Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-11-13
2004-11-16
Devi, S. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C436S518000
Reexamination Certificate
active
06818408
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a monoclonal antibody to phosphatidylinositol-3,4,5-triphosphate. The present invention also relates to a method for immunoassay of phosphatidylinositol-3,4,5-triphosphate using the monoclonal antibody.
BACKGROUND OF THE INVENTION
The basic system for survival of multicellular organisms, including humans, includes a “cellular signal transduction system,” in which an extracellular signal is transferred to the inside of the cell to induce a necessary cell response. It is becoming clear that abnormality in this system causes pathological conditions attributed to an inadequate cell response, such as neoplastic transformation of cells. Phosphatidylinositol (hereinafter abbreviated as PI), which is a phospholipid present in various biomembranes, changes the phosphorylated state of the inositol ring by the action of enzymes activated by extracellular stimulation to transduce an extracellular signal into cells. Phosphatidylinositol-3,4,5-triphosphate with a phosphorylated inositol group at the 3-position has been shown, inter alia, to be produced from phosphatidylinositol-4,5-biphosphate (hereinafter abbreviated as PI-4,5-P2) by the action of phosphatidylinositol-4,5-kinase (hereinafter abbreviated as PI3K) activated by stimulating various proliferation or differentiation factors and to participate not only in signal transduction into the cell nucleus but also in versatile and fundamental cell responses such as construction of cell backbone and vesicular transport of substances (Fukui, Y. et al., J. Biochem., 124, 1-7, 1998). Phosphatidylinositol-3,4,5-triphosphate used herein is abbreviated PIP3; if it is necessary to specify the phosphorylated position, the triphosphate is designated PI-3,4,5-P3. The structure of PIP3 is schematically shown below.
Studies on the kinetics of PIP3 both in cells and in living organisms help clarify the fundamental cellular signal transduction and shed light on pathological conditions of various diseases. The role of PIP3 in cells has been explored so far mainly by indirect means of using PI3K inhibitors or modifiers such as activated or inactivated PI3K. As a direct approach, a quantitative assay has been established for PIP3. However, the known assay for PIP3 requires using a radioisotope, which necessitates special facilities for the assay. Moreover, the assay itself is time-consuming due to complicated operations including extraction and chromatography in many steps. Therefore, a simple assay technique has been desired.
For instance, antibodies that can recognize PIP3 will be important tools for further investigations. These antibodies will be useful for purification and immunoassay of PIP3 or as inhibitors against PIP3. However, phospholipid antigens are generally known to have a poor antigenicity. It is thus difficult to obtain antibodies to the antigens. PIP3 has an additional problem in that it is difficult to produce in large quantities. Though attempts to produce the antibodies using elaborated adjuvants were reportedly successful, most of the antibodies produced have a poor ability to recognize the phosphorylated state of PI. In most cases, it is difficult to distinguish PIP3 from PI or PI-4,5-P2 due to their cross-reactivity.
It has been reported that a rabbit was immunized to produce a polyclonal antibody to PI-4,5-P2 (Molec. Immun., 16, 193-196, 1979). The polyclonal antibody exhibited cross-reactivity with other phospholipid antigens such as phosphatidylinositol-4-phosphate (hereinafter abbreviated as PI-4-P) or cardiolipin (hereinafter abbreviated as CL). A monoclonal antibody to PI-4-P was obtained by immunization with liposomes (Molec. Immun., 21, 863-868, 1984). However, it was confirmed that the antibody was also cross-reactive with other phospholipids, that is, the cross-reactivity with PI-4,5-P2 was observed by the complement-dependent liposome lysis assay.
A monoclonal antibody to PI-4,5-P2 was produced using as an immunogen a suspension of PI-4,5-P2 together with phosphatidylcholine or methylated BSA (Proc. Japan Acad., 59, 28-32, 1983). The thus-produced monoclonal antibody has been verified to be cross-reactive with an acidic phospholipid such as PI-4-P or PI. Furthermore, a monoclonal antibody specific to PI-4,5-P2 and showing a low cross-reactivity with other phospholipids was obtained using a Salmonella cell as an adjuvant (Miyazawa A., Molec. Immun., 26, 1025-1031, 1988). However, there has been no report of an antibody highly specific to PIP3 and non-cross-reactive with other phospholipid antigens being obtained. For this reason, no immunoassay technique for PIP3 has been found yet. Immunoassay is an excellent technique that can achieve a high sensitivity and accuracy in a simple operation. Accordingly, an immunological assay for PIP3 is strongly desired for further studies on cellular signal transduction.
SUMMARY OF THE INVENTION
An objective of the present invention is to provide an antibody specifically recognizing PIP3. Another objective of this invention is to provide an immunological assay method using the antibody. More specifically, the present invention seeks to provide a novel antibody specifically recognizing PIP3 and a simple method for determining PIP3 with high sensitivity, like enzyme immunoassay, without requiring any special facilities.
Producing an anti-PIP3 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 PIP3 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 PIP3 to dead Salmonella cells. In this way, the inventors have succeeded in producing a novel monoclonal antibody that binds specifically to PIP3. Using the antibody, an immunological assay specific to PIP3 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 PIP3 distribution in cells can be identified and inhibitors specific to the function of PIP3 can be developed, using the antibody of the present invention.
Specifically, the present invention relates to the following antibody and variable regions thereof as well as an immunological assay method using the antibody.
(1) An antibody specifically recognizing phosphatidylinositol-3,4,5-triphosphate.
(2) An antibody of (1), wherein the antibody is a monoclonal antibody.
(3) An antibody of (2), which recognizes an antigenic determinant comprising an inositol group and a glycerol backbone in phosphatidylinositol-3,4,5-triphosphate.
(4) An antibody of any one of (1) through (3), which is non-cross-reactive with phosphatidylinositol-4,5-diphosphate.
(5) A variable region of immunoglobulin heavy chain specifically binding to phosphatidylinositol-3,4,5-triphosphate, comprising an amino acid sequence shown by SEQ ID NO: 2 or an amino acid sequence of SEQ ID NO: 2 in which one or more amino acid residues have been substituted, deleted or added.
(6) A variable region of immunoglobulin light chain specifically binding to phosphatidylinositol-3,4,5-triphosphate, comprising an amino acid sequence shown by SEQ ID NO: 4 or an amino acid sequence of SEQ ID NO: 4 in which one or more amino acid residues have been substituted, deleted or added.
(7) CDR1 in immunoglobulin heavy chains specifically binding to phosphatidylinositol-3,4,5-triphosphate, comprising an amino acid sequence shown by SEQ ID NO: 5 or an amino acid sequence of SEQ ID NO: 5 in which one or more amino acid residues have been substituted, deleted or added.
(8) CDR2 in immunoglobulin heavy chains specifically binding to phosphatidylinositol-3,4,5-triphosphat
Fukui Yasuhisa
Nagata Satoshi
Shirai Ryuichi
Conlin David G.
Devi S.
Medical & Biological Laboratories Co. Ltd.
O'Day Christine C.
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