Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-07-08
2002-04-23
Swartz, Rodney P (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C424S130100, C424S137100, C424S141100, C424S150100, C424S163100, C424S264100, C435S007100, C435S034000, C536S055100, C536S123100
Reexamination Certificate
active
06376203
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a novel glycoglycerophospholipid originating from
Mycoplasma fermentans
, an antibody against the glycoglycerophospholipid specifically existing in
Mycoplasma fermentans
, a method for measuring the glycoglycerophospholipid based on the use of the antibody, and a method for detecting
Mycoplasma fermentans.
BACKGROUND ART
The present inventors have already found five species of glycoglycerophospholipids (phosphocholine-containing glycoglycerolipids) from MT-4 cells (human helper T cells infected with HTLV-I (human T lymphotropic retrovirus Type I), Miyoshi et al., Gann., 71, 155-156 (1980)). The present inventors have previously found that one of the five species of glycoglycerophospholipids is 6′-O-phosphocholine-&agr;-glucopyranosyl-(1′-3)-1,2-diacyl-sn-glycerol (Shishitsu-Seikagaku-Kenkyu (
Studies on Lipid Biochemistry
), Vol. 35, pp. 111-114, 1993).
On the other hand, it has been reported that
Macodlasma fermentans
is an exacerbation factor of human acquired immunodeficiency syndrome (AIDS) (Lo. S., -C. et al., 1991,
Science,
251: 1074-1076; U.S. Pat. No. 5,242,820), or
Mycoplasma fermentans
is a cause of rheumatism (Williams, M. H. et al., 1970
, Lancet ii:
277-280).
Various antibodies against mycoplasmas have been hitherto known, and they have been also used for clinical examination. However, the majority of them are antibodies against
Mycoplasma pneumoniae
or
Mycoplasma genitalium
. Monoclonal antibodies against these mycoplasmas have been also prepared. However, it is presumed that such an antibody is an antibody which recognizes a protein of a mycoplasma, or simultaneously recognizes a protein and a lipid of a mycoplasma. Further, any of such antibodies does not exhibit specificity to
Mycoplasma fermentans
(Japanese Patent Laid-open Nos. 63-298, 63-184064, 63-32496, and 5-304990, and U.S. Pat. Nos. 5,158,870, 4,945,041, and 5,242,820). An antibody, which exhibits specificity to
Mycoplasma fementans
, is disclosed in U.S. Pat. No. 5,242,820. However, this antibody is obtained by using an entire extract of mycoplasmal cells as an immunogen, and thus the antibody is regarded as an antibody which recognizes a protein. Accordingly, if a mycoplasma contained in a body fluid such as a serum which contains various proteins is detected by using this antibody, the antibody highly possibly makes nonspecific binding. Therefore, it may be impossible to expect a high sensitivity. Further, when an antigen is a protein, it is sufficiently assumed that antigenicity disappears due to mutation in an amino acid sequence of the protein.
As far as the present inventors know, it has not been reported that any mycoplasma has a glycoglycerophospholipid containing phosphocholine. Further, no instance has been known, in which a glycoglycerophospholipid originating from a mycoplasma is used as an immunogen to obtain an antibody which exhibits specificity to the glycoglycerophospholipid. Moreover, it has not been known at all as well that the antibody, which exhibits the specificity to the glycoglycerophospholipid, exhibits high specificity to
Mycoplasma fementans.
DISCLOSURE OF THE INVENTION
It has been reported that the process of pathology of a patient infected with a human immunodeficiency virus is accelerated to arrive at AIDS by infection of a mycoplasma (1991
, Science,
251: 4991). It has been also reported that
Mycoplasma fementans
is an exacerbation factor of AIDS as described above. However, there is no means to correctly detect such behavior of
Mycoplasma fermentans
in vivo. Therefore, it has been desired to provide an antibody capable of immunologically detecting
Mycoplasma fementans
in vivo.
The present invention has been made considering a viewpoint as described above, an object of which is to elucidate a glycoglycerophospholipid specifically existing in
Mycoplasma fementans
, and provide an antibody against the glycoglycerophospholipid, a method for measuring the glycoglycerophospholipid based on the use of the antibody, and a method for detecting
Mycoplasma fementans.
In order to elucidate abnormal proliferation of cells, destruction of cells, and immunological abnormality caused by infection with a retrovirus, the present inventors have analyzed lipids of such infected cells. During this process, the present inventors have confirmed that a glycoglycerophospholipid (phosphocholine-containing glycoglycerolipid) extracted from a human helper T cell strain is unexpectedly a glycoglycerophospholipid originating from
Mycoplasma fermentans
(phosphocholine-containing glycoglycerolipid: hereinafter simply referred to as “glycoglycerophospholipid). Further, the present inventors have elucidated a structure of the lipid. Moreover, the present inventors have prepared an antibody against the lipid, and confirmed specificity to various mycoplasmas. As a result, the present inventors have found out that the antibody specifically recognizes
Mycoplasma fementans
. Thus the present invention has been completed.
Namely, the present invention lies in a glycoglycerophospholipid extractable from
Mycoplasma fermentans
, having the following properties:
(A) the glycoglycerophospholipid is reactive with orcinol reagent, Dittmer reagent, Dragendorff reagent, and ninhydrin reagent;
(B) the glycoglycerophospholipid is degradable with alkali;
(C) the glycoglycerophospholipid is obtained as a non-adsorptive fraction upon fractionation with an anion exchanger having diethylaminoethyl group; and
(D) the glycoglycerophospholipid has a molecular weight of 1048+28n measured by using a mass spectrometer, wherein n is −1, 0, 1, or 2.
The glycoglycerophospholipid described above highly possibly comprises constitutional components of &agr;-glucopyranosyl-(1′-3)-1,2-diacyl-sn-glycerol, phosphocholine, and phosphoric ester of aminopropanediol.
In another aspect, the present invention lies in an anti-glycoglycerophospholipid antibody having reaction specificity to a glycoglycerophospholipid comprising at least phosphocholine, glucose, fatty acid, and glycerol, the lipid being non-adsorptive to an anion exchanger having diethylaminoethyl group, and unstable against alkali. The glycoglycerophospholipid includes, for example, a glycoglycerophospholipid specifically existing in
Mycoplasma fementans.
The present invention provides, as specified embodiments of the anti-glycoglycerophospholipid antibody, an anti-glycoglycerophospholipid polyclonal antibody which reacts with both of 6′-O-phosphocholine -&agr;-glucopyranosyl-(1′-3)-1,2-diacyl-sn-glycerol and the glycoglycerophospholipid extractable from
Mycoplasma fermentans
, having the following properties, and an anti-glycoglycerophospholipid monoclonal antibody which has reaction specificity to the glycoglycerophospholipid having the following properties:
(A) the glycoglycerophospholipid is reactive with orcinol reagent, Dittmer reagent, Dragendorff reagent, and ninhydrin reagent;
(B) the glycoglycerophospholipid is degradable with alkali;
(C) the glycoglycerophospholipid is obtained as a non-adsorptive fraction upon fractionation with an anion exchanger having diethylaminoethyl group; and
(D) the glycoglycerophospholipid has a molecular weight of 1048+28n measured by using a mass spectrometer, wherein n is −1, 0, 1, or 2.
In further aspects, the present invention provides a method for measuring a glycoglycerophospholipid, comprising the step of immunologically measuring the glycoglycerophospholipid having the foregoing properties contained in a specimen, by using the foregoing anti-glycoglycerophospholipid antibody, and a method for detecting
Mycoplasma fementans
, comprising the steps of measuring a glycoglycerophospholipid contained in a specimen in accordance with the foregoing method, and relating the presence or absence of the glycoglycerophospholipid or an existing amount thereof to the presence or absence of
Mycoplasma fementans
or an existing amount thereof in the specimen.
In still another aspect, the present invention provides
Matsuda Kazuhiro
Yamamoto Naoki
Knobbe Martens Olson & Bear LLP
Seikagaku Corporation
Swartz Rodney P
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