Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1999-05-13
2001-10-23
Ceperley, Mary E. (Department: 1641)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C436S800000, C530S391300, C530S402000, C530S404000, C530S405000
Reexamination Certificate
active
06307029
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a dye-labeled protein conjugate prepared by binding an antibody to a protein to form a protein complex or conjugate and labelling the conjugate with a cyanine dye, and further to a method for preparing the same.
The dye-labeled antibody, which is obtained by labeling an antibody with a dye, specifically reacts with an antigen included in a sample solution and is readily recognizable with naked eyes. The dye-labeled antibodies are accordingly applied for immunosensors, each of which takes advantage of an immunological antigen-antibody reaction to detect a target substance included in a sample solution, and are used for diagnoses in a variety of medical institutions.
Cyanine dyes having the high molar absorption coefficient and the high reactivity are often used to label antibodies (Bioconjugate Chemistry Vol. 4, No. 2, pp105-111, 1993).
The functional group of the cyanine dye reacts with and is covalently bound to an amino group or a carboxyl group included in an antibody, and 20 to 50 molecules of the dye are attached to one molecule of the antibody.
The cyanine dye-labeled antibody thus prepared generally has high visual recognizability, and is effectively applied for, for example, immunochromatography to detect a small amount of a specific substance, such as human chorionic gonadotropin (HCG) that is present only in the urine of pregnant women.
The antibody generally includes several hundreds to several thousands of amino group or carboxyl group. The antibody has a three-dimensional steric configuration and thereby has only 50 groups that are related to the reaction. Namely only 50 molecules of the dye are bound to one molecule of the antibody.
When the dye-labeled antibody is applied for an immunosensor, it is accordingly difficult to detect a target substance having a low concentration.
BRIEF SUMMARY OF THE INVENTION
The object of the present invention is thus to provide a dye-labeled protein conjugate that is labeled with a large number of dye molecules.
Another object of the present invention is to provide a method for preparing the dye-labeled protein conjugate.
The present invention provides a dye-labeled protein conjugate comprising a protein, an antibody bound to the protein via a disulfide bond to form a protein conjugate, and a cyanine dye represented by the formula (1) or the formula (2) given below, the protein conjugate being labeled with the cyanine dye.
where R
1
and R
2
denote hydrogen or an alkyl group, X denotes a halogen, M denotes hydrogen or an alkali metal, and n represents an integer of 1 to 4.
Binding a protein to the antibody extends the area of the antibody that can be linked with the cyanine dye, and thereby increases the number of cyanine dye molecules bound to the protein conjugate, compared with a single body of the antibody. The number of dye molecules bound to one molecule of the antibody in the protein conjugate is, for example, about 10 times that of the antibody in the single body. The dye-labeled protein conjugate accordingly has high visual recognizability.
When the dye-labeled protein conjugate of the present invention is applied for, for example, immunochromatography, the immunochromatography can detect a target substance (sample) with high sensitivity even when the sample has a low concentration. Because of the high sensitivity, the dye-labeled protein conjugate of the present invention is applicable for biosensors.
In accordance with one preferable application of the dye-labeled protein conjugate of the present invention, a skeleton of the cyanine dye is bound to the protein conjugate via a covalent bond of an acyl carbon originated from a succinimidyl group in the cyanine dye with a nitrogen originated from an amino group in the protein conjugate.
The present invention is also directed to a method for preparing a dye-labeled protein conjugate. The method comprises the steps of: reducing a protein in a neutral or weak alkaline phosphate buffer solution; adding an antibody to the buffer solution to prepare a protein conjugate; and adding a cyanine dye represented by the formula (1) or the formula (2) given above to the buffer solution to label the protein conjugate with the cyanine dye.
The present invention is further directed to another method for preparing a dye-labeled protein conjugate. The method comprises the steps of: reducing a protein in a neutral or weak alkaline phosphate buffer solution; adding a cyanine dye represented by the formula (1) or the formula (2) given above to the buffer solution to label the reduced protein with the cyanine dye; and adding an antibody to the buffer solution to make the antibody bound to the reduced protein.
In accordance with one preferable application, the method includes the step of labeling the antibody with succinimidyl pyridyl dithiopropionate represented by the formula (3) given below in a neutral or weak alkaline phosphate buffer solution, prior to the step of making the antibody bound to the reduced protein.
In any of the above methods, it is preferable that the phosphate buffer solution has a pH value in a range of 7.0 to 8.0.
The antibody used to prepare the dye-labeled protein conjugate of the present invention is not specifically restricted, but may have a variety of origins and sub-classes. Available examples of the antibody include immunoglobulins (Ig), such as mouse IgG, mouse IgM, mouse IgA, mouse IgE, rat IgG, rat IgM, rat IgA, rat IgE, rabbit IgG, rabbit IgM, rabbit IgA, rabbit IgE, goat IgG, goat IgM, goat IgE, goat IgA, sheep IgG, sheep IgM, sheep IgA, and sheep IgE. These antibodies may be of commercial origin or directly collected from the corresponding animals.
The protein bound to the antibody may be any protein that does not exert the function as the antibody. The protein having high solubility in water is especially preferable. For example, serum-originated albumin that does not inhibit the reaction of the antibody and has high water solubility is preferably used.
The cyanine dyes represented by the formula (1) and formula (2) which have blue colors are less affected by impurities due to their absorption at long wavelength. They are therefore effective for a sensor that detects a specific analyte based on its absorbance.
The halogen represented by X in the formula (1) or the formula (2) may be fluorine, chlorine, bromine, or iodine. The metal represented by M may be lithium, sodium, or potassium.
While the novel features of the invention are set forth particularly in the appended claims, the invention, both as to organization and content, will be better understood and appreciated, along with other objects and features thereof, from the following detailed description taken in conjunction with the drawings.
DETAILED DESCRIPTION OF THE INVENTION
The following describes the mechanism of binding the cyanine dye to the antibody.
When the antibody is mixed with the cyanine dye having a succinimidyl group, an amino group in the antibody approaches an ester bond of the succinimidyl group in the dye as shown by the formula (4).
The amino group reacts with the ester bond as shown by the formula (5), so that one hydrogen atom is released from the amino group. The hydrogen atom released from the amino group is attached to succinimide in the succinimidyl group. Succinimide is then changed to hydroxysuccinimide, which is released from the succinimidyl group. At the same time, the residue of the succinimidyl group and the hydrogen atom-released amino group combine to form an amide bond, through which the dye is linked with the antibody.
The following describes one exemplified process of synthesizing the cyanine dye represented by the formula (1) given above.
The process first dissolves hydrazinobenzenesulfonic acid (6) and isopropyl methyl ketone in an acidic solvent and heats the mixture to obtain indoleniumsulfonate (7). The process then adds a metal hydroxide-saturated alcohol solution into an alcohol solution of indoleniumsulfonate (7), so as to yield a metal salt of indoleniumsulfonate (8).
The process subsequen
Miyazaki Jinsei
Nakayama Hiroshi
Shigetou Nobuyuki
Ceperley Mary E.
Matsushita Electric - Industrial Co., Ltd.
McDermott & Will & Emery
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