Method for measuring beta-glucan, and beta-glucan-binding protein for use in the method

Abstract

Disclosed are a method for measuring βG comprising the steps of bringing a sample into contact with a βG-binding protein 1 and a βG-binding protein 2, each comprising an amino acid sequence which is identical or substantially identical to an amino acid sequence shown in any one of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20, and having the β-glucan binding activity, to form a complex of the βG-binding protein 1, βG in the sample and the βG-binding protein 2, measuring quantity of the complex, and determining βG concentration in the sample based on the quantity of the complex; a reagent and a kit for use in said method; a novel βG-binding protein; a nucleic acid molecule encoding the βG-binding protein; and a method for producing the aforementioned βG-binding protein.

1 - 21 . (canceled) 
     
     
         22 . A method for measuring β-glucan (hereinafter, abbreviated as “βG”), which comprises:
 (1) contacting a sample with protein 1 comprising an amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20, or an amino acid sequence having 95% or more of homology to the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20, and having a βG binding activity (hereinafter, abbreviated as “βG-binding protein 1”), and 
 protein 2 comprising an amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20, or an amino acid sequence having 95% or more of homology to the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20, and having a βG binding activity (hereinafter, abbreviated as “βG-binding protein 2”), to form a complex of the βG-binding protein 1, 13G in the sample and the βG-binding protein 2, with the proviso that both the βG-binding protein 1 and the βG-binding protein 2 comprising an amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10, or an amino acid sequence having 95% or more of homology to the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10, and having a βG binding activity, is excluded, 
 wherein the βG-binding protein 2 is labeled with a labeling substance; 
 (2) measuring a quantity of the labeling substance in the complex; and 
 (3) determining a βG concentration in the sample based on the quantity measured in (2). 
 
     
     
         23 . The method of measurement according to  claim 22 , wherein the βG-binding protein 1 is immobilized to an insoluble carrier. 
     
     
         24 . The method of measurement according to  claim 22 , wherein each of the βG-binding protein 1 and the βG-binding protein 2 is labeled with a labeling substance that is different from one another. 
     
     
         25 . The method of measurement according to  claim 22 , wherein the measurement comprises:
 (1) contacting a sample with the βG-binding protein 1 to form a complex-1 of βG in the sample and the βG-binding protein 1;   (2) contacting the complex-1 with the βG-binding protein 2 which is labeled with a labeling substance, to form a complex-2 of the βG-binding protein 1, βG in the sample and the βG-binding protein 2;   (3) measuring quantity of the labeling substance in the complex-2; and   (4) determining a βG concentration in the sample based on the quantity of the complex-2 obtained.   
     
     
         26 . The method of measurement according to  claim 22 , wherein the measurement comprises:
 (1) contacting a sample with βG-binding protein 1 which is immobilized to an insoluble carrier, to form a complex-1 of βG in the sample and the βG-binding protein 1 immobilized to the insoluble carrier;   (2) contacting the complex-1 with the βG-binding protein 2 which is labeled with a labeling substance, to form a complex-2 of the complex-1 and the labeled βG-binding protein 2;   (3) measuring quantity of the labeling substance in the complex-2; and   (4) determining βG concentration in the sample based on the quantity obtained.   
     
     
         27 . The method of measurement according to  claim 22 , wherein the βG-binding protein 1 and the βG-binding protein 2 are used without being immobilized to an insoluble carrier. 
     
     
         28 . The method of measurement according to  claim 27 , wherein after the complex is formed, the complex is separated from free βG-binding protein 1 and free βG-binding protein 2 bp capillary electrophoresis. 
     
     
         29 . The method of measurement according to  claim 22 , wherein the βG-binding protein 1 is not immobilized to an insoluble carrier, and is not labeled with a labeling substance. 
     
     
         30 . A method for measuring βG comprising:
 (1) contacting a sample with 
 βG-binding protein 1 comprising an amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20, or an amino acid sequence having 95% or more of homology to the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20, and having a βG binding activity (hereinafter, abbreviated as “βG-binding protein 1”), wherein the βG-binding protein 1 is immobilized to an insoluble carrier, and 
 βG-binding protein 2 comprising an amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20, or an amino acid sequence having 95% or more of homology to the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20, and having a βG binding activity (hereinafter, abbreviated as “βG-binding protein 2”), to form a complex of the βG-binding protein 1, βG in the sample and the βG-binding protein 2, with the proviso that both of the βG-binding protein 1 and the βG-binding protein 2 comprising an amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or an amino acid sequence having 95% or more of homology to the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, and having a βG binding activity, is excluded, 
 (2) measuring a quantity of the aforementioned complex by a turbidimetric method, a nephelometric method, or a latex aggregation method; and 
 (3) determining a βG concentration in the sample based on the quantity measured in (2). 
 
     
     
         31 . A method for measuring βG comprising:
 (1) contacting a sample with 
 βG-binding protein 1 comprising an amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20, or an amino acid sequence having 95% or more of homology to the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20, and having a βG binding activity, and 
 βG-binding protein 2 comprising an amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20, or an amino acid sequence having 95% or more of homology to the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20, and having a βG binding activity, to form a complex of the βG-binding protein 1, βG in the sample and the βG-binding protein 2, 
 with the proviso that both of βG-binding protein 1 and βG-binding protein 2 comprising an amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or an amino acid sequence having 95% or more of homology to the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, and having a βG binding activity, is excluded, 
 wherein the βG-binding protein 1 and βG-binding protein 2 are different each other, and 
 wherein at least one of the βG-binding protein 1 and the βG-binding protein 2 comprises an amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 10, or an amino acid sequence having 95% or more of homology to the amino acid sequence shown in SEQ ID NO: 4, or SEQ ID NO: 10, and having a βG binding activity and peroxidase activity; 
 (2) measuring quantity of the complex by measuring a peroxidase activity derived from the βG-binding protein 1 and/or βG-binding protein 2; and 
 (3) determining a βG concentration in the sample based on the quantity. 
 
     
     
         32 . A kit for measuring βG comprising the followings as constituents:
 (1) a reagent containing a protein 1 comprising an amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20, or an amino acid sequence having 95% or more of homology to the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20, and having a βG binding activity (hereinafter, abbreviated as “βG-binding protein 1”); and 
 (2) a reagent containing a protein 2 comprising an amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20, or an amino acid sequence having 95% or more of homology to the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20, and having a βG binding activity (hereinafter, abbreviated as “βG-binding protein 2”), 
 with the proviso that both of the βG-binding protein 1 and the βG-binding protein 2 comprising an amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or an amino acid sequence having 95% or more of homology to the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, and having a βG binding activity, is excluded;
 wherein the βG-binding protein 2 is labeled with a labeling substance. 
 
 
     
     
         33 . The kit according to  claim 32 , wherein the βG-binding protein 1 is immobilized to an insoluble carrier. 
     
     
         34 . The kit according to  claim 32 , wherein each of the βG-binding protein 1 and the βG-binding protein 2 is labeled with a labeling substance that is different from one another. 
     
     
         35 . The kit according to  claim 32 , wherein the βG-binding protein 1 and the βG-binding protein 2 are not immobilized to an insoluble carrier. 
     
     
         36 . The kit according to  claim 32 , wherein the βG-binding protein 1 is not immobilized to an insoluble carrier, and is not labeled with a labeling substance. 
     
     
         37 . A kit for measuring βG comprising the followings as constituents:
 (1) a reagent containing a protein 1 comprising an amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20, or an amino acid sequence having 95% or more of homology to the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20, and having a βG binding activity, wherein the βG-binding protein 1 is immobilized to an insoluble carrier, and 
 (2) a reagent containing a protein 2 comprising an amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20, or an amino acid sequence having 95% or more of homology to the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20, and having a βG binding activity, 
 with the proviso that both of the βG-binding protein 1 and the βG-binding protein 2 comprising an amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or an amino acid sequence having 95% or more of homology to the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, and having a βG binding activity, is excluded. 
 
     
     
         38 . A kit for measuring βG comprising the followings as constituents:
 (1) a reagent contacting a protein 1 comprising an amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20, or an amino acid sequence having 95% or more of homology to the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20, and having a βG binding activity, and 
 (2) a reagent containing a protein 2 comprising an amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20, or an amino acid sequence having 95% or more of homology to the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20, and having a βG binding activity, 
 with the proviso that both of the βG-binding protein 1 and the βG-binding protein 2 comprising an amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or an amino acid sequence having 95% or more of homology to the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, and having a βG binding activity, is excluded;
 wherein the βG-binding protein 1 and βG-binding protein 2 are different each other, and 
 wherein at least one of the βG-binding protein 1 and the βG-binding protein 2 comprises an amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 10, or an amino acid sequence having 95% or more of homology to the amino acid sequence shown in SEQ ID NO: 4, or SEQ ID NO: 10, and having a βG binding activity and peroxidase activity. 
 
 
     
     
         39 . An isolated protein comprising an amino acid sequence identical or substantially identical to an amino acid sequence shown in any one of SEQ ID NO: 14, or SEQ ID NO: 20, or an amino acid sequence having 95% or more of homology to the amino acid sequence shown in SEQ ID NO:14, or SEQ ID NO: 20, and having a βG binding activity. 
     
     
         40 . A nucleic acid molecule comprising a nucleotide sequence identical or substantially identical to a nucleotide sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 13, or SEQ ID NO: 19. 
     
     
         41 . A nucleic acid molecule encoding a protein comprising an amino acid sequence identical or substantially identical to an amino acid sequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, or SEQ ID NO: 20, and having a βG binding activity. 
     
     
         42 . The nucleic acid molecule according to  claim 40 , wherein the nucleic acid molecule is DNA. 
     
     
         43 . A recombinant construct incorporating therein the nucleic acid molecule according to  claim 40 . 
     
     
         44 . A transformant or a transductant, which is transformed or transduced by the recombinant construct according to  claim 43 . 
     
     
         45 . A process for producing a protein comprising an amino acid sequence identical or substantially identical to an amino acid sequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, or SEQ ID NO: 20, and having a βG binding activity, characterized in that the transformant or the transductant according to  claim 41  is cultured and the protein is separated from the resulting culture. 
     
     
         46 . The nucleic acid molecule according to  claim 41  wherein the nucleic acid molecule is DNA. 
     
     
         47 . A recombinant construct incorporating therein the nucleic acid molecule according to  claim 41 .