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
1999-05-05
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, C530S402000, C530S404000, C530S405000
Reexamination Certificate
active
06303757
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to an polymerized and labelled antibody prepared by polymerizing an antibody and binding the resultant polymerized antibody to a protein to form a protein complex or polymerized antibody conjugate, and labelling the conjugate with a cyanine dye, and further to a method for manufacturing the same.
A dye-labelled antibody prepared by labelling an antibody with a dye, which is visible optically, has a specific reaction with an immunogen or antigen contained in a sample solution. Therefore, such antibody has been used, for example, in an immunosensor for detecting a specific substance or antigen contained in a sample solution with the aid of immunological antigen-antibody reaction and has a wide application as a diagnostic means at various medical facilities.
Cyanine dyes are the most frequently used dyes for labelling antibodies, because they are highly reactive and have a high molar absorption coefficient (see Bioconjugate Chemistry Vol. 4, No. 2, pp. 105-111, 1993).
Those cyanine dyes have a functional group which reacts with an amino group or a carboxyl group present in the antibody and forms a covalent bond. The binding ratio of cyanine dye to antibody is 20 to 50 molecules per molecule of antibody.
The cyanine dye-labelled antibody thus produced has been applied to, for example, immunochromatography because of its generally high visibility and widely used for detecting a small amount of a specific substance such as human chorionic gonadotropin which is contained only in the urine of pregnant women.
Normal antibody includes several hundreds to several thousands of amino group or carboxyl group. However, the conventional interpretation is that, of those many amino or carboxyl groups, only 50 or so can participate in the reaction due to its three-dimensional structure, which limits the number of binding molecules of cyanine dye to 50 molecules per molecule of antibody.
Moreover, since the antibody has a limited number of reaction site with antigen only to 2 for one molecule, the binding sensitivity of antibody to antigen remains low.
The use of such a labelled antibody in an immunosensor or the like limits the sensitivity of the sensor, producing a significant problem of difficult detection of analyte, that is, antigen if its concentration in a sample solution is low.
In view of the above-mentioned problems, the object of the present invention is to provide a high-sensitive indirect polymerized and labelled antibody facilitating detection of a low concentration of analyte in a sample solution.
Another object of the present invention is to provide a method for manufacturing the polymerized and labelled antibody as stated above.
BRIEF SUMMARY OF THE INVENTION
The present invention provides an polymerized and labelled antibody comprising an antibody and a dye for labelling the antibody, the dye being a cyanine dye represented by one of the chemical formulae (1) to (4):
where R
1
and R
2
represent hydrogen or an alkyl group, X represents a halogen, M represents hydrogen or an alkali metal, and n represents an integer of 1 to 4,
wherein the antibody is polymerized via a multi-functional reagent and bound to a protein via a disulfide bond to form a polymerized antibody conjugate, the antibody conjugate being labelled with the cyanine dye.
A polymerized antibody prepared by polymerizing an antibody is multi-valent having many reaction sites with antigen and therefore has a high binding sensitivity to antigen compared to other usual divalent antibodies.
Moreover, when bound to a protein, the resultant polymerized antibody conjugate is increased in area for binding to the cyanine dye, which also increases the number of binding molecules of the dye to the polymerized antibody conjugate. As a result, the polymerized and labelled antibody obtained by the above-mentioned method can have excellent visibility.
The use of the polymerized and labelled antibody in accordance with the present invention in, for example, immunochromatography facilitates detection of a low concentration of analyte, that is, antigen with high sensitivity. The polymerized and labelled antibody in accordance with the present invention is also applicable to any type of biosensor because of its high sensitivity.
In a preferred mode of the present invention, the polymerized and labelled antibody has a structure where the skeleton of the cyanine dye is bound to the polymerized antibody conjugate via a covalent bond between acylcarbon derived from a succinimidyl group present in the cyanine dye and nitrogen derived from an amino group present in the polymerized antibody conjugate.
In the polymerized and labelled antibody in accordance with the present invention, desirable degrees of polymerization for the antibody may be in a range of 2 to 50.
The method for manufacturing the polymerized and labelled antibody in accordance with the present invention comprises the steps of
polymerizing an antibody using a multi-functional reagent in a neutral or weak alkaline phosphate buffer solution to form a polymerized antibody,
reducing a protein in a neutral or weak alkaline phosphate buffer solution to form a reduced protein, reacting the polymerized antibody with the reduced protein to form a polymerized antibody conjugate, and reacting the polymerized antibody conjugate with a dye thereby labelling the polymerized antibody conjugate with the dye, the dye being a cyanine dye represented by one of the chemical formulae (1) to (4).
In another mode of the present invention, the method may comprise the steps of polymerizing an antibody using a multi-functional reagent in a neutral or weak alkaline phosphate buffer solution to form a polymerized antibody,
reducing a protein in a neutral or weak alkaline phosphate buffer solution to form a reduced protein, reacting the reduced protein with a dye to form a labelled protein, and reacting the polymerized antibody with the labelled protein, the dye being a cyanine dye represented by one of the above-mentioned chemical formulae (1) to (4).
In either method, preferable pH value for the phosphate buffer solution is in a range of 7.0 to 8.0.
Applicable antibodies to the polymerized and labelled antibody in accordance with the present invention are not limited to particular ones and any antibody can be used regardless of its derivation and subclass. Examples of applicable antibodies are a variety of immunoglobulin (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, sheep IgE, etc. These antibodies may be commercially available or harvested directly from the animal of interest.
The multi-functional reagent may be exemplified as reagents having, in the same molecule, 2 or more functional groups, such as succinimidyl group, pyridildisulfide group, etc., which can bind to an antibody. Examples of such reagent may be dithiosulfosuccinimidyl propionate represented by the chemical formula (5), bis(sulfosuccinimidyl)suberate represented by the chemical formula (6), disuccinimidyl tartrate represented by the chemical formula (7), ethylene glycol bis(succinimidyl succinate) represented by the chemical formula (8), N-succinimidyl-3-(2-pyridildithio)propionate represented by the chemical formula (9), etc.
The protein to be bound to the polymerized antibody may be any one that does not function as an antibody. Highly water-soluble protein is more preferable. For example, serum-derived albumin is preferable because it has no inhibitory effect on the antigen-antibody reaction and is highly soluble in water.
Cyanine dyes represented by the chemical formula (1) or (2) are red dyes facilitating macroscopic confirmation.
Whereas the dyes represented by the chemical formula (1) are less in number of covalent carbon and have high solubility in water, those represented by the chemical formula (2) have an intense deep red color and are most easily confirmed macroscopically.
If a device, such as senso
Miyazaki Jinsei
Shigetou Nobuyuki
Ceperley Mary E.
Matsushita Electric - Industrial Co., Ltd.
McDermott & Will & Emery
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