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
1999-03-17
2001-06-26
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...
C435S091500, C435S188000, C435S960000, C530S391500
Reexamination Certificate
active
06252053
ABSTRACT:
DETAILED DESCRIPTION OF THE INVENTION
1. Technical Field of the Invention
The present invention relates to an enzyme-antibody complex which is covalently conjugated via a carrier and said complex is utilized in immunohistochemistry and in immunoassay such as an enzyme immunoassay.
2. Prior Art
As a result of a recent progress in immunochemistry, an immunoassay where substances in small amounts are detected with a high sensitivity using an antigen-antibody reaction have been widely used. In the field of immunohistochemistry for example, a specific substance on a tissue section can be detected using an antibody which recognizes said substance. The means therefor is that, at first, an antibody (the primary antibody) which specifically recognizes the specific substance to be detected is made to react on the tissue section. When enzyme is conjugated to the primary antibody at that time, a chromogenic substrate is colored due to its enzymatic activity whereby detection of the specific substance is possible. Practically however, in this system, the sensitivity is low and, in addition, labeling of the enzyme to various primary antibodies actually requires a lot of labor and is difficult whereby that is not a common method.
Therefore, usually, after the reaction with the primary antibody, a reaction with the secondary antibody which specifically binds to the antibody is conducted. As a result thereof, several kinds of the secondary antibody may be prepared corresponding to the animal species of the primary antibody. In that case, when a sample whereby an enzyme can be directly bound to the secondary antibody is used, the substrate is colored by the enzymatic activity whereby the detection can be conducted. However, even in such a system, sensitivity is still low and, therefore, attempts have been made.
Usually, the secondary antibody where a plurality of biotins are conjugated is used and is further made to react with a substance (an enzyme reagent) prepared by conjugating an enzyme with streptavidin which specifically binds to biotin whereupon numbers of the enzyme binding per molecule of the secondary antibody are amplified to detect the specific substance in good sensitivity. Another amplifying method which is used is that where a complex in which streptavidin is conjugated with much enzyme is previously produced and then made to react with the secondary antibody to which biotin is conjugated. As such, it is possible to detect a specific substance on a tissue section using an antibody. In that operation, the processes of 1) reaction with the primary antibody, 2) reaction with the secondary antibody, 3) reaction with an enzyme reagent and 4) coloration are necessary. If an enzyme-labeled secondary antibody of a high quality is available in that case, staining can be conducted by an operation consisting of 1) reaction with the primary antibody, 2) reaction with an enzyme-labeled secondary antibody and 3) coloration where one step is eliminated as compared with the conventional methods.
In addition, an enzyme immunoassay using two kinds of antibody makes the detection of a specific substance (analyte) with a good sensitivity possible. In detecting the specific substance by an enzyme immunoassay, the following operations are necessary. Thus, 1) antibody (the primary antibody) binding to a specific substance (analyte) is immobilized on a microtiter plate or on polystyrene beads, 2) then the plate or the beads is/are blocked by protein such as albumin, 3) a solution containing the specific substance (analyte) is added and made to react for a predetermined period, 4) enzyme-labeled antibody (the secondary antibody) specifically binding to a specific substance (analyte) is added and made to react for a predetermined period, and 5) a chromogenic substrate for the enzyme is added and the resulting degree of coloration is measured by a spectrophotometer. Incidentally, a washing operation is included between the respective steps.
Amount of the specific substance (analyte) is correlated with the enzymatic activity of the labeled enzyme of the secondary antibody bound to said substance and, therefore, it is possible to determine the amount of the specific substance (analyte) by means of the degree of coloration. The antibody used here may be any of monoclonal antibody and polyclonal antibody. In the case of monoclonal antibody however, it is necessary that the antibody used for immobilization and the antibody which is to be labeled bind to different sites of said specific substance (analyte). Unlike in the case of immunohistochemistry, it is common in an enzyme immunoassay that enzyme is directly labeled to the secondary antibody and no amplifying operation is conducted. Accordingly, sensitivity of that measuring system is greatly dependent upon the quality of the enzyme-labeled secondary antibody.
When an enzyme-labeled antibody of a high quality is obtained as such, it is now possible to stain in less steps in the field of immunohistochemistry and also to conduct a measurement with higher sensitivity in an enzyme immunoassay.
3. Problems to be Solved by the Invention
Accordingly, an object of the present invention is to newly offer an enzyme-labeled antibody of a high quality.
BRIEF EXPLANATION OF THE DRAWINGS
The attached drawing shows the result of Example 5 of the present invention.
1. Means to Solve the Problems
The present inventors have conducted an investigation on a method for the manufacture of an enzyme-antibody complex for obtaining an enzyme-labeled antibody of a high quality and have found that such an object can be achieved by conjugating the enzyme to the antibody via a carrier. As a result of further study, they have accomplished the present invention.
Thus, the present invention relates to an enzyme-antibody complex where one or more molecule(s) of enzyme into which a maleimide group or a thiol group is introduced is/are covalently conjugated to a carrier in which a thiol group when a maleimide group is introduced into the enzyme or a maleimide group when a thiol group is introduced into the enzyme via them, and a maleimide group is introduced into at least one amino group remaining in the above complex and is covalently conjugated to antibody or antibody fragment via thiol group obtained by reduction of them. The present invention also relates to a method for the manufacture of an enzyme-antibody complex comprising a step where enzyme into which a maleimide group or a thiol group is introduced is covalently conjugated to a carrier in which a thiol group when a maleimide group is introduced into the enzyme or a maleimide group when a thiol group is introduced into the enzyme via them, and a step where a maleimide group is introduced into at least one amino group remaining in the above complex and the reduced antibody or antibody fragment is covalently conjugated thereto via a thiol group. The present invention will be further illustrated as hereunder.
Although the present invention has been at first conducted during the course of investigating the enzyme-antibody complex which can be used in the field of immunohistochemistry and enzyme immunoassay, there is no restriction for applying it to other fields.
There is no particular limitation for the carrier of the present invention so far as it is a substance having at least two amino groups. However, in order to enhance the sensitivity of the immunoassay, it is recommended that many enzymes and antibodies are conjugated to a carrier. Accordingly, it is necessary that the carrier has a molecular weight to a satisfactory extent and has many amino groups. Examples of the preferred carrier achieving such an object are peptide polymer and/or polysaccharide having many amino groups preferably having a molecular weight of 5,000-500,000 or, more preferably, 10,000-300,000. However, the above-mentioned range of the molecular weight is just a rough yardstick. Therefore, substances having more molecular weight than the above range may be used so far as neither precipitation nor sedimentation is resulted in a solution and, if an object of th
Kitano Yuriko
Kitoh Takashi
Ohbayashi Hirokazu
Burns Doane Swecker & Mathis L.L.P.
Ceperley Mary E.
Nichirei Corporation
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