METHOD FOR DETECTING PROSTATE CANCER

Abstract

[Problem] To provide a method for detecting prostate cancer, the method having a higher degree of certainty than the PSA blood test and providing measurement values that correlate with risk classification.

[Solution] This method for detecting prostate cancer is characterized in that: the method includes reacting fucose 1.fwdarw.6 specific lectin and fucosylated PSA contained in a sample constituted of serum collected from a subject, and detecting the reacted lectin; and the pH in at least one step of the group consisting of the reaction step for reacting the fucosylated PSA and the lectin and processing steps thereafter is adjusted to above 8.5 and less than 11.0.

Claims

1. A method for detecting prostate cancer, comprising reacting a fucosylated PSA contained in a specimen consisting of serum sampled from a subject with a fucose 1.fwdarw.6 specific lectin to detect the reacted lectin, wherein a pH is adjusted within an alkaline region lower than 11.0 in at least one step of the group consisting of a step for reacting the fucosylated PSA with the lectin and subsequent processing steps.

2. The method for detecting prostate cancer according to claim 1, wherein the fucose 1.fwdarw.6 specific lectin is extracted from a basidiomycete belonging to Strophariaceae, Tricholomataceae, Amanitaceae or Polyporaceae.

3. The method for detecting prostate cancer according to claim 1, wherein the fucose 1.fwdarw.6 specific lectin is at least one of Pholiota squarrosa lectin, Pholiota terrestris lectin, Stropharia rugosoannulata lectin, Naematoloma sublateritium lectin, Lepista sordida lectin, and Amanita muscaria lectin.

4. The method for detecting prostate cancer according to claim 1, wherein the fucose 1.fwdarw.6 specific lectin is a protein or peptide containing: (a) a protein or peptide consisting of an amino acid sequence represented by any of SEQ ID Nos. 1 to 5; or (b) a protein or peptide consisting of an amino acid sequence represented by any of SEQ ID Nos. 1 to 5 in which one or more amino acids are deleted, inserted or substituted, and being functionally equivalent to a protein having the amino acid sequence represented by any of SEQ ID Nos. 1 to 5.

5. The method for detecting prostate cancer according to claim 1, wherein the fucose 1.fwdarw.6 specific lectin is labeled.

6. The method for detecting prostate cancer according to claim 1, wherein the fucosylated PSA is detected using the fucose 1.fwdarw.6 specific lectin and an antibody.

7. The method for detecting prostate cancer according to claim 1, wherein a reaction between the fucosylated PSA and the fucose 1.fwdarw.6 specific lectin is performed in presence of albumin.

8. The method for detecting prostate cancer according to claim 1, wherein when a subject shows a signal (reaction value) resulting from the reaction between the fucosylated PSA and the fucose 1.fwdarw.6 specific lectin higher than a signal (reference value) obtained from a person with a Gleason score of 6 or lower, it is suggested that the subject has a high-risk prostate cancer.

9. A diagnostic agent kit for detecting prostate cancer, containing: a fucose 1.fwdarw.6 specific lectin; and an alkaline reagent for adjusting a pH within an alkaline region lower than 11.0 in at least one step selected from a group of steps including a step for reacting a fucosylated PSA contained in a specimen consisting of serum sampled from a subject with the fucose 1.fwdarw.6 specific lectin and subsequent processing steps.

10. The diagnostic agent kit for detecting prostate cancer according to claim 9, further containing an anti-PSA antibody.

11. The method for detecting prostate cancer according to claim 1, wherein an alkaline region is not lower than pH 7.4.

12. The diagnostic agent for detecting prostate cancer according to claim 10, wherein an alkaline region is not lower than pH 7.4.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0041] FIG. 1 is a structural drawing of an 1.fwdarw.6 fucose oligosaccharide and a non-1.fwdarw.6 fucose oligosaccharide.

[0042] FIG. 2 is another structural drawing of the 1.fwdarw.6 fucose oligosaccharide and the non-1.fwdarw.6 fucose oligosaccharide.

DESCRIPTION OF EMBODIMENTS

[0043] Hereinafter, embodiments of the present invention will be explained in more detail. In the method for detecting prostate cancer according to the present invention (hereinafter, referred to as the method according to the present invention), a fucose 1.fwdarw.6 specific lectin is made to actuate on a fucosylated PSA contained in a specimen consisting a human serum under a specific condition to measure a signal (reaction value) based on a complex of the fucosylated PSA and the fucose 1.fwdarw.6 specific lectin.

[0044] A first candidate subject for the method according to the present invention is a human male who considers undergoing a PSA screening as a medical examination. The detection method according to the present invention detects prostate cancer with higher accuracy than that in the PSA screening, as demonstrated in the following Examples.

[0045] A second candidate subject for the method of the present invention is a person who exhibits an abnormally high blood PSA level of 4 ng/mL or more in a PSA screening. When a subject exhibits a blood PSA level of 4 ng/mL or more, he is suspected of prostate cancer and determined to be test-positive. Test-positive patients include patients who do not need treatment such as GS 6 patients and patients with advanced cancer such as GS 7 to 8 patients. In the method of the present invention, the reaction value of a complex of the fucosylated PSA and the fucose 1.fwdarw.6 specific lectin increases as the stage of prostate cancer progresses. Therefore, the method of the present invention can provide an indication for determining necessity of biopsy and information about cancer malignancy of a patient.

[0046] In Particular, a patient with a blood PSA level of 4-20 ng/mL is unlikely to have prostate cancer even if he is test-positive, or the patient is unlikely to require treatment because of being at GS 6 even if he suffers from prostate cancer. The method of the present invention can provide an indication of determining necessity of biopsy for such a patient.

[0047] The fucose 1.fwdarw.6 specific lectin can be defined by:

[0048] (1) a lower limit of a binding constant for the 1.fwdarw.6 fucose sugar chain; and

[0049] (2) an upper limit of a binding constant for sugar chains and glycolipid-type sugar chains excluding the 1.fwdarw.6 fucose.

[0050] More specifically, the fucose 1.fwdarw.6 specific lectin has all of the following properties [1]-[3].

[1] The fucose 1.fwdarw.6 specific lectin has affinity expressed by a binding constant of 1.010.sup.4 M.sup.1 or more (at 25 C.) for an 1.fwdarw.6 fucose sugar chain No. 405 having the following structural formula (1):

##STR00001##

[wherein Gal, GlcNAc, Man and Fuc refer to galactose, N-acetylglucosamine, mannose and fucose respectively.]
[2] The fucose 1.fwdarw.6 specific lectin has a binding constant of 1.010.sup.3 M.sup.1 or less (at 25 C.) for a sugar chain No. 003 excluding the 1.fwdarw.6 fucose and having the following structural formula (2):

##STR00002##

[wherein GlcNAc and Man refer to N-acetylglucosamine and mannose respectively.]
[3] The fucose 1.fwdarw.6 specific lectin has a binding constant of 1.010.sup.3 M.sup.1 or less (at 25 C.) for the glycolipid-type sugar chain No. 909 excluding the 1.fwdarw.6 fucose and having the following structural formula (3):

##STR00003##

[wherein Gal, GlcNAc, Fuc and Neu5Ac refer to galactose, N-acetylglucosamine, fucose, and N-acetylneuraminic acid respectively.]

[0051] In this specification, the binding constant means a value measured e.g., by means of a frontal affinity chromatography (FAC method) at an analysis temperature of 25 C. Details of the FAC method are described in Patent Document 2 filed by some of the present applicants, for example.

[0052] The binding constant (at 25 C.) of the fucose 1.fwdarw.6 specific lectin for the 1.fwdarw.6 fucose sugar chain No. 405 is preferably 5.010.sup.4 M.sup.1 or more, more preferably 1.010.sup.5 M.sup.1 or more, still more preferably 2.010.sup.5 M.sup.1 or more.

[0053] The binding constant (at 25 C.) for the sugar chain No. 003 and glycolipid-type sugar chain No. 909 excluding the 1.fwdarw.6 fucose is generally 1.010.sup.3 M.sup.1 or less, preferably 1.010.sup.2 M.sup.1 or less, particularly preferably 0.

[0054] Furthermore, the fucose 1.fwdarw.6 specific lectin may also have a high affinity for an 1.fwdarw.6 fucose sugar chain having a sialic acid at non-reduced terminal of the sugar chain No. 405. The term high affinity means that the binding constant (at 25 C.) is preferably 1.010.sup.4 M.sup.1 or more, more preferably 5.010.sup.4 M.sup.1 or more, and still more preferably 1.010.sup.5 M.sup.1 or more. On the other hand, some conventional lectins have a low affinity for the 1.fwdarw.6 fucose sugar chain having the sialic acid at the non-reduced terminal. Herein, the low affinity means that the binding constant (at 25 C.) is 1.010.sup.3 M.sup.1 or less.

[0055] The fucose 1.fwdarw.6 specific lectin further has an affinity expressed by a binding constant (at 25 C.) of preferably 1.010.sup.4 M.sup.1 or more, more preferably 5.010.sup.4 M.sup.1 or more, further preferably 1.010.sup.5 M.sup.1 or more for an N-linked single-, double-, triple- and/or quadruple-stranded sugar chain bound to the 1.fwdarw.6 fucose.

[0056] The molecular weight of the fucose 1.fwdarw.6 specific lectin based on SDS polyacrylamide electrophoresis is usually 4,000 to 40,000, preferably 4,000 to 20,000. Herein, the molecular weight based on SDS polyacrylamide electrophoresis is measured according to e.g. a method of Laemmi (Nature, vol. 227, page 680, 1976). The lectin may be generally formed by binding 2 to 10, preferably 2 to 6, more preferably 2 to 3 subunits to each other.

[0057] Fucose 1.fwdarw.6 specific lectins obtained from natural products will be outlined. The natural products are exemplified by mushrooms such as basidiomycetes and ascomycetes. Strophariaceae, Tricholomataceae, Polyporaceae and Amanitaceae belong to basidiomycetes. Examples of Strophariaceae include Pholiota squarrosa, Pholiota terrestris, Stropharia rugosoannulata, Naematoloma sublateritium, Pholiota aurivella, Pholiota adiposa and the like. Examples of Tricholomataceae include Lepista sordida and the like. Examples of Polyporaceae include Trichaptum elongatum, Microporus vemicipes and the like. Examples of Amanitaceae include Amanita muscaria and the like.

[0058] Methods of extracting and/or purifying the fucose 1.fwdarw.6 specific lectin from natural products are described in detail in Patent Document 2 filed by some of the present applicants and Non-Patent Document 5 submitted by the present applicants. Herein, Pholiota terrestris lectin (PTL) described in Patent Document 2 is replaced by Pholiota squarrosa lectin (PhoSL).

[0059] Among the above-mentioned basidiomycetes or ascomycetes, Strophariaceae, Tricholomataceae or Amanitaceae are preferred from the viewpoints of the specificity of the fucose 1.fwdarw.6 specific lectin for recognizing the 1.fwdarw.6 fucose sugar chain and the recovery efficiency of the lectin. Above all, Pholiota squarrosa lectin (PhoSL), Pholiota terrestris lectin (PTL), Stropharia rugosoannulata lectin (SRL), Naematoloma sublateritium lectin (NSL), Lepista sordida lectin (LSL) and Amanita muscaria lectin (AML) are particularly preferable. Amino acid sequences of the PhoSL, SRL, LSL and NSL are shown in Table 1.

[0060] The PhoSL shown in SEQ ID No. 1 is a lectin that can be extracted from Pholiota squarrosa. The Xaa at the 10th and 17th positions in SEQ ID No. 1 may be any amino acid residue, but is preferably Cys. The Xaa at the 20th, 23rd, 27th, 33rd, 35th and 39th positions are Tyr/Ser, Phe/Tyr, Arg/Lys/Asn, Asp/Gly/Ser, Asn/Ala and Thr/Gln, respectively.

[0061] The SRL shown in SEQ ID No. 2 is a lectin that can be extracted from Stropharia rugosoannulata. The Xaa at the 10th and 17th positions in SEQ ID No. 2 may be any amino acid residue, but is preferably Cys. The Xaa at the 4th, 7th, 9th, 13th, 20th, 27th, 29th, 33rd, 34th and 39th positions are Pro/Gly, Glu/Lys, Val/Asp, Asn/Asp/Glu, His/Ser, Lys/His, Val/Ile, Gly/Asn/Ser, Ala/Thr and Arg/Thr, respectively.

[0062] The LSL shown in SEQ ID No. 3 is a lectin that can be extracted from Lepista sordida. The Xaa at the 10th and 17th positions in SEQ ID No. 3 may be any amino acid residue, but is preferably Cys. The Xaa at the 1st, 4th, 7th, 8th, 9th, 13th, 16th, 20th, 22nd, 25th, 27th, 31st and 34th positions are Ala/Gln, Pro/Lys, Ala/Ser, Met/Ile/Val, Tyr/Thr, Asp/Asn, Lys/Glu, Ala/Asn, Val/Asp/Asn, Asp/Asn, Arg/His/Asn, Gln/Arg and Thr/Val, respectively.

[0063] The NSL shown in SEQ ID No. 4 is a lectin that can be extracted from Naematoloma sublateritium. The Xaa at the 10th and 17th positions in SEQ ID No. 4 may be any amino acid residue, but is preferably Cys. The Xaa at the 13th, 14th and 16th positions are Asp/Thr, Ser/Ala and Gln/Lys, respectively.

[0064] The NSL shown in SEQ ID No. 5 is also a lectin that can be extracted from Naematoloma sublateritium. The Xaa at the 10th and 18th positions in SEQ ID No. 5 may be any amino acid residue, but is preferably Cys. The Xaa at positions the 14th, 15th and 17th are Asp/Thr, Ser/Ala and Gln/Lys, respectively. Note that SEQ ID No. 5 can also be said to be a variant in which one Asn is inserted into the peptide of SEQ ID No. 4.

[0065] The fucose 1.fwdarw.6 specific lectin may be a protein or peptide which includes (a) a protein or peptide consisting of an amino acid sequence represented by any of SEQ ID Nos. 1 to 5; or (b) a protein or peptide consisting of an amino acid sequence represented by any of SEQ ID Nos. 1 to 5 in which one or more amino acids are deleted, inserted or substituted, and which is functionally equivalent to a protein having the amino acid sequence represented by any of SEQ ID Nos. 1 to 5.

[0066] The phrase one or more amino acids are deleted, inserted or substituted does not include amino acids added for other functions such as His tag, Flag tag, and GST tag, as well as spacers and the like for adding the amino acids. In addition, in a case of a protein or peptide in which a plurality of the sequences of (a) and (b) are linked, whether the phrase one or more amino acids are deleted, inserted or substituted is applied or not is determined with respect to a sequence corresponding to the (a) and (b), and spacers and the like for linking the amino acids are not related to the phrase one or more amino acids are deleted, inserted or substituted.

[0067] Herein, the functionally equivalent means that it has affinity expressed by a binding constant (at 25 C.) of 1.010.sup.4 M.sup.1 or more, preferably 5.010.sup.4 M.sup.1 or more, more preferably 1.010.sup.5 M.sup.1 or more, still more preferably 2.010.sup.5 M.sup.1 or more for the 1.fwdarw.6 fucose sugar chain No. 405. An example of a protein or peptide variant consisting of the amino acid sequence shown in SEQ ID No. 4 is a protein or peptide consisting of the amino acid sequence shown in SEQ ID No. 5.

[0068] The fucose 1.fwdarw.6 specific lectin may also be a peptide or protein obtained by not only extraction from the natural products but also chemical synthesis based on amino acid sequences of a naturally occurring lectin. Furthermore, the chemically synthesized peptide and protein may be a peptide in which one or several amino acids in amino acid sequences of a naturally occurring lectin are substituted with lysine and/or arginine and which has a carbohydrate-binding activity. A method for obtaining the peptides of the fucose 1.fwdarw.6 specific lectin is described in detail in Patent Document 3 filed by some of the present applicants. An amino acid sequence (SEQ ID No. 6) of the PhoSL peptide is shown in Table 1. The PhoSL peptide represented by SEQ ID No. 6 has an amino acid sequence in which the 1st Ala, the 20th Tyr, and the 39th Thr are substituted with Lys, and the 40th Gly is deleted in the specific example of PhoSL represented by SEQ ID No. 1 (APVPVTKLVC DGDTYKCTAY LDFGDGRWVA QWDTNVFHTG).

TABLE-US-00001 TABLE1 SEQ ID Lectin Aminoacidsequence No. PhoSL Ala-Pro-Val-Pro-Val-Thr-Lys- 1 Leu-Val-Xaa-Asp-Gly-Asp-Thr- Tyr-Lys-Xaa-Thr-Ala-Xaa-Leu- Asp-Xaa-Gly-Asp-Gly-Xaa-Trp- Val-Ala-Gln-Trp-Xaa-Thr-Xaa- Val-Phe-His-Xaa-Gly SRL Ala-Pro-Val-Xaa-Val-Thr-Xaa- 2 Leu-Xaa-Xaa-Asp-Gly-Xaa-Ser- Tyr-Lys-Xaa-Thr-Ala-Xaa-Leu- Asp-Tyr-Gly-Asp-Gly-Xaa-Trp- Xaa-Ala-Gln-Trp-Xaa-Xaa-Asn- Val-Phe-His-Xaa LSL Xaa-Pro-Val-Xaa-Val-Lys-Xaa- 3 Xaa-Xaa-Xaa-Asp-Gly-Xaa-Thr- Tyr-Xaa-Xaa-Thr-Ala-Xaa-Leu- Xaa-Tyr-Gly-Xaa-Gly-Xaa-Trp- Val-Ala-Xaa-Trp-Ser-Xaa-Ala- Val-Phe-His-Gln-Ser NSL Ala-Pro-Val-Pro-Val-Thr-Lys- 4 Leu-Val-Xaa-Asp-Gly-Xaa-Xaa- Phe-Xaa-Xaa-Thr-Ala-Asn-Leu- Asp-Phe-Gly-Asp-Gly-Asn-Trp- Val-Ala-Gln-Trp-Ser-Thr-Asn- Val-Phe-His-Asn NSL Ala-Pro-Val-Pro-Val-Thr-Lys- 5 Leu-Val-Xaa-Asp-Asp-Gly-Xaa- Xaa-Phe-Xaa-Xaa-Thr-Ala-Asn- Leu-Asp-Phe-Gly-Asp-Gly-Asn- Trp-Val-Ala-Gln-Trp-Ser-Thr- Asn-Val-Phe-His-Asn PhoSL Lys-Pro-Val-Pro-Val-Thr-Lys- 6 Pep- Leu-Val-Cys-Asp-Gly-Asp-Thr- tide Tyr-Lys-Cys-Thr-Ala-Lys-Leu- Asp-Phe-Gly-Asp-Gly-Arg-Trp- Val-Ala-Gln-Trp-Asp-Thr-Asn- Val-Phe-His-Lys

[0069] The fucose 1.fwdarw.6 specific lectin may be not only an extract from the natural product but also a recombinant artificially developed in a known host different from natural origins by using nucleic acids encoding an amino acid sequence of a naturally occurring lectin.

[0070] Binding constants (at 25 C.) of PhoSL, SRL, NSL and LSL belonging to the fucose 1.fwdarw.6 specific lectin for various sugar chains are shown in Tables 2 to 5. For comparison, binding constants (at 25 C.) of Aleuria aurantia lectin (AAL), Aspergillus oryzae lectin (AOL), Lens culinaris lectin (LCL), and Pisum sativum lectin (PSL) for various sugar chains (FIGS. 1 and 2) are also shown in Tables 2 to 5, these lectins having affinity for the fucose 1.fwdarw.6 but being not specific to the fucose 1.fwdarw.6.

TABLE-US-00002 TABLE 2 (1) Binding constant for sugar chains having 1.fwdarw.6 fucose (at 25 C.) Sugar chain PhoSL SRL NSL LSL AAL AOL LCL PSL No. (M.sup.1) (M.sup.1) (M.sup.1) (M.sup.1) (M.sup.1) (M.sup.1) (M.sup.1) (M.sup.1) 015 5.0 10.sup.5 4.9 10.sup.4 3.6 10.sup.4 1.9 10.sup.5 9.1 10.sup.4 1.3 10.sup.5 5.6 10.sup.4 1.2 10.sup.5 201 4.6 10.sup.5 6.4 10.sup.4 3.8 10.sup.4 2.3 10.sup.5 5.1 10.sup.4 1.2 10.sup.5 5.0 10.sup.5 1.0 10.sup.5 202 4.0 10.sup.5 6.1 10.sup.4 3.9 10.sup.4 2.3 10.sup.5 5.4 10.sup.4 1.2 10.sup.5 1.1 10.sup.5 4.4 10.sup.4 203 3.3 10.sup.5 5.2 10.sup.4 3.0 10.sup.4 2.0 10.sup.5 6.6 10.sup.4 1.3 10.sup.5 7.3 10.sup.5 2.5 10.sup.4 401 3.5 10.sup.5 5.7 10.sup.4 5.1 10.sup.4 3.5 10.sup.5 5.5 10.sup.4 1.4 10.sup.5 4.2 10.sup.4 9.2 10.sup.4 402 2.0 10.sup.5 4.4 10.sup.4 3.6 10.sup.4 2.0 10.sup.5 1.1 10.sup.4 1.6 10.sup.5 5.9 10.sup.4 4.8 10.sup.4 403 3.4 10.sup.5 5.6 10.sup.4 5.2 10.sup.4 3.4 10.sup.5 5.2 10.sup.4 1.3 10.sup.5 9.9 10.sup.4 3.8 10.sup.4 404 3.9 10.sup.5 5.8 10.sup.4 4.3 10.sup.4 3.9 10.sup.5 6.5 10.sup.4 1.5 10.sup.5 5.8 10.sup.4 4.5 10.sup.4 405 3.2 10.sup.5 5.4 10.sup.4 5.0 10.sup.4 3.2 10.sup.5 5.6 10.sup.4 1.3 10.sup.5 4.7 10.sup.4 3.6 10.sup.4 406 2.2 10.sup.5 3.8 10.sup.4 2.3 10.sup.4 2.2 10.sup.5 4.7 10.sup.4 1.1 10.sup.5 1.8 10.sup.4 1.3 10.sup.4 407 2.8 10.sup.5 1.0 10.sup.4 N.T 2.8 10.sup.5 4.5 10.sup.4 1.1 10.sup.5 1.0 10.sup.3 <1.0 10.sup.3 410 2.2 10.sup.5 3.9 10.sup.4 2.2 10.sup.5 2.2 10.sup.5 3.8 10.sup.4 1.0 10.sup.5 <1.0 10.sup.3 <1.0 10.sup.3 413 2.8 10.sup.5 N.T 2.8 10.sup.5 2.8 10.sup.5 3.2 10.sup.4 7.9 10.sup.4 <1.0 10.sup.3 <1.0 10.sup.3 418 2.2 10.sup.5 4.3 10.sup.3 2.2 10.sup.5 2.2 10.sup.5 <1.0 10.sup.3 6.3 10.sup.4 <1.0 10.sup.3 <1.0 10.sup.3 601 2.4 10.sup.5 1.0 10.sup.4 2.4 10.sup.5 2.4 10.sup.5 6.1 10.sup.4 1.4 10.sup.5 3.0 10.sup.4 3.1 10.sup.4 602 1.2 10.sup.5 3.2 10.sup.4 1.2 10.sup.5 1.2 10.sup.5 5.2 10.sup.4 1.4 10.sup.5 2.4 10.sup.4 2.9 10.sup.4 N.T: not analyzed

TABLE-US-00003 TABLE 3 (2) Binding constant for sugar chains having fucose other than 1.fwdarw.6 fucose (at 25 C.) Sugar chain PhoSL SRL NSL LSL AAL AOL LCL PSL No. (M.sup.1) (M.sup.1) (M.sup.1) (M.sup.1) (M.sup.1) (M.sup.1) (M.sup.1) (M.sup.1) 419 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 420 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 718 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 6.5 10.sup.4 1.7 10.sup.s <1.0 10.sup.3 <1.0 10.sup.3 719 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 720 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 721 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 722 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 5.6 10.sup.4 <1.0 10.sup.3 <1.0 10.sup.3 723 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 3.5 10.sup.4 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 726 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 727 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 1.3 10.sup.5 7.6 10.sup.4 <1.0 10.sup.3 <1.0 10.sup.3 728 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 1.2 10.sup.4 <1.0 10.sup.3 <1.0 10.sup.3 729 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 730 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 731 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 739 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 909 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 3.1 10.sup.5 4.4 10.sup.4 <1.0 10.sup.3 <1.0 10.sup.3 910 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 9.0 10.sup.4 5.6 10.sup.4 <1.0 10.sup.3 <1.0 10.sup.3 931 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 9.9 10.sup.4 <1.0 10.sup.3 <1.0 10.sup.3 932 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 933 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 2.1 10.sup.5 3.1 10.sup.4 <1.0 10.sup.3 <1.0 10.sup.3

TABLE-US-00004 TABLE 4 (3) Binding constant for sugar chains having no fucose (at 25 C.) Sugar chain PhoSL SRL NSL LSL AAL AOL LCL PSL No. (M.sup.1) (M.sup.1) (M.sup.1) (M.sup.1) (M.sup.1) (M.sup.1) (M.sup.1) (M.sup.1) 001 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 002 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 003 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 7.2 10.sup.3 7.8 10.sup.3 004 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 005 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 7.3 10.sup.3 <1.0 10.sup.3 7.4 10.sup.3 006 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 1.4 10.sup.4 7.1 10.sup.3 007 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 7.8 10.sup.3 008 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 6.9 10.sup.4 1.8 10.sup.4 1.0 10.sup.4 009 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 1.8 10.sup.4 7.9 10.sup.3 010 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 1.3 10.sup.4 <1.0 10.sup.3 011 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 1.4 10.sup.4 <1.0 10.sup.3 012 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 2.5 10.sup.4 1.7 10.sup.4 013 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 1.7 10.sup.4 <1.0 10.sup.3 014 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 1.7 10.sup.4 8.6 10.sup.3 101 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 103 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 104 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 105 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 107 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 108 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 301 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 1.8 10.sup.4 <1.0 10.sup.3 <1.0 10.sup.3 304 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 2.0 10.sup.4 <1.0 10.sup.3 <1.0 10.sup.3 305 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 307 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 2.4 10.sup.3 308 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 2.0 10.sup.4 <1.0 10.sup.3 <1.0 10.sup.3 313 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 314 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 323 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 501 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 502 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 503 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 504 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3

TABLE-US-00005 TABLE 5 (4) Binding constant for sugar chains having no fucose (at 25 C.) Sugar chain PhoSL SRL NSL LSL AAL AOL LCL PSL No. (M.sup.1) (M.sup.1) (M.sup.1) (M.sup.1) (M.sup.1) (M.sup.1) (M.sup.1) (M.sup.1) 701 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 702 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 703 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 704 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 705 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 706 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 707 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 708 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 709 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 710 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 711 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 712 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 713 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 715 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 716 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 717 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 724 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 725 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 728 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 732 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 733 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 734 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 735 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 736 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 737 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 738 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 901 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 902 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 903 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 905 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 906 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 907 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3 <1.0 10.sup.3

[0071] The AAL and AOL bind to the fucose 1.fwdarw.6 sugar chains (sugar chains No. 015, 201 to 203, and 401 to 418), as well as to the glycolipid-type sugar chain excluding the fucose 1.fwdarw.6 (sugar chains No. 718, 722, 723, 727, 909, 910 and 933). The LCL and PSL bind to the fucose 1.fwdarw.6 sugar chain, as well as to sugar chain excluding the 1.fwdarw.6 fucose (sugar chains No. 003, and 005 to 014).

[0072] The fucose 1.fwdarw.6 specific lectin such as PhoSL firmly binds to the fucose 1.fwdarw.6 sugar chain and does not bind to the sugar chain excluding the 1.fwdarw.6 fucose at all. Moreover, its coupling constant (at 25 C.) is larger than that of the conventional fucose 1.fwdarw.6 affinitive lectin (coupling constant is 1.010.sup.4 M.sup.1 or more). Furthermore, the binding constant of the fucose 1.fwdarw.6 specific lectin is not decreased even if a sialic acid is added to the fucose 1.fwdarw.6 sugar chain (sugar chains No. 601 and 602). In addition, the fucose 1.fwdarw.6 specific lectin also strongly binds to the triple-strand (sugar chains No. 407 to 413) and the four-strand (sugar chains No. 418) of the fucose u1.fwdarw.6 sugar chain.

[0073] The method according to the present invention specifically includes the following steps:

[0074] (A) reacting a fucosylated PSA contained in a specimen consisting of serum sampled from a subject with a fucose 1.fwdarw.6 specific lectin to obtain a fucosylated PSA-fucose 1.fwdarw.6 specific lectin complex; and

[0075] (B) detecting the complex by an appropriate means.

[0076] As described in the following Reference Example 1, when the fucose 1.fwdarw.6 specific lectin is made to actuate on the fucosylated PSA in serum sampled from a subject, there are problems such as high noise due to serum impurities. The method according to the present invention improves the sensitivity of the fucosylated PSA-fucose 1.fwdarw.6 specific lectin complex by adjusting a pH to within a specific alkaline range in at least one of the steps (A) and (B).

[0077] Specifically, the pH is adjusted to within a specific alkaline range, for at least one solution selected from: a solvent for the lectin reaction step of reacting the fucosylated PSA with the fucose 1.fwdarw.6 specific lectin to obtain the fucosylated PSA-fucose 1.fwdarw.6 specific lectin complex; a lavage fluid for a washing step of washing the complex; a solvent for a probe reaction step for reacting the complex with a secondary probe or subsequent probes; and a lavage fluid for washing the complex after the probe reaction. Preferably, the pH in the lectin reaction step is adjusted to within a specific alkaline range.

[0078] The lower limit of the pH in the alkaline region is higher than 8.5, preferably 8.6 or higher, more preferably 8.8 or higher, further preferably 9.0 or higher. The upper limit of the pH in the alkaline region is lower than 11.0, preferably 10.5 or lower. If the pH is 8.5 or lower or 11.0 or higher, a signal/noise ratio of the complex cannot be improved in some cases.

[0079] The pH is adjusted by adding an alkaline reagent, preferably an alkaline solution, more preferably an alkaline buffer. Examples of the alkaline buffer include a glycine-sodium hydroxide (NaOH) buffer; a carbonate-bicarbonate buffer; a Good's buffer such as TAPS, Tricine, Bicine, CHES, CAPSO, and CAPS; a sodium borate buffer; an ammonium chloride buffer; a wide range buffer such as Britton-Robinson buffer; and the like. At least one selected from the glycine-NaOH buffer, the carbonate-bicarbonate buffer, and the TAPS buffer is preferable, and at least one selected from the glycine-NaOH buffer and the TAPS buffer is more preferable. Preparation of these buffers is based on conventionally known methods.

[0080] It is preferable to add albumin, e.g., bovine serum albumin (BSA) to the reaction between the fucosylated PSA and the fucose 1.fwdarw.6 specific lectin in the step (A). An albumin concentration in the reaction solution may be normally 0.01 to 10% and is preferably 0.1 to 3%, particularly preferably 0.5 to 1%.

[0081] Preferably, a labeling means is previously incorporated in the lectin for detecting the complex in the step (B). The labeling means is not particularly limited but a known labeling method can be applied, and examples of the method include labeling with a radioisotope, binding of a labeling compound, and the like. Examples of the radioisotope include .sup.14C, .sup.3H and .sup.32P. Also, an anti-lectin antibody capable of binding to the lectin may be used for detection.

[0082] Example of the labeling compound include an enzyme label (horseradish peroxidase, alkaline phosphatase, etc.), a biotin label, a digoxigenin label, and a fluorescent label (fluorescein isothiocyanate, CyDye (registered trademark), ethyl 4-aminobenzoate (ABEE), aminopyridine, allophycocyanin, phycoerythrin, Alexa Fluor (registered trademark), etc.). These labeling compounds can be bound to the lectin by a ordinary method. In particular, the biotin label is preferred for its high sensitivity.

[0083] In the step (B), the means for detecting the complex is not particularly limited. As the detection means, ELISA (direct adsorption method, sandwich method and competition method), lectin affinity chromatography, lectin staining, lectin chip, flow cytometry (FACS) method, coagulation method, surface plasmon resonance method (e.g., Biacore (registered trademark) system), electrophoresis, beads, and the like can be used. Several representative detection methods are outlined below.

[0084] In the direct adsorption ELISA method, a specimen (serum) is added to a plate and immobilized. Then, the biotin-labeled lectin is added to allow the PSAto react with the lectin. As a secondary labeling compound, an HRP (horseradish peroxidase)-labeled streptavidin solution is added to allow the biotin to react with the streptavidin. Subsequently, a chromogenic substrate for HRP is added to develop color, and the coloring intensity is measured with an absorptiometer. The sugar chain can also be quantified by previously graphing a calibration curve with a standard sample containing a known concentration of the sugar chain.

[0085] In the sandwich ELISA method, at least one selected from lectins and antibodies (e.g., anti-PSA antibody) or fragments thereof having affinity for the fucosylated PSA are added to a plate and immobilized. The antibody may be either a monoclonal antibody or a polyclonal antibody. Then, a specimen (serum) is exposed to the plate. Then, the biotin-labeled fucose 1.fwdarw.6 specific lectin is added to allow the fucosylated PSA in the serum to react with the fucose 1.fwdarw.6 specific lectin. This reaction produces the complex of the fucosylated PSA and the fucose 1.fwdarw.6 specific lectin. As a secondary labeling compound, an HRP-labeled streptavidin solution is added to allow the biotin to react with the streptavidin. Subsequently, a chromogenic substrate for HRP is added to develop color, and the coloring intensity is measured with an absorptiometer. The 1.fwdarw.6 fucose sugar chain can also be quantified by previously graphing a calibration curve with a standard sample of a known concentration.

[0086] The lectin affinity chromatography is an affinity chromatography utilizing the property that a lectin immobilized on a carrier specifically binds to a sugar chain. High throughput can be expected by combining with HPLC.

[0087] As a carrier for immobilizing the lectin, gel materials such as agarose, dextran, cellulose, starch and polyacrylamide are commonly used. For these materials, commercial products can be used without special limitation, and exemplified by Sepharose 4B and Sepharose 6B (both from GE Healthcare Biosciences Corp.). Examples of a column used for the lectin chromatography also include a column prepared by immobilizing the lectin on a microplate or a nanowell.

[0088] A concentration of a lectin to be immobilized is generally 0.001 to 100 mg/mL, preferably 0.01 to 20 mg/mL. When the carrier is an agarose gel, it is activated with CNBr or the like and then coupled with the lectin. The lectin may be immobilized on a gel into which the activated spacer has been introduced. Furthermore, the lectin may be immobilized on a gel into which a formyl group has been introduced and then reduced with NaCNBH.sub.3. Alternatively, a commercial activated gel such as NHS-Sepharose (from GE Healthcare Biosciences Corp.) may be used.

[0089] The specimen (serum) is put in a column, to which subsequently a buffer solution is shed for the purpose of washing. Alternatively, the specimen in the buffer solution is put in the column. The buffer solution can be exemplified by a phosphate buffer solution, a tris buffer solution, a glycine buffer solution and the like, and it has a molar concentration of generally 5 to 500 mM, preferably 10 to 500 mM, and a pH of generally 4.0 to 10.0, preferably 6.0 to 9.0. In addition, it is a buffer solution in which a content of NaCl is generally 0 to 0.5 M, preferably 0.1 to 0.2 M, and a content of CaCl.sub.2, MgCl.sub.2 or MnCl.sub.2 is generally 0 to 10 mM, preferably 0 to 5 mM.

[0090] After washing the affinity column, the sugar chain is eluted in a neutral non-modified buffer solution capable of effectively eluting the sugar chain using a desorbent such as sodium chloride and hapten sugar. This buffer solution may be the same as described above. The concentration of the desorbent is preferably 1 to 500 mM, particularly preferably 10 to 200 mM.

[0091] In step (B), a signal (reaction value) from the complex of the fucosylated PSA in the serum and the fucose 1.fwdarw.6 specific lectin is compared with a signal (reference value) obtained in a person having a Gleason score of 6 or less, preferably 6, to evaluate with higher accuracy the presence or absence of high-risk prostate cancer development and, if cancer has been developed, its malignancy. That means, when a signal (reaction value) of a specimen is higher than a signal (reference value) obtained from a person with a Gleason score of 6 or lower, it is suggested that a subject of the specimen has a high-risk prostate cancer.

[0092] The level of the signal (reaction value) from the complex of the fucosylated PSA in the serum and the fucose 1.fwdarw.6 specific lectin depends on the reaction condition of the lectin, the PSA concentration of the blood fucosylated PSA, and the type of the lectin. A calibration curve expressing the relationship between the fucosylated PSA concentration and the signal value is graphed using a fucosylated PSA reference standard (known concentration) so as to quantify the signal. For each lectin, a reaction value corresponding to the fucosylated PSA concentration of 10 ng/mL is taken as 10 U/mL.

[0093] The present invention also provides a diagnostic agent for detecting prostate cancer containing: a fucose 1.fwdarw.6 specific lectin; and an alkaline reagent for adjusting a pH to within a specific alkaline region in at least one step selected from a group of steps including a step for reacting a fucosylated PSA contained in a specimen consisting of serum sampled from a subject with a fucose 1.fwdarw.6 specific lectin, and subsequent processing steps. Explanation of the fucose 1.fwdarw.6 specific lectin and the alkaline reagent is as described above.

[0094] The diagnostic agent may appropriately include agents generally used for detection, such as various labeling compounds, a buffer, a plate, beads and a reaction-stopping liquid. The diagnostic agent preferably includes a reagent for extracting a fucosylated PSA contained in a specimen consisting of serum (e.g., an anti-PSA antibody, or a fragment or analogue thereof).

EXAMPLE

[0095] Hereinafter, the present invention will be described in more detail with reference to Examples of the present invention. However, the present invention is not limited to the following Examples.

1. Preparation of Sample

[0096] Reagents used for the detection method of the present invention were prepared by the following procedure.

(1) Anti-PSA Antibody for Solid-Phasing

[0097] An anti-PSA antibody was purchased from HyTest, Ltd. and used as an anti-PSA antibody for solid phasing after removing sugar chains thereof in accordance with the method described in Non-Patent Document 5.

(2) Fucosylated PSA Reference Standard

[0098] A fucosylated PSA was purified from a PSA (reference standard, from BBI Solutions) using an LCA (Lens culinaris lectin) column (from J-OIL MILLS, Inc.) to obtain a fucosylated PSA reference standard.

(3) Biotin-Labeled Lectin

[0099] As the fucose 1.fwdarw.6 specific lectin used in the method according to the present invention, Pholiota squarrosa lectin (PhoSL), Stropharia rugosoannulata lectin (SRL), Naematoloma sublateritium lectin (NSL), Amanita muscaria lectin (AML), and Pholiota squarrosa lectin peptide (hereinafter referred to as PhoSL peptide, SEQ ID No. 6) were prepared. This PhoSL peptide was synthesized on the basis of the description of Example 6 in Patent Document 3 (with the proviso that PTL in Patent Document 3 is replaced with PhoSL). These lectins were weighed out, to which a 0.1 M sodium bicarbonate solution was added for dissolution (concentration: 5 mg/mL). A biotinylating reagent dissolved in dimethylsulfoxide was added to the lectin solution and reacted. The reaction solution was subjected to solvent substitution with water using ultrafiltration (molecular weight cut-off: 3 K). This solution was lyophilized to obtain a biotin-labeled lectin. In addition, a biotin-labeled Aleuria aurantia lectin (AAL, from J-OIL MILLS, Inc.) was prepared as an 1.fwdarw.6 fucose affinitive lectin.

(4) Reagents and the Like for Use (4-1) Phosphate Buffered Saline (PBS)

[0100] 5.75 g of disodium hydrogenphosphate, 1.0 g of potassium dihydrogenphosphate, 1.0 g of potassium chloride, and 40.0 g of sodium chloride were dissolved in 5 L of water to obtain PBS (pH 7.4).

(4-2) 100 mM Glycine-Sodium Hydroxide Buffer (Glycine-NaOH, pH 10)

[0101] 3.76 g of glycine was dissolved in about 400 mL of water. 5N sodium hydroxide was added thereto to adjust the pH to 10. Then, the volume was adjusted to 500 mL by adding water to prepare a buffer of pH 10.

(4-3) 1% Bovine Serum Albumin (BSA)/PBS

[0102] 1 g of bovine serum albumin (BSA, from Sigma-Aldrich Co. LLC) was dissolved in 100 mL of PBS to obtain a PBS solution with a BSA concentration of 1% (hereinafter referred to as 1% BSA/PBS).

(4-4) 0.1% Bovine Serum Albumin (BSA)/PBS

[0103] 0.1 g of bovine serum albumin was dissolved in 100 mL of PBS to obtain a PBS solution with a BSA concentration of 0.1% (hereinafter referred to as 0.1% BSA/PBS).

(4-5) 0.1% BSA/10-Fold Diluted PBS+Glycine-NaOH (pH 9.6)

[0104] To 5 mL of a solution obtained by 5-fold dilution of 1% BSA/PBS, 5 mL of the glycine-NaOH was added to obtain 0.1% BSA/10-fold diluted PBS+glycine-NaOH (pH 9.6).

(5-1) Subject Sample A

[0105] Human serum specimens sampled from 9 patients diagnosed with prostate cancer and 7 healthy persons were purchased from KAC Co., Ltd. and used as subject samples A. Blood PSA levels of the respective specimens, as well as blood PSA levels and prostate cancer risk classification of the respective prostate cancer patients are shown in Table 6.

TABLE-US-00006 TABLE 6 Blood PSA value Diagnosis of Histological TNM Name Age (ng/mL) patient diagnosis GS classification Staging Healthy 1 U/A U/A Healthy Healthy 2 U/A U/A Healthy Healthy 3 U/A U/A Healthy Healthy 4 U/A U/A Healthy Healthy 5 U/A U/A Healthy Healthy 6 U/A U/A Healthy Healthy 7 U/A U/A Healthy Prostate 76 4.49 Prostate Adenocarcinoma 4 (2 + 2) T2NxM0 II cancer 1 cancer Prostate 79 19.00 Prostate Adenocarcinoma 5 (2 + 3) T2NxM0 II cancer 2 cancer Prostate 80 2.82 Prostate Adenocarcinoma 6 (3 + 3) T2NxM0 II cancer 3 cancer Prostate 66 24.11 Prostate Adenocarcinoma 6 (3 + 3) T2cN0M0 IIB cancer 4 cancer Prostate 67 16.95 Prostate Adenocarcinoma 7 (3 + 4) T2cN0M0 IIB cancer 5 cancer Prostate 80 156 Prostate Adenocarcinoma 7 (3 + 4) T2N0M1 IV cancer 6 cancer Prostate 51 3.54 Prostate Adenocarcinoma 8 (3 + 5) T2cN0M0 IIB cancer 7 cancer Prostate 64 7.82 Prostate Adenocarcinoma 8 (4 + 4) T2cN0M0 IIB cancer 8 cancer Prostate 54 477 Prostate Adenocarcinoma 8 (4 + 4) T2N0M1 IV cancer 9 cancer U/A: not available

(5-2) Subject Sample B

[0106] Informed consent was gotten from patients with prostate cancer at Osaka University Hospital, and then their collected serum was used as subject samples B. Information on the respective specimen is shown in Table 7 with classification on the basis of their Gleason scores. The Negative in Table 7 refers to a group in which the blood PSA level was high but prostate cancer was not detected in the prostate biopsy.

TABLE-US-00007 TABLE 7 Group n Age GS Negative 9 59-76 G6 9 60-78 6 G7 11 55-74 7 G8-9 6 59-76 8-9

[Example 1] Fucosylated PSA Detecting Test Using Pholiota squarrosa Lectin (PhoSL) (I)

[0107] For the purpose of detecting the serum fucosylated PSA of prostate cancer patients with high sensitivity, a test was executed in which the pH was changed during the reaction between the serum fucosylated PSA and PhoSL.

1. Sandwich ELISA

(1) Antibody Immobilization

[0108] The anti-PSA antibody from which sugar chains had been removed was diluted to 5 g/mL with PBS. 25 L of this diluted solution was added to each well of an ELISA plate and allowed to stand at 37 C. for 12 hours, and then the additive solution was discarded.

(2) Washing

[0109] 150 L of PBS containing 0.05% Tween 20 (product name: polyoxyethylene sorbitan monolaurate, from Nacalai Tesque, Inc.) was added to each well, and then the additive solution was discarded. This manipulation was repeated three times in total.

(3) Blocking

[0110] 25 L of 1% BSA/PBS was added to each well and allowed to stand at 37 C. for 1 hour, and then the additive solution was discarded.

(4) Washing

[0111] 150 L of PBS containing 0.05% Tween 20 was added to each well, and then the additive solution was discarded. This manipulation was repeated three times in total.

(5) Antigen-Antibody Reaction

[0112] To graph a calibration curve, 25 L of the fucosylated PSA reference standard diluted with 1% BSA/PBS to a concentration of 0 to 200 ng/mL was added to each well and allowed to stand at room temperature for 1 hour, and then the additive solution was discarded. In addition, to detect the fucosylated PSA in the serum of the subject by the lectin, 25 L of serum of Healthy 1 and Prostate cancer 4 2-fold diluted with PBS was added to each well and allowed to stand at room temperature for 1 hour, and then the additive solution was discarded.

(6) Washing

[0113] 150 L of PBS containing 0.05% Tween 20 was added to each well, and then the additive solution was discarded. This manipulation was repeated three times in total.

(7) Reaction of Labeled Lectin

[0114] In Reference Example 1, 25 L of a biotin-labeled PhoSL diluted to 0.1 g/mL with 0.1% BSA/PBS (pH 7.4) was added to each well and allowed to stand at 4 C. for 30 minutes, and then the additive solution was discarded. In Example 1, 25 L of a biotin-labeled PhoSL diluted to 0.1 g/mL with 0.1% BSA/10-fold diluted PBS+glycine-NaOH (pH 9.6) was added to each well and allowed to stand at 4 C. for 30 minutes, and then the additive solution was discarded.

(8) Washing

[0115] 150 L of PBS containing 0.05% Tween 20 was added to each well, and then the additive solution was discarded. This manipulation was repeated three times in total.

(9) HRP-Labeled Streptavidin Reaction

[0116] 25 L of a horseradish peroxidase (HRP)-labeled streptavidin solution (from Vector Laboratories, Inc., concentration: 0.4 g/mL, in 1% BSA/PBS) was added to each well and allowed to stand at room temperature for 30 minutes, and then the additive solution was discarded.

(10) Washing

[0117] 150 L of PBS containing 0.05% Tween 20 was added to each well, and then the additive solution was discarded. This manipulation was repeated three times in total.

(11) Coloring Reaction

[0118] 25 L of chromogenic substrate for HRP (product name: TMB, from Kirkegaard & Perry Laboratories, Inc.) was added to each well and allowed to stand at room temperature for 10 minutes.

(12) Termination of Reaction

[0119] 25 L of 1M phosphoric acid was added to terminate the reaction.

(13) Absorbance Measurement

[0120] Absorbance (Ab) at 450 nm and 630 nm was measured using a plate reader, and a measurement (Ab.sub.450-630) was obtained.

(14) Graphing of Calibration Curve

[0121] A calibration curve was graphed by plotting signals (reaction values, Ab.sub.450-630) of the fucosylated PSA reference standard with the biotin-labeled PhoSL. A Signal (Ab.sub.450-630) of the biotin-labeled PhoSL corresponding to the fucosylated PSA concentration of 10 ng/mL was defined as 10 U/mL.

(15) Calculation of Reaction Value

[0122] The signals of biotin-labeled PhoSL (Ab.sub.450-630) for the serum fucosylated PSA in the subject samples A were assigned to the calibration curve to calculate reaction values (unit: U/mL).

[0123] Table 8 shows results of measuring the PhoSL reaction values in tests (Reference Example 1 and Example 1) in which the pH in the lectin reaction was changed.

TABLE-US-00008 TABLE 8 Reference Example 1 Example 1 (7) pH in lectin reaction 7.4 9.6 PhoSL reaction value A of 108.4 U/mL 178.8 U/mL Prostate cancer 4 (GS6) PhoSL reaction value B of 35.8 U/mL 51.9 U/mL Healthy 1 (=A B) 72.6 U/mL 126.9 U/mL

[0124] Table 8 shows that in Example 1 in which the pH was adjusted to within a specific alkaline range according to the present invention in the reaction step between the serum fucosylated PSA and the PhoSL, the PhoSL reaction value A of prostate cancer increases, meanwhile the PhoSL reaction value B of healthy 1 decreases, compared to Reference Example 1. As a result of increase in the difference A between the reaction values A and B, it turned out that a prostate cancer patient (GS6) could be detected with high sensitivity in Example 1 according to the present invention.

[Example 2] Fucosylated PSA Detecting Test Using PhoSL (II)

[0125] The PhoSL reaction values for the subject samples A were measured by the same operation as in Example 1. The results are shown in Table 9. From the PhoSL reaction values, an average value and a median value were determined. Furthermore, a standard cutoff value (89.7 U/mL) was used to determine a detection rate (positive rate) of the prostate cancer patients, and a false detection rate (false positive rate) of the healthy persons. The results are shown in Table 10.

[0126] Comparative Example 1 in Table 9 indicates results of measuring the blood PSA values. A cut-off value of the serum PSA value was 4 ng/mL. As in Example 2, an average value, a median value, a positive rate and a false positive rate were determined. The results are shown in Table 10.

[0127] Comparative Example 2 in Table 9 indicates results of detecting the serum fucosylated PSA using AAL (AAL reaction values) in the subject samples A. Specifically, in Reference Example 1, AAL reaction values were measured in the same manner as in Reference Example 1 except that the biotin-labeled PhoSL was replaced with a biotin-labeled AAL. A standard cut-off value for the AAL reaction values was 894.8 U/mL. As in Example 2, an average value, a median value, a positive rate and a false positive rate were determined from the AAL reaction values. The results are shown in Table 10.

TABLE-US-00009 TABLE 9 Comparative Comparative Example 2 Example 2 Example 1 AAL PhoSL Blood PSA reaction reaction value value value GS (ng/mL) (U/mL) (U/mL) Healthy Healthy 1 N/A 1099.6 51.9 person Healthy 2 N/A 854.6 55.9 group Healthy 3 N/A 1062.7 63.9 Healthy 4 N/A 115.0 74.9 Healthy 5 N/A 32.3 55.8 Healthy 6 N/A 1193.1 39.7 Healthy 7 N/A 1028.8 59.0 Prostate Prostate 4 (2 + 2) 4.49 831.2 115.8 cancer cancer 1 patient Prostate 5 (2 + 3) 19.00 1032.3 116.7 group cancer 2 Prostate 6 (3 + 3) 2.82 1152.7 104.5 cancer 3 Prostate 6 (3 + 3) 24.11 926.5 178.8 cancer 4 Prostate 7 (3 + 4) 16.95 1025.0 115.8 cancer 5 Prostate 7 (3 + 4) 156.00 961.5 167.5 cancer 6 Prostate 8 (3 + 5) 3.54 1211.9 125.6 cancer 7 Prostate 8 (4 + 4) 7.82 1010.0 129.8 cancer 8 Prostate 8 (4 + 4) 477.00 988.1 251.4 cancer 9

TABLE-US-00010 TABLE 10 Comparative Comparative Example 2 Example 2 Example 1 AAL PhoSL Blood PSA reaction reaction value value value Healthy Average value 769.4 U/mL 57.3 U/mL person Median value 1028.8 U/mL 55.9 U/mL group False positive 57% 0% rate Prostate Average value 69.5 ng/mL 1015.5 U/mL 145.1 U/mL cancer Median value 12.4 ng/mL 1010.0 U/mL 125.6 U/mL patient Positive rate 77% 89% 100% group

[0128] As shown in Table 10, a detection rate (positive rate) of the prostate cancer patient group based on the blood PSA values was 77%. A detection rate (positive rate) of the prostate cancer patient group based on the AAL reaction values was 89%. However, also a false positive rate obtained by judging a healthy group to have prostate cancer was as high as 57%. In Example 2 according to the present invention, a positive rate of the prostate cancer patient group was 100%, and a false positive rate of the healthy group was 0%. From the above description, it was revealed that when the fucose 1.fwdarw.6 specific lectin was used under a specific condition according to the present invention, prostate cancer could be detected with high sensitivity and high specificity.

[0129] Next, the measurement results of the prostate cancer patient group in Table 9 were classified into Negative (G5 or lower), GS6, GS7, and GS8 or higher. An average value and a median value of each group are shown in Table 11.

TABLE-US-00011 TABLE 11 Comparative Comparative Example 2 Example 2 Example 1 AAL PhoSL Blood PSA reaction reaction value value value Subgroup n (ng/mL) (U/mL) (U/mL) Negative 2 11.7 931.8 116.3 (GS5 or less) 11.7 931.8 116.3 GS6 2 13.5 1039.6 141.7 13.5 1039.6 141.7 GS7 2 86.5 993.3 141.7 86.5 993.3 141.7 GS8 or more 3 162.8 1070.0 129.8 7.8 1010.0 168.9 Upper line: Mean value Lower line: Median value

[0130] As shown in Table 11, no correlation is observed between the median blood PSA value or the median AAL reaction value and the GS. In Example 2, there is a tendency of increase in the GS (malignant progression) and in the median PhoSL reaction value.

[Example 3] Fucosylated PSA Detecting Test Using PhoSL (III)

[0131] For the purpose of confirming the presence of a correlation between the median PhoSL reaction value and the GS in Example 2, the PhoSL reaction values were measured in subject samples B having an n number more than that in the subject samples A in the same operation as in Example 2. The results are shown in Table 12. For comparison, the results of measuring the blood PSA values are also shown in Table 12.

TABLE-US-00012 TABLE 12 Comparative Example 3 Example 3 Blood PSA value PhoSL reaction value Subgroup n (ng/mL) (U/mL) Negative 9 8.4 203.8 7.8 178.0 4.0 84.3 GS6 9 17.4 181.7 6.8 188.9 29.4 31.88 GS7 11 23.1 119.5 9.6 210.6 39.0 27.2 GS8-9 6 75.3 232.9 12.9 239.8 156.4 52.5 Upper line: Mean value Middle line: Median value Lower line: Standard deviation

[0132] As shown in Table 12, there is no correlation between the blood PSA values and the GS. On the other hand, in Example 3 according to the present invention, the median PhoSL reaction value tends to increase as the GS increases. From the results in Tables 11 and 12, the GS can be predicted from the reaction value by using the fucose 1.fwdarw.6 specific lectin such as PhoSL under a specific condition. Consequently, the method according to the present invention is expected to predict the GS without biopsy and to predict a high-risk prostate cancer at GS7 or higher.

[Example 4] Fucosylated PSA Detecting Test Using Stropharia rugosoannulata Lectin (SRL)

[0133] Two types of SRL reaction values were measured by the same operation as in Example 1 and Reference Example 1 except that the PhoSL in Example 1 and Reference Example 1 was changed to the SRL. The results are shown in Table 13.

TABLE-US-00013 TABLE 13 Reference Example 2 Example 4 (7) pH in lectin reaction 7.4 9.6 SRL reaction value A 146.3 U/mL 234.5 U/mL of Prostate cancer 4 SRL reaction value B 20.0 U/mL 16.8 U/mL of Healthy 1 (=A B) 126.3 U/mL 217.7 U/mL

[0134] Table 13 shows that in Example 4 in which the serum fucosylated PSA was reacted with the SRL in a specific alkaline region, the reaction values of the prostate cancer patients increase and meanwhile the reaction values of the healthy persons decrease compared to Reference Example 2. Thus, even if the SRL is used under a specific condition, the serum fucosylated PSA can be detected with high sensitivity. Therefore, the SRL is effective for detecting prostate cancer.

[Example 5] Fucosylated PSA Detecting Test Using Naematoloma sublateritium (NSL)

[0135] Two types of NSL reaction values were measured by the same operation as in Example 1 and Reference Example 1 except that the PhoSL in Example 1 and Reference Example 1 was changed to the NSL. The results are shown in Table 14.

TABLE-US-00014 TABLE 14 Reference Example 3 Example 5 (7) pH in lectin reaction 7.4 9.6 NSL reaction value A of 399.7 U/mL 282.6 U/mL Prostate cancer 4 NSL reaction value B of 177.7 U/mL 42.3 U/mL Healthy 1 (=A B) 222 U/mL 240.3 U/mL

[0136] Table 14 shows that in Example 5 in which the serum fucosylated PSA was reacted with the NSL in a specific alkaline region, a difference A between the reaction values of the prostate cancer patients and the reaction values of the healthy persons increases compared to Reference Example 3. Thus, even if the NSL is used under a specific condition, the serum fucosylated PSA can be detected with high sensitivity. Therefore, the NSL is effective for detecting prostate cancer.

[Example 6] Fucosylated PSA Detecting Test Using Amanita muscaria Lectin (AML)

[0137] Two types of AML reaction values were measured by the same operation as in Example 1 and Reference Example 1 except that the PhoSL in Example 1 and Reference Example 1 was changed to the AML. The results are shown in Table 15.

TABLE-US-00015 TABLE 15 Reference Example 4 Example 6 (7) pH in lectin reaction 7.4 9.6 AML reaction value A of 141.3 U/mL 150.9 U/mL Prostate cancer 4 AML reaction value B of 21.2 U/mL Below detection limit Healthy 1 (=A B) 120.1 U/mL 150.9 U/mL

[0138] Table 15 shows that in Example 6 in which the serum fucosylated PSA was reacted with the AML in a specific alkaline region, the reaction values of the prostate cancer patients increase and meanwhile the reaction values of the healthy persons decrease compared to Reference Example 4. Even if the AML is used under a specific condition, the serum fucosylated PSA can be detected with high sensitivity. Therefore, the AML is effective for detecting prostate cancer.

[Example 7] Fucosylated PSA Detecting Test Using PhoSL Peptide

[0139] Two types of PhoSL peptide reaction values were measured by the same operation as in Example 1 and Reference Example 1 except that the PhoSL in Example 1 and Reference Example 1 was changed to the PhoSL peptide. The results are shown in Table 16.

TABLE-US-00016 TABLE 16 Reference Example 5 Example 7 (7) pH in lectin reaction 7.4 9.6 PhoSL peptide reaction 101.5 U/mL 234.4 U/mL value A of Prostate cancer 4 PhoSL peptide reaction 1.0 U/mL Below detection limit value B of Healthy 1 (=A B) 100.5 U/mL 234.4 U/mL

[0140] Table 16 shows that in Example 7 in which the serum fucosylated PSA was reacted with the PhoSL peptide in a specific alkaline region, the reaction values of the prostate cancer patients increase and meanwhile the reaction values of the healthy persons decrease compared to Reference Example 5. Even if the PhoSL peptide is used under a specific condition, the serum fucosylated PSA can be detected with high sensitivity. Therefore, the PhoSL peptide is effective for detecting prostate cancer.

[Sequence Listing]

NP2360 SEQUENCEYNP2360PCT_ST25.txt

[0141]