Monoclonal antibody recognizing phosphatidylinositol-3,4-diphosphate

Abstract

A novel monoclonal antibody that specifically recognizes phosphatidylinositol-3,4-biphosphate (PI-3,4-P2) but does not cross-react with structurally similar phospholipid antigens is advantageous for PI-3,4-P2-specific immunoassay. The gene in the variable regions of the monoclonal antibody has been identified, which enables producing recombinant antibodies.

1 . An antibody specifically recognizing phosphatidylinositol-3,4-biphosphate.  
     
     
         2 . The antibody of  claim 1 , wherein the antibody is a monoclonal antibody.  
     
     
         3 . The antibody of  claim 2 , which recognizes an antigenic determinant comprising an inositol group and a glycerol backbone in phosphatidylinositol-3,4-biphosphate.  
     
     
         4 . The antibody of claims  1 , which is substantially non-cross-reactive, with at least one compound selected from the group consisting of phosphatidylinositol-4,15-bisphospate, phosphatidylinositol-3,4,5-triphosphate phosphatidylinositol-1,4,5-triphosphate, and phisphatidylinositol-1,3,4,5-tetraphosphate.  
     
     
         5 . A hybridoma producing the antibody of  claim 2 .  
     
     
         6 . The hybridoma of  claim 5 , which has the properties of the deposit identified by the accession No. FERM-BP-6849.  
     
     
         7 . A method of producing the antibody of  claim 2 , the method comprising culturing the hybridoma of  claim 5 .  
     
     
         8 . A variable region of immunoglobulin heavy chain specifically binding to phosphatidylinositol-3,4-biphosphate, comprising an amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence of SEQ ID NO: 2 in which one or more amino acid residues are substituted, deleted or added.  
     
     
         9 . A variable region of immunoglobulin light chain specifically binding to phosphatidylinositol-3,4-biphosphate, comprising an amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence of SEQ ID NO: 4 in which one or more amino acid residues are substituted, deleted or added.  
     
     
         10 . CDR1 in immunoglobulin heavy chains specifically binding to phosphatidylinositol-3,4-biphosphate, comprising an amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence of SEQ ID NO: 5 in which one or more amino acid residues are substituted, deleted or added.  
     
     
         11 . CDR2 in immunoglobulin heavy chains specifically binding to phosphatidylinositol-3,4-biphosphate, comprising an amino acid sequence set forth in SEQ ID. NO: 6 or an amino acid sequence of SEQ ID NO: 6 in which one or more amino acid residues are substituted, deleted or added.  
     
     
         12 . CDR3 in immunoglobulin heavy chains specifically binding to phosphatidylinositol-3,4-biphosphate, comprising an amino acid sequence set forth in SEQ ID NO: 7 or an amino acid sequence of SEQ ID NO: 7 in which one or more amino acid residues are substituted, deleted or added.  
     
     
         13 . CDR1 in immunoglobulin light chains specifically binding to phosphatidylinositol-3,4-biphosphate, comprising an amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence of SEQ ID NO: 8 in which one or more amino acid residues are substituted, deleted or added.  
     
     
         14 . CDR2 in immunoglobulin light chains specifically binding to phosphatidylinositol-3,4-biphosphate comprising an amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence of SEQ ID NO: 9 in which one or more amino acid residues are substituted, deleted or added.  
     
     
         15 . CDR3 immunoglobulin light chains specifically binding to phosphatidylinositol-3,4-biphosphate, comprising an amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence of SEQ ID NO: 10 in which one or more amino acid residues are substituted, deleted or added.  
     
     
         16 . An immunogen composition for use in producing an antibody specifically recognizing phosphatidylinositol-3,4-biphosphate, comprising a mixture of a dead  Salmonella  cell as an adjuvant and phosphatidylinositol-3,4-biphosphate.  
     
     
         17 . A method for producing an antibody specifically recognizing phosphatidylinositol-3,4-biphosphate, the method comprising using an immunogen composition of  claim 16  for immunization.  
     
     
         18 . An immunoassay method which comprises the steps of reacting the antibody specifically recognizing phosphatidylinositol-3,4-biphosphate or a variable region thereof with phosphatidylinositol-3,4-biphosphate present in a sample, and detecting the binding based on an immunological reaction between the antibody or a variable region thereof and the biphosphate.  
     
     
         19 . An immunoassay method of  claim 18 , which comprises observing the degree to which the immunological reaction between the antibody or a variable region thereof and an antigenic determinant recognized thereby is inhibited by phosphatidylinositol-3,4-biphosphate present in a sample.  
     
     
         20 . A kit for immunoassay for phosphatidylinositol-3,4-biphosphate comprising the antibody of specifically recognizing phosphatidylinositol-3,4-biphosphate or a variable region thereof.