Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Patent
1997-09-11
2000-07-25
Wortman, Donna C.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
435332, 435339, 5303881, 5303883, 530413, C12Q 170, C12N 520, C07K 1608
Patent
active
060935340
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
This invention relates to a novel monoclonal antibody. More particularly, it relates to a monoclonal antibody specifically recognizing the adeno-associated virus CAP protein, a hybridoma cell line producing this monoclonal antibody, and a method of detecting the adeno-associated virus and a method of purifying a virus vector for gene therapy by using the above-mentioned monoclonal antibody.
BACKGROUND ART
There have been made a number of attempts to transfer genes for gene therapy. At the present time, it is the most accustomed method therefore to use virus vectors, since a high gene transfer efficiency can be achieved thereby. For example, retrovirus vectors and adenovirus vectors are now under development. However, it is reported that use of these vectors is accompanied by various problems such that the gene thus transferred can be expressed only at a low efficiency or hardly integrated into chromosome, or there arises a cytotoxicity, etc.
The adeno-associated virus (AAV) vector may be cited as an example of the virus vectors attracting public attention in recent years. AAV is also called an adeno-satellite virus and falls into the category of the genus Parvovirus B, i.e., the smallest particles among animal viruses. It is known that this virus is a defective virus lacking the ability to self-proliferate and depends on the adenovirus in proliferation. Different from double-stranded DNA viruses, namely, the adeno-associated virus is a single-stranded DNA virus characterized by encoding exclusively a capsid protein. Since the transcription and replication of this virus per se depend on a cell system, it can proliferate exclusively in cells infected with adenovirus.
It is also reported that AAV tends to be integrated into a specific region in the long arm of the human 19th chromosome, which makes this virus further noteworthy.
As described above, the adeno-associated virus cannot replicate except in cells infected with adenovirus. To use an AAV vector as a virus vector for gene therapy, it is therefore needed to purify the AAV vector. That is to say, a general method for constructing an AAV vector comprises co-transfecting appropriate cells such as HeLa cells with a packaging plasmid prepared by deleting ITR (inverted terminal repeat) from the wild type AAV gene and a vector plasmid containing a gene having IDR introduced thereinto, simultaneously infecting these cells with adenovirus and then repeating freeze-thawing to thereby give the virus vector thus produced. Since not only the AAV recombinant but also the adenovirus are produced in this process, it is required to establish a method for purifying the AAV recombinant alone. Although it is suggested to carry out the purification with the use of a monoclonal antibody specific to AAV, no monoclonal antibody specific to AAV has been obtained so far.
DISCLOSURE OF THE INVENTION
Accordingly, an object of the present invention is to develop a monoclonal antibody which specifically recognizes the adeno-associated virus CAP protein. Another object of the present invention is to provide a hybridoma cell line capable of producing the above-mentioned monoclonal antibody, and a method of detecting the adeno-associated virus and a method of purifying a virus vector by using the above-mentioned monoclonal antibody.
Accordingly, the present invention provides a monoclonal antibody specifically recognizing the adeno-associated virus CAP protein, which is produced by hybridomas obtained by fusing lymphocytes prepared from a mammal which has been immunized with the adeno-associated virus CAP protein or a recombinant thereof as an antigen with a myeloma cell line.
The present invention further provides a hybridoma cell line capable of producing the above-mentioned monoclonal antibody specifically recognizing the adeno-associated virus CAP protein.
The present invention furthermore provides a method of detecting the adeno-associated virus and a method of purifying a recombinant adeno-associated virus vector for gene therapy by using the above-mention
REFERENCES:
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Kuma Hidekazu
Shimada Takashi
Suzuki Yosuke
Brumback Brenda G.
Hisamitsu Pharmaceutical Co. Inc.
Wortman Donna C.
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