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
2001-10-16
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
Reexamination Certificate
active
06303758
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a polymerized antibody labeled with a cyanine dye and a method for preparing the same.
The dye-labeled antibody prepared 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.
One molecule of the antibody generally has only two sites reacting with the antigen and thereby has relatively low reaction sensitivity to the antigen.
When the conventional dye-labeled antigen is used for an immunosensor, the immunosensor accordingly does not have sufficient sensitivity. In the case that the sample solution contains a low concentration of a target substance (antigen), it is difficult to detect the target substance.
BRIEF SUMMARY OF THE INVENTION
The object of the present invention is thus to provide a highly sensitive dye-labeled and polymerized antibody that enables detection of even a low concentration of a target substance.
Another object of the present invention is to provide a method for preparing such a dye-labeled and polymerized antibody.
The present invention provides a dye-labeled and polymerized antibody comprising an antibody and a cyanine dye represented by the formula (1) or the formula (2) given below:
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 in a range of 1 to 4,
wherein the antibody is polymerized via a polyfunctional reagent and the polymerized antibody is labeled with the cyanine dye.
The polymerized antibody is a polyvalent antibody having a large number of sites reacting with an antigen and thereby has a higher binding sensitivity to the antigen, compared with the conventional divalent antibody.
The number of dye molecules bound to one molecule of the polymerized antibody is greater than the number of dye molecules bound to one molecule of the conventional antibody. This improves the visual recognizability.
When the dye-labeled and polymerized antibody 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 and polymerized antibody of the present invention is applicable for biosensors.
In accordance with one preferable application of the dye-labeled and polymerized antibody of the present invention, a skeleton of the cyanine dye is bound to the polymerized antibody via a covalent bond between an acyl carbon originated from a succinimidyl group in the cyanine dye and a nitrogen originated from an amino group in the polymerized antibody.
The degree of polymerization of antibody in the dye-labeled and polymerized antibody of the present invention is generally in a range of 2 to 50.
The present invention is also directed to a method for preparing a dye-labeled and polymerized antibody. The method includes the steps of: polymerizedizing an antibody using a polyfunctional reagent in a neutral or a weak alkaline phosphate buffer solution; and adding a cyanine dye represented by the formula (1) or the formula (2) given above to the buffer solution, so as to label the polymerizedized antibody. It is here 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 and polymerized antibody 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 polyfunctional reagent used in the present invention has two or more functional groups (for example, succinimidyl group, pyridyldisulfide group), which can be bound to an antibody, in one identical molecule. Available examples of the polyfunctional reagent include dithiobis(sulfosuccinimidyl propionate) represented by the formula (3), bis (sulfosuccinimidyl) suberate represented by the formula (4), disuccinimidyl tartrate represented by the formula (5), ethylene glycol bis(succinimidyl succinate) represented by the formula (6), and N-succinimidyl-3-(2-pyridyldithio) propionate represented by the formula (7).
Cyanine dyes represented by the formula (1) and the 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.
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Spector, D.L. “Flourescent labeling of antibodies and DNA probes” 199, Cold Spring Harbour Laboratory Press, US vol. 2 of “Cells: a Laboratory Manual”; “Light Microscopy and Cell Structure” p. 82.1 p p. 82.7; table 82.1.
Mujumdar R B, et al: “Cyanine Dye Labeling Reagents: Sulfoindocyanine Succinimidyl Esters” Bioconjugate Chemistry, US, American, Chemical Society, Washington, vol. 4, No. 2, p. 105-111 ISSN 1043-1802.
Hirai Mahito
Miyazaki Jinsei
Shigetou Nobuyuki
Ceperley Mary E.
Matsushita Electric - Industrial Co., Ltd.
McDermott & Will & Emery
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