PSAの分析方法により、試料中のレクチンに親和性のあるPSAの量を分析することにより、前立腺癌と前立腺肥大症とを鑑別することができる。
前記PSAの分析方法により、レクチンに親和性のあるPSAの量を測定し、前立腺肥大症又は健常人から採取した血液等中のレクチンに親和性のあるPSAの量と比較することにより、前記患者が前立腺癌であるか否かを判定することができる。より具体的には、前立腺肥大症又は健常人の試料中に存在するレクチンに親和性のあるPSAと比べて、前記患者の試料中に存在するレクチンに親和性のあるPSAが有意に多い場合に前記患者は前立腺癌であると判定することができる。
前記分析方法(A)においては、β-N-アセチルガラクトサミン残基及びフコースα(1,2)ガラクトース残基を認識するTJA-IIへの結合率と、β-N-アセチルガラクトサミン残基のみを認識するWFAへの結合率が異なっている。従って、使用するレクチンの測定値によって、前立腺癌と前立腺肥大症とを鑑別することのできるカットオフ値を、それぞれ決定することが好ましい。カットオフ値は、前立腺癌患者の100%を陽性と判定でき、前立腺肥大症患者の100%を陰性と判定できる値が最も好ましい。もし、分析した前立腺癌と前立腺肥大症の母集団が増加することによって、前立腺癌患者と前立腺肥大症患者との間の測定値が、オーバーラップした場合は、前立腺癌患者が100%陽性と判断される値をカットオフ値として選択することが好ましいが、オーバーラップした範囲の任意の値をカットオフ値とすることも可能である。具体的には、後述の実施例のTJA-II結合画分中のPSA量を測定した場合、前立腺癌患者の検出のためのカットオフ値は、前立腺癌患者のPSAを検出することのできる値であれば、限定されないが、例えば200pg/mLを超え、240pg/mL未満に設定することが可能であり、好ましくは、220pg/mLである。
また、後述の実施例のレクチンへのPSAの結合率は、下記の式で得ることのできる百分率から決定することができる。
PSAの結合率=(結合画分中のPSA量/分別前の試料中のPSA量)×100%
PSAの結合率のカットオフ値も、前立腺癌患者の100%を陽性と判定でき、前立腺肥大症患者の100%を陰性と判定できる値が最も好ましい。もし、分析した前立腺癌と前立腺肥大症の母集団の増加によって、前立腺癌患者と前立腺肥大症患者との間で、レクチンへのPSAの結合率が、オーバーラップする場合は、前立腺癌患者が100%陽性と判断される値が好ましいが、オーバーラップした範囲の任意の値をカットオフ値とすることも可能である。具体的には、後述の実施例のPSAのTJA-II結合率のカットオフ値は前立腺癌を検出することのできる値であれば、限定されないが、例えば1.8%を超え、3%未満に設定することが可能であり、好ましくは2.4%である。
本発明の診断キットは、β-N-アセチルガラクトサミン残基と親和性のあるレクチンを含む。また、本発明の診断キットは、フコースα(1,2)ガラクトース残基と親和性のあるレクチンを含むこともできる。更に、本発明の診断キットは、β-N-アセチルガラクトサミン残基(GalNAcβ1→R)及びフコースα(1,2)ガラクトース残基と親和性を有するレクチンを含むこともできる。本発明の診断キットに含まれるレクチンは、上記担体に結合させた状態のものも含まれる。
例えば、標識化抗体を用いる免疫学的手法、例えば、酵素免疫測定法、化学発光免疫測定法、蛍光抗体法、又は放射免疫測定法などの場合には、標識物質で標識した標識化抗体又は標識化抗体断片の形態で含むことができる。標識物質の具体例としては、酵素としてペルオキシダーゼ、アルカリフォスファターゼ、β-D-ガラクトシダーゼ、又はグルコースオキシダーゼ等を、蛍光物質としてフルオレセインイソチアネート又は希土類金属キレート等を、放射性同位体として3H、14C、又は125I等を、その他、ビオチン、アビジン、又は化学発光物質等を挙げることができる。酵素又は化学発光物質等の場合には、それ自体単独では測定可能なシグナルをもたらすことはできないことから、それぞれ対応する適当な基質等を選択して含むことが好ましい。
グレーゾーンのPSA値(トータルPSA値が4~10ng/mL)を示す患者においては、前記のように血清試料のフリーPSA/トータルPSA比(F/T値)の測定が行われている。表1に示すように、前立腺肥大症患者9例中7例において、F/T値が25%以下を示しており、これらの患者は、確定診断のための生検の対象となるが、実際には前立腺肥大症であり、患者にとって過度の負担となっている。
本発明に用いることのできるTJA-IIは糖鎖の非還元末端に存在するフコースα(1,2)ガラクトース残基及びβ-N-アセチルガラクトサミン残基を認識するレクチンである。WFAは糖鎖の非還元末端に存在するβ-N-アセチルガラクトサミン残基を認識するレクチンである。また、UEA-Iは糖鎖の非還元末端に存在するフコースα(1,2)ガラクトース残基を認識するレクチンである。本明細書によって、前立腺癌患者の体内にTJA-II及びWFAと結合するPSAが存在することが示され、すなわち、癌化に伴ってPSA糖鎖に、これらの糖鎖構造が出現することが、初めて示されたものである。前記のように前立腺癌由来のLNCaP細胞のPSAは、非還元末端側にHexNacβ1-HexNAc残基持つことが報告されている。HexNAcには、β-N-アセチルガラクトサミン(GalNAcβ)及びβ-N-アセチルグルコサミン(GlcNAcβ)が含まれるが、質量分析では分子量が同一となり、区別することができない。また、このLNCaP細胞から分泌されるPSAの糖鎖は、シアル酸を欠如していることなどから、前立腺癌患者の生体のPSAを反映していないと考えられていた。従って、本明細書において、TJA-IIカラム、WFAカラム及びUEA-Iカラムを用いることによって、前立腺癌患者の血清PSAにおける非還元末端β-N-アセチルガラクトサミン、及びフコースα(1,2)ガラクトース残基を初めて同定できたものである。癌化に伴うPSA糖鎖構造の変化、すなわちフコースα(1,2)ガラクトース残基あるいはβ-N-アセチルガラクトサミン残基の出現は、前立腺癌の発症によって、糖鎖の合成に関わる糖転移酵素活性量の変化が背景にあるものと考えられる。
一方、本発明で用いることのできるTJA-IIはPSA糖鎖の非還元末端に存在するフコースα(1,2)ガラクトース残基又はβ-N-アセチルガラクトサミン残基を認識し、WFAはPSA糖鎖の非還元末端に存在するβ-N-アセチルガラクトサミン残基を認識するが、これらの糖鎖構造は血清α1-アンチキモトリプシン上には存在しないため、トータルPSA測定キットを用いて定量することが可能である。一般的にフリーPSA量はトータルPSA量の20%以下であり、本発明の分析方法においてはトータルPSA測定キットを用いることできるため、非特許文献2及び特許文献1に記載の発明と比較して感度の上で有利である。
キカラスウリ(Trichosanthes japonica)の根塊20gより、既報(Yamashita et al., J. Biol. Chem., 267, 25441-25422, 1992)に従って、TJA-IIを精製した。具体的には、キカラスウリの塊根を細かく細断し、ワーリングブレンダーを用いて、0.15M NaClを含む10mMリン酸緩衝液(pH7.4)16mLでホモジェナイズした。1000xgで30分遠心し、得られた上清の35~55%飽和の硫安分画沈殿物を水に溶解し、蒸留水で透析した。凍結乾燥後、435mgの35~55%飽和の硫安沈殿物を6mLのPBSに溶解し、PBSで平衡化したporcine stomach mucinセファロース4B(10mg/mLゲル)カラム10mLにアプライした。カラムを洗浄した後、0.1Mラクトースを含むPBSで溶出し、TJA-IIを得た。
精製したTJA-IIを用いて、セファロースカラムにカップリングさせ、TJA-IIカラム及びWFAカラムを作製した。具体的には、CNBr-Sepharose(GE healthcare社製)を用い、メーカーの推奨する添付のプロトコールに従い、ゲル容積1mLあたり3mgの密度でTJA-II結合させ、TJA-IIカラムを作製した。
また、WFA(EYラボラトリー社製)及びCNBr-Sepharose(GE healthcare社製)を用いて、同様にWFAカラムを作製した。
本実施例では、前記カラム作製例において作製したTJA-IIカラムを用いて、前立腺癌と診断された15人の患者及び前立腺肥大症と診断されたトータルPSAが4.0ng/mL以上の9人の患者について、本発明のPSAの分析方法によりPSAの量を測定した。
4℃の条件下で、TJA-IIカラム(1mL容量)を0.1%牛血清アルブミン(BSA)含有リン酸緩衝液で平衡化した。血清試料1μL~50μLをリン酸緩衝液で希釈し、200μLとして、カラムにアプライし、30分保持した。その後、5倍容量の洗浄用緩衝液(0.1%BSA含有リン酸緩衝液)でカラムを洗浄し、各1mLずつ分画してTJA-II非結合画分を得た。カラムを室温に戻した後、5倍容量の溶出緩衝液(10mMラクトース、0.1%BSA含有リン酸緩衝液)で、1mLずつ分画して溶出し、TJA-II結合画分を得た。TJA-IIカラムによる分別前の血清試料、TJA-II非結合画分、及びTJA-II結合画分について、トータルPSA量を、アクセスハイブリテックトータルPSA(ベックマンコールター社製)を用いて測定した。TJA-IIカラムからのPSA回収率は常に97%~100%であった。表1に分別前の血清試料のPSA量、TJA-II結合画分のPSA量、及びPSAのTJA-II結合率を示す。なお、TJA-II結合率は次の式により計算した。
TJA-II結合率=(TJA-II結合画分中のPSA量/TJA-II非結合画分及びTJA-II結合画分中のPSA量)×100%
また、分別前の血清試料のフリーPSAを、アクセスハイブリテックフリーPSA(ベックマンコールター社製)を用いて測定した。前記トータルPSAの量及びフリーPSAの量から、フリーPSA/トータルPSA比を計算した。結果を表1、図2及び図3に示す。なお、癌患者の、年齢、病期(ステージ)、グリソンスコアは表2に示した。病期(ステージ)は、前立腺癌の進展度を示したものである。また、グリソンスコアは、前立腺癌の悪性度を、病理学上の分類によって5段階に分けたものである。「1」が最もおとなしい癌で、「5」が最も悪い癌を意味するが、前立腺癌の多くは、複数の、悪性度の異なる成分を有しているため、最も多い成分と次に多い成分を足し算してスコア化したものが、グリソンスコアである。例えば、最も多い成分が「3」で次に多い成分が「4」の場合、グリソンスコアは「3」+「4」=「7」となる。グリソンスコアが「6」以下は悪性度の低いもの、「7」は中程度の悪性度、「8」~「10」は悪性度の高い癌と考えられている。
本実施例では、前記カラム製造例で製造したWFAカラムを用いて、前立腺癌と診断された3人の患者について、本発明のPSAの分析方法によりPSAの量を測定した。
具体的には、TJA-IIカラムと溶出液(10mMラクトース、0.1%BSA含有リン酸緩衝液)に代えて、WFAカラムと溶出緩衝液(10mM GalNAc、0.1%BSA含有リン酸緩衝液)を用いたことを除いては、前記実施例1の操作を繰り返し、前立腺癌患者の3人の血清試料を分析し、WFA結合画分中のPSA量及びWFA結合率を得た。WFA結合率の結果を表3に示す。
なお、WFA結合率は次の式により計算した。
WFA結合率=(WFA結合画分中のPSA量/WFA非結合画分及びWFA結合画分中のPSA量)×100%
また、比較のためにTJA-II結合率を表3に示す。
但し、WFA結合率は、TJA-II結合率よりやや低く、84.0%~93.8%である。このことは、前立腺癌患者の血液中には、TJA-IIのみに親和性のあるPSAが存在する可能性を示している。すなわち、β-N-アセチルガラクトサミン残基(GalNAcβ1→R)を有さず、フコースα(1,2)ガラクトース残基(Fucα1→2Galβ1→R)のみを有しているPSAが存在する可能性を示唆している。
本実施例では、前立腺肥大症の患者血清中のPSA、前立腺癌の患者血清中のPSA、及び精漿中のPSAについて、TJA-IIカラム、UEA-Iカラム、又はWFAカラムへのそれぞれのPSAの結合率を調べた。
TJA-IIカラムへの結合率の測定は、実施例1に記載の方法に従った。WFAカラムへの結合率の測定は、実施例2に記載の操作に従った。
また、UEA-Iカラムへの結合率の測定は、以下のように行った。ハリエニシダアグルチニン-I(Ulex europaeus agglutinin-I:UEA-I)の結合したアガロース(UEA-Iアガロース:Jオイルミルズ)を用いて、UEA-Iに結合するPSAの量を測定した。具体的には、TJA-IIカラムと溶出液(10mMラクトース、0.1%BSA含有リン酸緩衝液)に代えて、UEA-Iアガロースと溶出緩衝液(50mM fucose、0.1%BSA含有リン酸緩衝液)を用いたことを除いては、前記実施例1の操作を繰り返し、UEA-I結合率を測定した。結果を表4に示す。
本比較例では、特許文献1に記載のレクチンであるMAAを用いて、前立腺癌患者のPSAの分別及び測定を行った。
MAAの精製は、既報(Kawaguchi et al., J. Biol. Chem., 249, 2786-2792, 1974)の方法に従った。具体的には、イヌエンジュマメ(Maackia amurensis)の種子50gを数百mLのPBSでホモジナイザーにより細かくなるまでホモジナイズし、更に一晩攪拌後、9000rpm30分遠心分離にて沈殿物を除いた。この抽出液(210mL)の50~80%の硫安分画画分をPBSに透析し、遠心操作で沈殿を除いた後、一部(30mL)をチログロブリンセファロース(19mg/mL、15mL)に添加し、PBSで洗浄後、0.1Mラクトース、0.075M NaCl含有0.15Mグリシン塩酸緩衝液(pH2.5)で溶出する。レクチン活性の高い部分をプールし濃縮後、50mMリン酸緩衝液(pH4.5)に透析にて置換する。遠心操作で沈殿を除去後、50mMリン酸緩衝液(pH4.5)で平衡化したSPセファデックスC-50(100mL)に添加し、非吸着画分を集め濃縮し、精製MAAを得た。
一方、前立腺癌患者のPSAは、2番目のMAA非結合分画に検出される未吸着のPSA、3番目のMAA非結合分画の肩の部分に検出されるわずかに吸着するPSA、及び400mMラクトースで溶出される吸着PSAに分かれた。すべての画分のPSA量を合計すると4ng/mLであり、分別前の血清試料のPSA量である10ng/mLに対して、40%程度しか回収されなかった。この回収率は、溶出溶液として、0.1M酢酸溶液を用いても改善されなかった。更に、用いた前立腺癌患者のPSAは、フリーPSA/トータルPSA比が3.6であり、96.4%のPSAがα-アンチキモトリプシンと結合したPSA-ACTの状態で存在している。α-アンチキモトリプシンは、1分子あたりシアル酸α(2,3)ガラクトース残基を1残基有しているため、PSA-ACTは、MAAカラムと結合するはずであるが、多くのPSAが2番目のMAA非結合分画に検出される未吸着のPSA、及び3番目のMAA非結合分画の肩の部分に検出されるわずかに吸着するPSAとして検出された。
以上、本発明を特定の態様に沿って説明したが、当業者に自明の変形や改良は本発明の範囲に含まれる。
Claims (19)
- β-N-アセチルガラクトサミン残基に親和性のあるレクチンと、
PSAを含む可能性のある試料とを接触させ、前記レクチンに親和性のあるPSAの量を判定することを特徴とする、PSAの分析方法。 - (a)前記β-N-アセチルガラクトサミン残基に親和性のあるレクチンとPSAを含む可能性のある試料とを接触させることにより、前記レクチンに親和性のあるPSAと前記レクチンに親和性のないPSAとを分別する段階、及び
(b)前記試料中のレクチンに親和性のあるPSAの量を判定する段階
を含む、請求項1に記載のPSAの分析方法。 - 前記レクチンに親和性のあるPSA量の判定が、
分別されたレクチンに親和性のあるPSA量の測定による判定、
分別前の試料中のPSA量の測定、及び分別されたレクチンに親和性のあるPSA量の測定による判定、又は
分別前の試料中のPSA量の測定、及び分別されたレクチンに親和性のないPSA量の測定による判定
である請求項2に記載のPSAの分析方法。 - 前記PSA量の測定が、トータルPSA又はフリーPSAの測定である、請求項3に記載のPSAの分析方法。
- 前記レクチンが、キカラスウリレクチン-II又はノダフジレクチンである、請求項1~4のいずれか一項に記載のPSAの分析方法。
- 前記レクチンが、更にフコースα(1,2)ガラクトース残基と親和性を有するレクチンである、請求項1~4のいずれか一項に記載のPSAの分析方法。
- 前記試料が、前立腺癌の疑いのある患者から得られた試料である、請求項1~6のいずれか一項に記載のPSAの分析方法。
- 前立腺癌の診断用である、請求項1~7のいずれか一項に記載のPSAの分析方法。
- フコースα(1,2)ガラクトース残基と親和性を有するレクチンと、
PSAを含む可能性のある試料とを接触させ、前記レクチンに親和性のあるPSAの量を判定することを特徴とする、PSAの分析方法。 - (a)前記フコースα(1,2)ガラクトース残基と親和性を有するレクチンとPSAを含む可能性のある試料とを接触させることにより、前記レクチンに親和性のあるPSAと前記レクチンに親和性のないPSAとを分別する段階、及び
(b)前記レクチンに親和性のあるPSAの量を判定する段階
を含む、請求項9に記載のPSAの分析方法。 - β-N-アセチルガラクトサミン残基に親和性のあるレクチン及びフコースα(1,2)ガラクトース残基と親和性を有するレクチンと、PSAを含む可能性のある試料とを接触させ、前記レクチンに親和性のあるPSAの量を判定することを特徴とする、PSAの分析方法。
- (a)前記β-N-アセチルガラクトサミン残基に親和性のあるレクチン及びフコースα(1,2)ガラクトース残基と親和性を有するレクチンと、PSAを含む可能性のある試料とを接触させることにより、前記レクチンに親和性のあるPSAと前記レクチンに親和性のないPSAとを分別する段階、及び
(b)前記レクチンに親和性のあるPSAの量を判定する段階
を含む、請求項11に記載のPSAの分析方法。 - 請求項1~12のいずれか一項に記載のPSAの分析方法により、試料中のレクチンに親和性のあるPSAの量を分析することを特徴とする、前立腺癌と前立腺肥大症との鑑別方法。
- β-N-アセチルガラクトサミン残基と親和性のあるレクチンを含む、PSAの分析キット。
- 抗PSA抗体を更に含む、請求項14に記載のPSAの分析キット。
- 前記レクチンが、キカラスウリレクチン-II又はノダフジレクチンである、請求項14又は15に記載のPSAの分析キット。
- 前記レクチンが、更にフコースα(1,2)ガラクトース残基と親和性を有するレクチンである、請求項14又は15に記載のPSAの分析キット。
- フコースα(1,2)ガラクトース残基と親和性を有するレクチンを含む、PSAの分析キット。
- β-N-アセチルガラクトサミン残基に親和性のあるレクチン及びフコースα(1,2)ガラクトース残基と親和性を有するレクチンを含む、PSAの分析キット。
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CN102317785B (zh) | 2015-01-14 |
US20110294141A1 (en) | 2011-12-01 |
JPWO2010090264A1 (ja) | 2012-08-09 |
CN102317785A (zh) | 2012-01-11 |
US10866240B2 (en) | 2020-12-15 |
CA2751400C (en) | 2018-04-17 |
EP2395357B1 (en) | 2015-05-06 |
EP2395357A4 (en) | 2012-10-10 |
JP5754767B2 (ja) | 2015-07-29 |
EP2395357A1 (en) | 2011-12-14 |
CA2751400A1 (en) | 2010-08-12 |
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