Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Animal cell – per se – expressing immunoglobulin – antibody – or...
Reexamination Certificate
1997-12-02
2001-01-16
Venkat, Jyothsna (Department: 1627)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Animal cell, per se, expressing immunoglobulin, antibody, or...
C435S325000, C435S326000, C435S346000, C435S352000, C435S354000, C530S388100, C530S388850, C530S388900, C546S129000, C546S130000, C546S131000
Reexamination Certificate
active
06174723
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a cocaine-protein conjugate for use as an antigen to produce an anti-cocaine antibody, a cocaine derivative as a starting material for the conjugate, a novel monoclonal antibody producing cell line obtained from the antigen and a monoclonal antibody produced by the cell line. The monoclonal antibody is useful in immunochemically detecting cocaine or its derivative out of blood or air with high sensitivity.
BACKGROUND OF THE INVENTION
J. Pharmacol. Exp. Ther., vol.199, p171 (1976) proposes a synthesis of an anti-cocaine antibody for use in an immunoassay for cocaine, and an antigen necessary in preparing the antibody. The antibody reported is, however, a polyclonal antibody which can be obtained from refined blood of a goat or rabbit immunized using an antigen of ecgonine of Formula (1).
To obtain an anti-cocaine antibody highly specific for cocaine, a cocaine-protein conjugate must be formed by using a derivative keeping the cocaine framework as precise as possible. Unfortunately, ecgonine lacks a methoxycarbonyl group and benzoyl group each characteristic of the cocaine molecule.
The polyclonal antibodies of the prior art can simply be prepared; on the other hand, it is less reproducible because the properties of the product depend on individual experimental animals. Thus, it is quite difficult to provide antibodies of the same quality.
Polyclonal antibodies are a mixture of a diversity of antibodies having respective affinities. All the affinities they have prevent the use of a polyclonal antibody in a sensitive detection system because of their low affinity for the molecule of interest. In accordance with the above, we tried to obtain a monoclonal antibody having high affinity for cocaine; however, we could not succeed because ecognine as an antigen is too different in structure from cocaine.
SUMMARY OF THE INVENTION
The invention aims to provide a method for easily preparing a cocaine-protein conjugate based on cocaine characterized by a methoxy carbonyl group and a benzoyl group.
The invention further aims to provide a monoclonal antibody of high affinity for cocaine or cocaine derivatives by immunizing with the cocaine-protein conjugate.
The invention further aims to provide a cell line for producing the monoclonal antibody.
To attain the above-mentioned aims, the inventors brought about an amino derivative of cocaine based on norcocaine substituted with an amino alkyl group in its secondary amino group, a pyridyl dithio derivative of an amino cocaine derivative substituted with a 3-(2-pyridyl dithio)propynoyl group in its amino group, and a cocaine-protein conjugate. Further, the inventors established a monoclonal antibody producing cell line by fusing a spleen cell derived from mouse immunized with an antigenic cocaine-protein conjugate and a cell line derived from a myeloma cell culture and cloning the fusion.
A first compound of the invention is shown in Formula (2):
wherein n is an integer of 1 or more and Ph is a benzene ring.
A first cocaine-protein conjugate is the above cocaine derivative conjugated to a protein.
A second compound of the invention is shown in Formula (3):
wherein n is an integer of 1 or more and Ph is a benzene ring.
A second cocaine-protein conjugate is the second cocaine derivative conjugated to a protein by a disulfide bond.
The method for preparing a monoclonal antibody producing cell line comprises fusing a spleen cell derived from a mouse immunized with an antigen of the first cocaine-protein conjugate and a cell line derived from a myeloma cell line and cloning the fusion.
It is preferable in the above method that the protein is keyhole limpet hemocyanin.
It is also preferable in the above method that the cell line derived from a myeloma cell line is P3X63-Ag8.653.
It is also preferable in the above method that the mouse is derived from an A/J strain.
The cell line of the invention is preferably a monoclonal antibody producing cell line deposited in the National Institute of Bioscience and Human Technology 1-3, Higashi l-chrome, Tsukuba-shi, Ibaraki-ken 305 Japan as No.BP-4177 produced with the above method, characterized by producing an antibody capable of specifically binding to cocaine or cocaine derivatives.
The invention further aims to provide a monoclonal antibody from the above monoclonal producing cell line.
With the amino derivatives of cocaine of the invention, one can readily prepare a pyridyl dithio derivative of an amino cocaine derivative having a disulfide group capable of linking to a protein, keeping the cocaine framework as it is. By immunizing a test animal with a cocaine-protein conjugate made from the above pyridyl dithio derivative, one can get a cell line for producing an antibody of higher affinity for cocaine because the cocaine-protein conjugate holds the cocaine framework.
After immunoreaction, antibody-producing cells are stored in the spleen. Although a spleen is unable to reproduce by itself, it can proliferate and produce a hybridoma cell line by fusing with a cell line derived from myeloma. The hybridoma cell line vigorously produces antibodies as it proliferates. By selecting a hybridoma cell which has much multiplication ability and produces an antibody having the highest affinity for a certain molecule and cloning the hybridoma, one can produce a desired monoclonal antibody. The monoclonal antibody thus obtained is a single kind of antibody, and of high-affinity. A constant quality of monoclonal antibody can permanently be provided by culturing the hybridoma.
REFERENCES:
patent: 5233042 (1993-08-01), Buechler et al.
patent: 5808074 (1998-09-01), Gowda et al.
Collison et al.,J. Forensic Science, 43(2): 390-394 (1998) (Abstract).
O'Connell et al. J. Immunological Meth. vol. 225, No. 1-2, pp. 157-169. Abstract only, AN 1999291914, MEDLINE, May 1999.
American Type Culture Collection (ATTC) Catalogue of Cell Lines and Hybridomas, 7th Edition. Editors: Hay et al. ATTC: Maryland, USA, 1992.
Harlow, E. and Lane, D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory: New York, USA, 1988.
Mitsumata Tadayasu
Miyazaki Jinsei
Sigetoh Nobuyuki
Yugawa Keiko
Garcia Maurie E.
Matsushita Electric - Industrial Co., Ltd.
Morrison & Foerster / LLP
Venkat Jyothsna
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