Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1997-10-14
2001-06-19
Duffy, Patricia A. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007950, C435S007100, C436S518000, C436S071000, C436S176000
Reexamination Certificate
active
06248545
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a reliable assay of denatured lipoproteins which are considered to involve arteriosclerosis and present in the living body.
2. Description of the Background Art
Arteriosclerosis is known to be the main cause of ischemic heart diseases, cerebral infarction and the like. The mechanism causative of arteriosclerosis is reported to form atheroma due to accumulation of smooth muscle cells, connective tissues and a great amount of lipids (mainly, cholesterol esters) on a vascular subendothelium, resulting in the hypertrophy of the arterial wall and advanced induration, which finally brings about the hypofunction of the artery. In particular, it is considered that the atheroma recognized in the initial lesion of this disease is formed from foam cells produced by the accumulation of a denatured low density lipoprotein (LDL) into macrophages. However, the true character of the denatured LDL which transforms the macrophages into the foam cells in the living body is unknown to date. However, it has recently been explicated that there is a possibility that two kinds of denatured LDLS one of which is a denatured LDL formed by the interaction between a vascular endothelial cell which is reported to play an important role in arteriosclerosis and LDL [Quinn, M. T., Parthasarathy, S., Fong, L. G. & Steingerg, D., Proc. Natl. Acad. Sci. USA, 84, 2995-2998 (1987)], and the other of which is an LDL modified with malondialdehyde (MDA) known as an end metabolic product of lipid peroxides and thromboxane A
2
, which also play an important role in arteriosclerosis [Fogelman, A. M., Shechter, I., Seager, J., Hokom, M., Child, J. S. & Bdwards, P. A., Proc. Natl. Acad. Sci. USA, 77, 2214-2218 (1980)], may deeply participate in the initial lesion of arteriosclerosis. An assay of these denatured LDLs in the living body has hence been regarded as important.
Some methods have heretofore been reported as methods for assaying denatured LDLs. For example, Gonen et al., and Salmon et al. apply an EIA competition analysis in which MDA-modified LDL coated on the plate, and an antibody which recognizes the MDA-modified LDL are used [Gonen, B., Fallon, J. J. & Baker, S. A, Atherosclerosis, 65, 265-272 (1987)] and an EIA sandwich technique in which an immobilized antibody which recognizes MDA-modified LDL and an enzyme-labeled antibody which recognizes LDL are used [Salmon, S., Maziere, C., Theron, L., Beucler, I., Ayrault-Jarrier, M., Goldstein, S. & Polonovski, J., Biochim. Biophys. Acta, 920, 215-22 (1987)], respectively, to assay extracts from a focus of arteriosclerosis or the MDA-modified LDL in the blood
However, Gonen et al. have failed to detect the MDA-modified LDL in the arteriosclerosis tissue according to this analysis. On the other hand, Salmon et al. have reported that although the assay of the MDA-modified LDL in the blood was performed as to 65 specimens, only 14 specimens were able to be detected, but the residual 51 specimens were unable to be detected.
As described above, it has heretofore been known to assay MDA-modified LDL artificially synthesized. It has however been impossible to effectively assay MDA-modified LDL, i.e., a denatured LDL, in vital samples.
On the other hand, it has been known that lipoprotein (a) [Lp(a)] involves the causation of the coronary artery disease independent of other risk factors in the causation of arteriosclerotic diseases. In recent years, attention has been paid to the participation of a denatured Lp(a) as a mechanism causative of this disease [Loscalzo. J., Arteriosclerosis, 10, 672-679 (1990)], that is to say, a mechanism that the denatured Lp(a) accumulates on the coronary artery, and macrophages attracted thereto incorporate it therein, whereby they are transformed into foam cells. As recent reports supporting this hypothesis, may be mentioned a report that when Lp(a) is treated with copper or modified with malondialdehyde (MDA) to be converted to a denatured Lp(a) having a minus charge, its engorgement into macrophages increases [Haberland, M. E., Fless, G. M., Scanu, A. M. & Fogelman, A. M., J. Biol. Chem., 267, 4143-4151 (1992)], and a report that Lp(a) accumulates on a focus of arteriosclerosis, and this Lp(a) has a more negative charge compared with native (normal) Lp(a) as determined by agarose electrophoresis [O'Neil, J. A., Pepin, J. M., Smejkal, G., et al, Arteriosclerosis, 10, 812a (1990)].
As described above, it has been explicated that denatured Lp(a)s have a possibility of deeply participating in the causation of arteriosclerosis, and an assay of the denatured Lp(a)s in the living body has hence been regarded as important.
A great number of methods have heretofore been reported as methods for assaying denatured Lp(a)s in the living body. For example, Fless et al. have established an EIA sandwich technique making good use of a likeness of Lp(a) to a product obtained by binding LDL with apo(a), in which an antibody which recognizes apoB, i.e., a main structural protein, and another antibody which recognizes apo(a) are used [Fless, G. M., Snyder, M. L. & Scanu, A. M., Lipid Res., 30, 651-662 (1989)].
As described above, it has heretofore been known to assay Lp(a). However, these methods are not intended to specifically detect denatured Lp(a)s in vital samples, which are considered to have a possibility of more deeply participating in the causation of arteriosclerosis.
On the other hand, among various kinds of lipoproteins, those in which the existence of denatured lipoproteins has been recognized include high density lipoprotein (HDL) and the like. However, no assay has been yet developed even on these denatured lipoproteins [Morel, D. W., Biochem. Biophys. Res. Commun., 200, 408-416 (1994); Maziere, J. C., Myara, I., Santus, R. & Mazier E. C., Atherosclerosis, 104, 213-219 (1993)].
SUMMARY OF THE INVENTION
Therefore, it is an object of the present invention to provide a method of selectively assaying denatured lipoproteins in vital samples.
In view of the foregoing circumstances, the present inventor has carried out an extensive investigation. As a result, it has been found that the fact that even when an antibody (hereinafter referred to as “anti-denatured lipoprotein antibody”) which recognizes a denatured lipoprotein is used, the denatured lipoprotein in the living body can not be assayed is attributed to the fact that the denatured site of the denatured lipoprotein is hidden in the interior of its lipoprotein particle, whereby the denatured lipoprotein is inactivated, and hence that if this denatured site is exposed to the surface of the lipoprotein particle, the denatured lipoprotein can react with the anti-denatured lipoprotein antibody to selectively assay it, thus leading to completion of the present invention.
In an aspect of the present invention, there is thus provided an assay of a denatured lipoprotein, which comprises exposing the denatured site of the denatured lipoprotein contained in a vital sample to the surface of its lipoprotein particle upon the reaction of an anti-denatured lipoprotein antibody with the vital sample containing the denatured lipoprotein.
According to the present invention, the denatured site of a denatured lipoprotein, which has been hidden in the interior of its lipoprotein particle, can be exposed to the surface of the lipoprotein particle to succeed in reaction with the anti-denatured lipoprotein antibody, whereby the denatured lipoprotein in the living body can be reliably assayed.
REFERENCES:
patent: 4376110 (1983-03-01), David et al.
patent: 5270165 (1993-12-01), Von Nostrand et al.
patent: 5766590 (1998-06-01), Founds et al.
patent: 0 361 468 A1 (1990-04-01), None
patent: 0484863 (1992-05-01), None
Nakamura et al, Handbook of Experimental Immunology, 1986 Blackwell Scientific Publications, pp. 27.1-27.20.*
Dubois et al., Journal of Immunological Methods, 125:215-223, 1989.*
Marcel et al. J. Biol. Chem, vol. 259
Kobayashi Noriko
Kondo Akira
Manabe Mitsuhisa
Nozawa Masayuki
Toda Naoko
Bacon & Thomas
Daiichi Pure Chemicals Co. Ltd.
Duffy Patricia A.
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