Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-03-03
2004-03-23
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007210, C435S007230, C435S007920, C435S070210, C435S452000, C435S326000, C435S343000, C435S975000, C436S519000, C436S548000, C436S071000, C530S387300, C530S388200, C530S388700, C530S389600
Reexamination Certificate
active
06709833
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a monoclonal antibody to phosphatidylinositol-3,4-diphosphate and a method for immunoassay using the monoclonal antibody.
BACKGROUND OF THE INVENTION
Formerly, phospholipids in living organisms was merely appreciated as constituents of cell membranes. Recently, however, inositol phospholipid, a member of phospholipids, has been found to play an important role in the intracellular signal transduction system. In particular, metabolism of phosphatidylinositol (PI), a phospholipid present in biomembranes, has been extensively investigated because abnormality of this system is known to induce aberrant cell proliferation and causes cancers.
It was commonly believed that inositol phospholipids are synthesized as follows: the 4-position of PI is phosphorylated by the action of phosphatidylinositol-4-kinase (PI4K) to generate phosphatidylinositol-4-monophosphate, the 5-position thereof is then phosphorylated by the action of phosphatidylinositol-4-monophosphate-5-kinase to generate phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2), which is degraded into inositol triphosphate (IP3) and diacylglycerol (DG) by extracellular stimulation. However, Cantley et al. (Rameh, L. E. and Cantley, L. C., J. Biol. Chem. Vol. 274, 8347-8350, 1999) discovered phosphatidylinositol-3-kinase (PI3K) that mediates phosphorylation at the 3-position of the inositol ring. They also demonstrated the products of this enzyme reaction, phosphatidylinositol-3-monophosphate (PI-3-P), phosphatidylinositol-3,4-bisphosphate, and phosphatidylinositol-3,4,5-triphosphate (PI-3,4,5-P3). Phosphatidylinositol-3,4-bisphosphate is also generated by dephosphorylation of the 5-position of PI-3,4,5-P3. Herein, phosphatidylinositol-3,4-bisphosphate is abbreviated as PI-3,4-P2 in principle. The structure of PI-3,4-P2 is schematically shown below.
PI3K is known to be involved in the signal transduction system of insulin. It is becoming clear that PI-3,4,5-P3 and PI-3,4-P2, which are produced by the action of PI3K, an enzyme activated by a stimulus such as insulin, also activate kinases such as phosphoinositidin kinase-1 (PKD-1) and Akt/PKB to generate a survival signal that suppresses the cell death (apoptosis) (Coffer, P. J. et al., Biochem. J., Vol. 335, 1-13, 1998). This means that PI-3,4-P2 suppresses apoptosis by activating Akt/PKB and thus takes part in survival of cells.
Based on this knowledge, specific detection of these phospholipids in the cells and clarification of the dynamics thereof have been demanded to shed light on not only mechanisms of intracellular signal transduction and apoptosis but also pathogenesis of cancers and other diseases. However, no method of detecting and measuring PI-3,4-P2 exclusive of other phosphatidylinositol-polyphosphates is known.
Antibodies that specifically recognize PI-3,4-P2 are useful for purification and immunoassay of PI-3,4-P2, and serve as inhibitors of PI-3,4-P2. However, in general, poor antigenicity of phospholipids make it difficult to obtain antibodies against them. Furthermore, PI-3, 4-P2 is difficult to be obtained in large quantities. These problems have prevented the development of an immunoassay technique for PI-3,4-P2. Immunoassays are so excellent analytical methods as to achieve a high sensitivity and accuracy by a simple manipulation. For further investigation of signal transduction, an immunological assay method for PI-3,4-P2 has been strongly desired.
SUMMARY OF THE INVENTION
An objective of the present invention is to provide an antibody specifically recognizing PI-3,4-P2 and an immunological assay method using the antibody. More specifically, the present invention seeks to provide a novel antibody specifically recognizing PI-3,4-P2 and a simple method for determining PI-3,4-P2 with high sensitivity, like enzyme immunoassay, without requiring any special facilities.
Producing an anti-PI-3,4-P2 antibody is a problem because it is difficult to obtain a large quantity of antigens and the poor antigenicity of phospholipids used as antigens makes it difficult to produce an antibody of high titer. The inventors have solved the former problem by chemical synthesis of PI-3,4-P2 that enables it to be produced in large quantities. The inventors have also overcome the problem of poor antigenicity by enhancing the antigenicity using an immunogen obtained through adsorption of PI-3,4-P2 to dead Salmonella cells. In this way, the inventors have succeeded in producing a novel monoclonal antibody that binds specifically to PI-3,4-P2. Using the antibody, an immunological assay specific to PI-3,4-P2 in the living organism can be performed successfully. Furthermore, the inventors have isolated a gene encoding the amino acid sequence that constitutes variable regions of the antibody and have determined the nucleotide sequence, which will enable producing recombinant antibodies. The inventors have also found that topological PI-3,4-P2 distribution in cells can be identified and inhibitors specific to the function of PI-3,4-P2 can be developed, using the antibody of the present invention.
Specifically, the present invention relates to the following antibody, monoclonal antibody, variable regions thereof, hybridoma producing the antibody, and an immunological assay method using the antibody.
(1) An antibody specifically recognizing phosphatidylinositol-3,4-biphosphate.
(2) The antibody of (1), wherein the antibody is a monoclonal antibody.
(3) The antibody of (2), which recognizes an antigenic determinant comprising an inositol group and a glycerol backbone in phosphatidylinositol-3,4-biphosphate.
(4) The antibody of (1), which is substantially non-cross-reactive with at least one compound selected from the group consisting of phosphatidylinositol-4,5-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 (2).
(6) The hybridoma of (5), which has the properties of the deposit identified by the accession No. FERM-BP-6849.
(7) A method of producing the antibody of (2), the method comprising culturing the hybridoma of (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 whi
Fukui Yasuhisa
Nagata Satoshi
Saito Naoaki
Shirai Ryuichi
Corless Peter F.
Edwards & Angell LLP
Grun James L.
O'Day Christine C.
Yasuhisa Fukui
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