Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
2000-04-14
2003-06-10
Warden, Jill (Department: 1743)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S466000, C204S467000, C204S456000, C204S606000, C204S616000, C204S618000, C356S039000, C356S344000
Reexamination Certificate
active
06576106
ABSTRACT:
This application is based on Patent Application Nos. 11-107258 (1999) filed on Apr. 14, 1999 in Japan and 2000-107103 (2000) filed on Apr. 7, 2000, the content of which is incorporated hereunto by reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to separation and assay of lipids in a sample such as serum or plasma in the field of medical research, biological research and the like. More specifically, the present invention relates to a method for separating and assaying lipoproteins contained in serum or plasma by means of electrophoresis; an assembly for performing such a method; and a system including such an assembly for separating and assaying lipoproteins, using electrophoresis to estimate the modific ation of lipoprotein by detecting quantitative and qualitative abnormalities of apoprotein in the lipoprotein.
2. Description of the Related Art
Lipoprotein in serum or plasma is a complex of lipid and protein, which includes cholesterol, phospholipid, and neutral fat. Therefore, an abnormality of lipoprotein reflects an abnormality of lipid metabolism, which can be estimated by a process including the steps of separating lipoproteins contained in serum or plasma and performing quantitative and qualitative analyses of the lipoprotein fraction with respect to any abnormalities thereof. Such a method for separating and assaying lipoprotein is one of the important clinical tests for the diagnosis and treatment of a disease affecting lipid metabolism such as hyperlipemia, coronary arteriosclerosis, hypothyroidism, obstructive liver disease, diabetes mellitus, and renal insufficiency.
As an approach to fractionating lipoproteins in a sample, there have been known methods using ultracentrifugal and electrophoretic techniques. The electrophoresis method uses an agarose film, a cellulose-acetate film, or a polyacrylamide gel(PAG) as a plate.
In the ultracentrifugal separation method, lipoproteins in serum can be separated from each other with respect to their respective specific gravities. The resulting fractions are generally referred as: High Density Lipoprotein (HDL); Low Density Lipoprotein(LDL); Very low Density Lipoprotein(VDL); and Chylomicron(CM), respectively.
Regarding the ultracentrifugal separation method, however, it takes much time to prepare a sample to be subjected to an ultracentrifugal machine and to fractionate such a sample. In addition, even finer fractionation of the sample is necessary for finer and detailed separation of lipoproteins to understand conditions during the separation. For this reason, it is difficult to perform the method of separating and assaying lipoprotein by means of ultracentrifugation in a clinical examination as a routine way to detect any abnormality in the target fraction or characterize the results of the separation for a specific disease.
In the electrophoretic separation method, on the other hand, lipoproteins in the solution can be fractionated mainly with respect to differences in their electrophoretic mobilities. In this case, the conditions of lipoprotein separation can be understood visually. For this reason, electrophoretic separation is often used in routine examination for separating and assaying lipoproteins in a sample. It is noted that the results of both electrophoretic and ultracentrigual procedures for separating lipoproteins show good correlation with each other. When a lipoprotein is fractionated using the electrophoretic technique, lipoprotein is separated into the fractions of &agr;(HDL), pre &bgr;(VLDL), &bgr;(LDL), and the point of origin(cylomicron). These fractions correspond to lipid species visualized by a lipid stain such as fat red 7B for estimating the amount of lipid in each fraction. For the convenience of one of skill in the art, the name of each lipoprotein fraction obtained by the prior art ultracentrifugal separation is described in the parentheses attached to each fraction name of the above electrophoresis.
The fundamental structure of lipoprotein is composed of a core part formed of a neutral fat(triglyceride) and cholesterol ester, a single hydrophilic lipid membrane that covers the core part, and one or more apoproteins adhered to the surface of such a membrane. The type of the apoprotein differs for each fraction of lipoprotein. That is, mainly, apoproteins in HDL are apoprotein A-I, A-II, A-IV, C-I, C-II, C-III, and E. Apoproteins in VLDL are apoprotein C-I, C-II, E, and B-100. Apoprotein in LDL is apoprotein B-100. Apoproteins in chylomicron are apoprotein A-I, A-II, A-IV, C-I, C-II, C-III, E, and B-48. In the electrophoretic process, a difference in the isoelectric point of each apoprotein contained in the lipoprotein is reflected as a difference in mobility of each fraction.
Since arteriosclerosis is a cause of adult diseases such as coronary arteriosclerosis, it is an important problem in basic and clinical medicine to prevent or treat arteriosclerosis. In particular, because of the recent increase in the number of patients exhibiting arteriosclerosis at young ages, and because patients with diabetes are liable to develop arteriosclerosis, management of lipid in serum is required to be stricter. For example, from epidemiological investigations conducted by the Framingham Institute and the like, it has been shown that the serum cholesterol value, especially the cholesterol value of low-density lipoprotein(LDL), is considered to be a most important,factor in atherosclerosis, which is a cause of coronary artery diseases.
The basis of cholesterol in LDL causing arteriosclerosis is that a macrophage is foamed by taking in modified LDL through a particular receptor, and the remnant adheres onto the vascular wall. Therefore, a screening examination of the modified LDL is necessary.
Here, “modified LDL” is a generic name of variously modified LDL such as oxidized LDL, acetyl LDL, saccharized LDL, and MDA-LDL(malondialdehyde LDL). Further, the term “modification” used in the present specification means modification in a broad sense as normally used by one of skill in the art for modification of lipid or protein. For example, for a lipoprotein component contained in a blood sample, it includes not only modification caused by changes in physical or chemical environment in vivo such as a lipid metabolic anomaly in the body but also modification caused by changes in physical or chemical environment in vitro after sampling, which will be obvious to one of ordinary skill in the art without describing practical examples.
In general, it is said that small-dense LDL is liable to be doxidized. Therefore, as a screening examination of modified LDL, several methods have been tried to detect the ratio of small-dense LDL in lipoprotein. For example, there is a method by the electrophoretic process using PAG having a predetermined pore size and a high molecular sieving effect of PAG, first the smallest-particle (the largest mobility) HDL is separated, and then LDL, VLDL, and chylomicron are separated in order. Specifically, in the lipoprotein fractionation by the PAG electrophoretic process, lipoprotein is fractionated according to the difference in sizes of the particles contained in the lipoproteins, rather than by the difference in isoelectric points of particles contained in the lipoproteins.
For detecting the ration of small-dense LDL using the PAG electrophoretic process, there is a method using the relative distance between each fraction with respect to the position of the VLDL fraction. In this method, electrophoresis using a polyacrylamide gel is carried out, where the electrophoretic migration distance from the central position of the VLDL fraction to the central position of the LDL fraction is defined as “x”, and the electrophoretic migration distance from the central position of the VLDL fraction to the central position of the HDL fraction is defined as “y”; a relative electophoretic migration distance(Rf value or Rm value) is determined by the ratio(x/y) of these distances. The obtained value is compared with a value obtained for a normal standard sample in
Axelrod Nancy J.
Brown Jennine
Frank Robert J.
Helena Laboratories Co., Ltd.
Venable LLP
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