Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Monoclonal antibody or fragment thereof
Reexamination Certificate
1998-10-02
2001-01-30
Allen, Marianne P. (Department: 1631)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
Monoclonal antibody or fragment thereof
C424S130100, C424S139100, C424S141100, C424S145100, C424S158100, C435S005000, C435S007100, C435S070200, C435S070210, C435S326000, C530S388100, C530S386000, C530S809000
Reexamination Certificate
active
06180102
ABSTRACT:
TECHNICAL FIELD
This invention relates to a monoclonal antibody specifically reactive with human Mx protein which is useful for the diagnosis of various diseases and to a hybridoma capable of producing the antibody.
BACKGROUND ART
It has been revealed by experiments using mice that Mx protein is induced when cells are stimulated with interferon, and that it is an important protein in exerting resistance to influenza virus infection [
Proc. Natl. Acad. Sci. USA,
80, 1910-1914 (1983),
Cell,
44, 147-158 (1986) and
Cell,
62, 51-61 (1990)]. Thereafter, it was found that two types of protein, MxA and MxB, homologous to the mouse Mx protein, are also induced in human when cells are stimulated with interferon [
Mol. Cell. Biol.,
9, 5062-5072 (1989) and
J. Virol.,
64, 1171-1181 (1990)]. Infection experiments using influenza virus A and vesicular stomatitis virus have revealed that MxA inhibits viral growth in cells but MxB does not have the inhibition activity [
J. Virol.,
64, 3370-3375 (1990)].
A monoclonal antibody to MxA was first prepared by immunizing mice with the purified protein [
J. Interferon Res.,
7, 331-343 (1987)]. Thereafter, Towbin et al. prepared 5 monoclonal antibodies using recombinant MxA expressed in
Escherichia coli
as an immunogen [
J. Interferon Res.,
12, 67-74 (1992)]. It was confirmed by enzyme immunoassay (ELISA) and Western blotting that the monoclonal antibodies of Towbin et al. reacted with MxA, and they could be divided into those which recognize an epitope corresponding to amino acids 1 to 429, counting from the N-terminus, and those which recognize another epitope corresponding to amino acids 430 to 662.
Recently, with the advance in studies on the structure and function of Mx protein, it has been revealed that the Mx protein has a GTP-binding domain and GTPase activity, which are now regarded as important factors in relation to the anti-virus activity [
Trends in Cell Biology,
3, 268-272 (1993)]. Also, the possibility has been suggested that Mx protein has a self-assembly motif and forms a large complex body through association of its molecules making use of the motif, thereby exerting its anti-virus function [
J. Biol. Chem.,
268, 15033-15038 (1993)].
Detailed elucidation of relationship between structure and function of Mx protein will make possible the accurate diagnosis of viral infectious conditions which are reflected by such a relationship. In the case of the known monoclonal antibodies which recognize MxA, it is difficult to elucidate the relationship of their functions with the GTP-binding domain and self-assembly motif because of the vague specificity of the binding site. Thus, accurate diagnosis of viral infectious conditions cannot be made. In addition, there are no reports of monoclonal antibodies capable of recognizing MxA which is intracellularly induced solely by viral infection, not by artificial stimulation with interferon.
The present invention relates to a monoclonal antibody which recognizes an epitope corresponding to amino acids 10 to 220, amino acids 221 to 297 or amino acids 469 to 662, counting from the N-terminus of a human Mx protein MxA, that specifically reacts with the human Mx protein by western blotting, immunoprecipitation or immunocyte staining and to a hybridoma which produces that antibody.
DISCLOSURE OF THE INVENTION
The following outlines the process for producing the anti-human Mx protein MxA monoclonal antibody of the present invention.
An MxA-encoding cDNA is cloned into a plasmid and introduced into
E. coli
cells in accordance with a known method [
J. Interferon Res.,
12, 67-74 (1992)]. Since MxA is expressed and accumulated as intracellular granules in
E. coli,
the cells are disrupted, for example, by an ultrasonication and MxA is solubilized by treating the sonicate with a surface active agent or a protein denaturing agent. The solubilized MxA is purified by repeated centrifugation. Splenocytes of an animal immunized with the purified recombinant MxA are fused with mouse myeloma cells to obtain hybridoma strains from which a hybridoma strain capable of producing a monoclonal antibody that reacts with MxA but not with MxA-free protein contaminant derived from
E. coli
cells is subsequently selected. The selected hybridoma strain is cultured or administered to an animal to produce ascites tumor in the animal, and monoclonal antibodies are collected from the resulting culture medium or ascitic fluid. From the monoclonal antibodies obtained, those which specifically react with MxA by western blotting are selected. Also selected are those which positively stain interferon-stimulated cells and virus-infected cells when used as the first antibody of immunocyte staining.
The sites recognized by the monoclonal antibodies can be determined in the following manner.
The MxA gene is digested into small fragments using restriction enzymes in order to synthesize mRNA by in vitro transcription (
Molecular Cloning, A Laboratory Manual,
2nd edition, published by Cold Spring Harbor Laboratory Press, 1989). Next, in vitro translation (
Molecular Cloning, A Laboratory Manual,
2nd edition, published by Cold Spring Harbor Laboratory Press, 1989) is carried out in the presence of
35
S-methionine to synthesize
35
S-methionine-labeled protein fragments which are successively shortened from the C-terminal end. These labeled protein fragments are allowed to react with monoclonal antibodies, and the reaction products are precipitated, for example, using beads labeled with a second antibody. The precipitate is separated by SDS-polyacrylamide electrophoresis, and the molecular weight of the precipitated protein is measured by autoradiography. The binding site of each monoclonal antibody is determined based on the measured molecular weight.
Some of the monoclonal antibodies which recognize certain domains of the human Mx protein MxA can be used, for example, in the diagnosis of viral infectious conditions.
The process for producing the anti-human Mx protein MxA monoclonal antibody of the present invention is described in detail below.
(1) Preparation of Antigen
Recombinant MxA is obtained by transforming
E. coli
cells with an expression vector which contains MxA-encoding cDNA or MxA is purified from cultured cells stimulated with interferon or the like.
(2) Immunization of Animals and Preparation of Antibody-Producing Cells
Mice, rats or hamsters of 3 to 20 weeks of age are immunized with the antigen prepared in step (1) above, and antibody-producing cells are collected from the spleen, lymph node or peripheral blood of the animals. The immunization may be carried out by administering the antigen together with an appropriate adjuvant, such as complete Freund's adjuvant or aluminum hydroxide gel plus pertussis vaccine, to the animals subcutaneously, intravenously or intraperitoneally. Following the first antigen administration, the antigen is administered repeatedly 5 to 10 times at one- to two-week intervals. Three to seven days after each administration, a blood sample is taken from the venous plexus of the fundus of the eye, and the resulting serum is examined, for example, by enzyme-linked immunosorbent assay (
Antibodies—A Laboratory Manual,
Cold Spring Harbor Laboratory, 1988) to determine whether it is reactive with the antigen. Mice, rats or hamsters whose serum shows a sufficient antibody titer against the antigen used for immunization are submitted for use as a splenocyte source.
For submission to fusion between splenocytes and myeloma cells, the spleen of the immunized mouse, rat or hamster is excised 3 to 7 days after the final administration of the antigen and splenocytes are collected. That is, the spleen is cut into pieces in MEM medium (Nissui Pharmaceutical Co., Ltd.) and dissociated using forceps, and, after centrifugation (1,200 rpm, 5 minutes), the supernatant is discarded and the sediment is treated with Tris-ammonium chloride buffer (pH 7.65) for 1 to 2 minutes to eliminate erythrocytes and then washed three
Furuya Akiko
Hanai Nobuo
Kusano Akira
Nagata Kyosuke
Taniguchi Noboru
Allen Marianne P.
Kyowa Hakko Kogyo Co. Ltd.
Nixon & Vanderhye P.C.
Zeman Mary K
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