• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    1. Process for the determination of low density lipoproteine (LDL) in body fluids, characterised in that one adds to the high density lipoproteins (HDL) antibodies, which have been obtained with purified complete (HDL) fraction as immunogen, separates off insolubles formed and determines the (LDL) or one of its components in the supernatant.
    公开号:SU1296019A3
    申请号:SU833585413
    申请日:1983-04-22
    公开日:1987-03-07
    发明作者:Й. Цигенхорн；З. Шифер；Б. Дрэгер
    申请人:Берингер Маннхайм Гмбх (Фирма)；
    IPC主号:
    专利说明:

    The invention relates to a method and reagent for the determination of low density lipoproteins (LDL).
    The purpose of the invention is the acceleration of the method.
    Hypercholesterolemia and hypertriglyceridemia are beneficial for the occurrence of atherosclerosis and heart attack. The determination of cholesterol and tri- | glycerides in serum (blood) therefore refers to the most frequently performed test in a clinical clinical laboratory.
    The method is carried out as follows.
    Antibody - high density lipoprotein (LBP) is added to the test samples, the insoluble part is separated and in the supernatant
    Bones are determined by LDL or its compound 20 is used either in the form of LBP-antitank.
    It has been established that HDL antibodies all, without exception, precipitate lipoprotein fractions that interfere with LDL determination, along with the samim HDL, especially VLDL and chylomicrons, however, LDL does not precipitate. Both LDL, and HDL, VLDL, and quomicrons contain a proportion of protein, which in turn contains the same apoproteins, also in very different concentrations. Antibodies to HDL are quantitatively precipitated not only by HDL, but also by VLDL and hylomncrony, without affecting LDL
    Deposition of VLDL and chylomicrons of mole- 35 animals, respectively, comparable
    but accelerated using additionally known precipitating agents for these substances. -.
    The residual in the supernatant of the LDL reagent - fraction is then determined by the method usual for this purpose. The bound cholesterol contained therein is determined using methods known for this purpose. For example, it is determined by saponification with an alcohol solution of potassium hydroxide and chemical determination by Liebermann-Burchard. However, it is preferable to carry out an enzymatic determination using cholesterol oxidase and cholesterol-cleaving enzyme or enzyme systems, such as, in particular, cholesterol esterase.

    Yu
    960192
    this method Fahmann method, the proposed method by removing fractions of VLDL and chylomicrons prevent the appearance of opacities that interfere with the optical measurement of the amount of cholestenone or H „0
    , „2 in p
    color reactions. Therefore, the method is suitable in combination with colorimetric methods for determining cholesterol.
    In addition, instead of cholesterol or other LDL components, such as apolipoprotein B, phospholipids and trigly-15 periods, contained in the LDL fraction, the LDL fraction is determined using neofelinometric determination or turbidimetric determination.
    LDL antibodies according to the invention
    0
    cranks, in the form of defatted HDL antisera, or in the form of purified HDL antibodies. Also used are fragments of HDL antibodies, for example, Fab, Fabj and Fab fragments, or antibodies to apolipoproteins A, C and / or E, HDL or its fragments, or monoclonal HDL antibodies.
    The antibodies used are prepared using pure HDL or one of these apolipoproteins as immunogens. Rabbits and sheep are used to produce antibodies. In addition to those mentioned
    Cultures of cells are also used by creatures.
    The separation of immunoaggregates formed with the addition of HDL antibodies is carried out using conventional methods. If soluble HDL antibodies are used, the separation is carried out by centrifugation. If immobilized, carrier-bound HDL antibodies are used, the immune aggregates are separated by simply separating the liquid phase from a compact solid phase, such as solids,
    covered with antibodies. If apolipoprotein-derived antibodies are used as an immunogen of an antibody, they are used when used as a polypeptide protein as an immunogen.
    has a lipid membrane. For this, after the usual lipid removal (de-lipidation) and subsequent fractionation of the apolipoproteins, the selected apolipoprotein of fraction A, C
    and / or E again re-lipidate (relipidate).
    As immunogens, it is preferable to use a fully purified HDL fraction. Purification is carried out by isolation in an ultracentrifuge. Depending on the conditions, further purification is carried out through immobilized concavalin A using affinity chromatography or electrophoresis methods.
    Example 1. A. Obtaining purified LEI. After separation of the VLDL and LDL, a narrowly selected HDL fraction is selected in the ultracentrifuge (, 080- .1,210) (V.P. Skipski Lipid. Composition of Lipoproteins in normal and diseased states, b: Blood Lipids and

    uyuschie
    Intramuscularly Subcutaneously Intramuscularly Subcutaneously
    The first blood sample is taken after 45 days.
    B, 50 μl of serum is mixed with 150 μl of the antiserum prepared according to claim B. After incubation for 30 minutes at room temperature, the precipitate formed is separated by centrifugation (2 minutes at 10,000 d).
    50 {4CL of clear supernatant is mixed with 2 ml of reagent, which contains 0.1 mol / l Tris buffer, pH 7.7i, 0.05 mol / l magnesium apparatus, 1 mmol / l 4-aminophenazone 6 mmol / l phenolJ 4 mmol / l 3,4-dichlorophenol, 0.3% polyglycolic lipoproteins: Quantitation, Composition and Metabolism. Hrsg: Nelson, Wiley, New York, 1972, p. 471-583). Then they sediment twice more,
    accordingly, the fraction is floated at density x 1.080 and 1.210. The LDL fraction is purified by affinity chromatography through immobilized concavalin A (Febs. Lett, 1974,
    91, 174-198) or through Geon-Pevi SOP-B1 electrophoresis electrophoresis according to R. W. Mahley, K.S. Holcombe (1977), J, Lipid. Res 18, 314-324.
    B. Getting anti-short. Type of animals: sheep or rabbits.
    When using the immunogen obtained from A.A., the immunization schedule given in Table 2 is used. one.
    Also
    a junction of fatty alcohol esters, 400 units / l cholesterol esterase, 250 units / l cholesterol oxidase, and 200 units / l peroxidase.
    After incubation for 20 minutes at room temperature, the extinction of the sample is measured as compared to the blank value of the reagent (the blank value of the reagent contains 50 µl of antisera and takes into account the cholesterol content in the short-run).
    There is a single value for the reagent of LDL cholesterol a (mg / dL) 1.385 X.
    In tab. 2 shows the results.
    The NIH method serves as a standard method (after the separation of VLDL and chylomicrons, LDL precipitates in an ultracentrifuge). From the difference between the amount of cholesterol before and after precipitation, values for LDL-cholesterol are obtained.
    Manual of laboratory Operations, Lipid Research Clinics Program, Lipid and Lipoprotein Analysis, DHEW Publication N 65-628.
    权利要求:
    Claims (2)
    [1]
    Invention Formula
    A method for determining low density lipoproteins (LDL) in serum by precipitating impurities with a precipitating agent followed by determining the content of LDL in the supernatant, characterized in that, in order to accelerate the method.
    VNIIPI
    Order 631/64 Edition 777
    Random polygons pr-tie, the city of Uzhgorod, st. Project, 4
    table 2
    Example
    [2]
    2. A solution of 50 µl of VLDL, HDL, and LDL, respectively, of chilo-microns, is mixed with 150 µl of rabbit antiserum, which is obtained, using HDL as an immunogen. After centrifugation in the supernatant, the cholesterol content is determined as described in Example 1.
    In tab. 3 shows the results. Tabli.tsa 3
    As a precipitating agent, a purified fraction of antibodies against ly- are used. high density poproteins (HDL) or antisera to HDL, antibody fragments of HDL sludge: and defatted LBP anti-short, taken at a concentration of 10 -10 mol / l, and cholesterol, phospholipids and triglycerides are determined.
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    同族专利:
    公开号 | 公开日
    EP0092801A1|1983-11-02|
    FI831359A0|1983-04-21|
    JPH0375825B2|1991-12-03|
    DE3215310A1|1983-10-27|
    IE54314B1|1989-08-16|
    DK159840C|1991-04-29|
    EP0092801B1|1986-01-08|
    ZA832842B|1984-01-25|
    ES8402426A1|1984-02-01|
    FI77538C|1989-03-10|
    AT17404T|1986-01-15|
    US5407836A|1995-04-18|
    NO162094B|1989-07-24|
    FI831359L|1983-10-24|
    DK175383D0|1983-04-21|
    DE3361763D1|1986-02-20|
    DK175383A|1983-10-24|
    AU1343783A|1983-10-27|
    NO162094C|1989-11-01|
    FI77538B|1988-11-30|
    US5532172A|1996-07-02|
    HU190836B|1986-11-28|
    CA1211707A|1986-09-23|
    AU539195B2|1984-09-13|
    JPS58191967A|1983-11-09|
    IE830823L|1983-10-23|
    NO831428L|1983-10-24|
    DK159840B|1990-12-10|
    ES521757A0|1984-02-01|
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    优先权:
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