• 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:
    This invention relates to clinical biochemistry, namely, a reagent for determining lipase, especially by the turbidimetric method. The purpose of the invention is to increase the sensitivity of the reagent for lipase determination. The dry turbidimetric lipase reagent contains a protective colloid and liquid triglyceride (in which the fatty acid residues have 8–20 carbon atoms and 1–8 carbon – carbon double bonds). As a protective colloid, monomeric pentose or hexose, or a polymer containing pentose or hexose groups, or solid polyethylene glycol is used. The protective colloid also includes sugar alcohol, polyethylene glycol. Liquid triglyceride also contains triolein, glycerol-tri-octanoin, glycerol tri-2-octanoin, glycerin-tri-tetradelano-in, glycerol-tri-2,4,6,8-tetradecanoin, glycerin-tri-eicosanoin. As a protective colloid, the reagent further comprises bovine albumin. The reagent additionally contains sodium azide, potassium or sodium salt of bile acid, colipase, buffer substance pH 6.0-10.0 from the group of di- or tri-ethanol buffer substances, activator (from the group of chlorides). The reagent has an increased sensitivity and has a higher reproducibility and stability of the results due to the stabilization of extinction during photometric measurement. CO 05 vl 00 O5 4j
    公开号:SU1367867A3
    申请号:SU802872054
    申请日:1980-01-23
    公开日:1988-01-15
    发明作者:Нойманн Ульрих；Книч Карл-Вольфганг；Цигенхорн Йоахим；Редер Альберт；Цвец Вернер；Кремер Вернер
    申请人:Берингер Маннхайм Гмбх (Фирма)；
    IPC主号:
    专利说明:

    The invention relates to clinical biochemistry, in particular to a reagent for determining lipase, especially by a turbidimetric method.
    The purpose of the invention is to increase the sensitivity of the reagent for lipase determination.
    The method is carried out as follows.
    - A dry reagent for the turbidimetric determination of lipase, containing a protective colloid and liquid triglycerol, while as a protective colloid it contains monomeric pentose or hexose, or a polymer containing 2-10 pentose or hexose groups, or solid polyethylene glycol as a liquid triglyceride use is one in which the fatty acid residues have 8–20 carbon atoms and 1–8 carbon-carbon double bonds, additionally contains an alkali metal azide, a bile acid potassium salt, a colipase, urea, a buffer agent ETS pH 6.0 in the following ratio
    Liquid triglyceride 0,47-6,58
    Kaliev salt

    about
    13.5-34.4 0.0065-0.05
    0.36-0.87
    3.82-28.29
    4.85-12.6
    0.22-3.25
    Rest
    Preferred examples of suitable polyhydroxy compounds are mannitol and similar polyatomic.
    alcohols, glucose oligosaccharide, mannose, maltoheptose, polyethylene glycol with an average molecular weight of between 3,500 and 7,000, and others.
    Protective colloids are amino acids such as apanine, as well as gum arabic. The preferred amount of protective colloid, respectively, of the mixture of protective colloids is 50-70% by weight. A mixture of sugar alcohol n polyalkylene glycol has proved to be particularly suitable. As bile acids, known surface active bile acids are taken into account, as
    cholic acid, taurocholic acid, deoxycholic acid, respectively, their alkali metal salts, in particular the sodium salt. The preferred amount is
    10-15 wt.%.
    A further essential part of the proposed reagent is a colipase. Particularly suitable is free of impurities.
    colipase The preferred amount is 0.003-0.01% by weight.
    As preservatives are used such that do not violate the enzymatic activity of the lipase to be determined. Alkali metal azides are particularly suitable.
    fishing, especially sodium azide. Od
    As a triglyceride, both natural and synthetic trigliderides with fatty acid residues between 4 and 22 C-atoms are used. Tributyrin is also suitable. Most preferred are triglycerides with long-chain unsaturated fatty acid residues, especially triglycerides, the fatty acid residues of which have 8–20 ° C atoms and 1–8, mostly 1-3, carbon-carbon double bonds. Due to its easy availability, triolein is particularly preferred, and olive oil can also be used. The preferred amount of triglyceride is 0.2-2.0 wt%.
    Polyhydroxy compounds, serum albumin, polyvinyl pyrrolidone, and solid poly (ethylene oxide) are used as a protective colloid.
    However, other preservatives are also suitable, such as thiozide, and other sulfur-containing preservatives. The preferred amount of preservative is 0.2-0.7% by weight.
    The reagent also contains urea, preferably 10-15 wt.%.
    All known buffers are suitable as a buffer substance and, as part of the proposed reagent, can set the pH between 0.6 and 10.5. PH range preferred
    between 8.3 and 9.5. Examples of a suitable buffer are diethanrlamine buffer J, triethanolamine buffer, Tris buffer, and Coo (1 buffer as Hepes buffer (suitable for addition before lyophilization). Tara buffer and Bicine. Preferred is a tris buffer. A preferred amount of buffer is 3 and 10% by weight. Further activators are calcium ions.
    with deoxycholic acid, they give insoluble compounds, in the presence of calcium, tauro deoxycholic acid is preferred as bile acid, since it allows higher concentrations of calcium in the range of 1-5 mmol.
    The proposed reagent also contains an activator for lipase. Lipase activators are known. Chlorides are preferred, especially sodium chloride, but also other alkali metal or alkaline earth metal chlorides, since they do not lead to the formation of insoluble compounds with other components of the reagent or sample. Magnesium ions also act as an activating agent.
    Dry reagent is obtained by lyophilization of one of the components of the emulsion obtained by the usual method. In this case, the buffer substance, urea, and in this case also part of the protective colloid and preservative, as well as the activator, are predominantly added to the dry emulsion only after freezing. The lyophilized emulsion can be dispensed with a conventional method, for example, by incorporating all of the constituents into an aqueous solvent, emulsifying using conventional methods, such as ultrasound, etc. and closing freezing at about -40 ° C and drying in a vacuum at
    at
      t
    normal conditions ().
    When lyophilization, the bile salt, colipase, at least 20 wt.% Of the protective colloid, at least part of the preservative and in this case the activator must be added. The preferred method for preparing the aqueous emulsion determined for lyophilization is that the above components, excluding triglyceride, are dissolved in water and then, with stirring, a solution of triglyceride in a volatile organic solvent is injected into the solution with a strong jet. In particular, aliphatic alcohols and ketones with 1–4 carbon atoms are suitable as a volatile organic solvent.
    The remaining constituents of the proposed reagent, namely the buffer substance, urea and in this case the rest of the protective colloid, preservative and activator, can be added.
    to the lyophilisate immediately after its preparation or a little later.
    about
    0 5
    Example 1.0.06 g CaCl
    2
    0
    five
    22.72 g of sodium deoxycholate, 25.35 g of polyethylene glycol with a molecular weight of 4,000, 1.1 g of sodium azide, 0.02 g of colipase and 50.8 g of mannitol are dissolved in 1.0 l of distilled water. Under stirring with a pressure of 2 bars, 1.42 g of triolanine dissolved in 32 ml of n-propanol is injected into the resulting solution through a nozzle with a diameter of 1.5 mm. The resulting triolein emulsion
    5 is frozen at -40 ° C and dried by LIOFSHI of ation.
    8.95 g of sodium deoxycholate, 7.25 g of Tris, 1.20 g of Tris-HCl, 7.5 g of polyethylene glycol with a molecular weight of 4,000, 1.97 g of NaCl, 16.2 g of urea and 56.9 g of mannitol are mixed each other in a state of 2 ma. the resulting buffer mixture using a spiral mixer is mixed with 1 wt.h. lyophipisate in a homogeneous reagent.
    To determine the lipase, 150 ml of this reagent is added with 2.5 ml of water, mixed with O, 1 ml of the product and measured photometrically at
    365 nm at 25 ° C relative to water. Suitable samples include, for example, short blood, the contents of the duodenum, urine and
    5 other body fluids,
    The correctness of the method is verified by comparison with the Ricca titrametric method. When studying the 60 series of human lipase of different activity, the following data was obtained: correlation: (x Rick, y - test according to the invention) at 0.93, x-20 ,, g-0.96.
    The linearity of the dough remains up to about 1300 Ricca units.
    One unit according to Ricca 1 mol FFA / min - Iv, FFA - liberated fatty acids.
    Example 2, 1,500 g of mannitol, 0.900 g of polyethylene glycol with a molecular weight of 4000, 0.524 g of sodium deoxycholate, 1.5 ml of colloid and 8 mg of sodium azide are dissolved in 50 ml of distilled water B while stirring through a fine nozzle Inject 150 mg of triolein dissolved in 5.0 ml of propanol. Half 20
    25
    valuable emulsion is frozen at and lyophilized.
    3.50 g of urea, 3.73 g of sodium deoxycholate, 0.40 g of NaCl, 0.10 g of sodium azide, 2 mg of calcium carbonate, 2.355 g of Tris, and 0.200 g of Tris-HCl in-. mixed vigorously with each other. To this is added crushed dry lyophilisate and homogeneously stirred.
    A complex mixture of 8.219 g contains; mannitol 1,500 g; polyethylene glycol 0.900 g total 2.4 g 29.2 wt.% 5 sodium deoxycholate 4.254 g 51.7 wt.%; colipase 1.5 mg; sodium azide 9 mg; Tris buffer 11.355 g; Tris-HCl 0,200 g amount of 1, 9 wt.%; urea 3.5 g 42.5 wt.%.
    37 mg of this mixture are dissolved in 2.5 ml of distilled water and measured photometrically with 200 ml of sample (serum) at 340 nm and 25 or relative to water or air. From the difference in attenuation in minutes, lipase activity is determined,
    Example 3. 8.41 g of albumin: cattle shorts, 12.2 g of sodium deoxycholate, 0.02 g of sodium azide and 0.034 g of colipase are diluted with 100 ml of distilled water. With stirring, a solution of 3.5 g of triolein in 7 ml of propanol is injected into this solution under pressure. The resulting emulsion is frozen at -40 ° C and lyophilized.
    17.4 g mannitol, 4 g solid polyethylene glycol, 7 g urea, 7.47 g sodium deoxycholate, 0.82 g HaCl, 0.2 g sodium azide, 2.71 g Tris, and 0.4 g Tris-HCl grind and intensively mixed with each other. To this is added the ground dry emulsion, and the whole is mixed homogeneously.
    90 mg of this powdered reagent is added to 2 ml of distilled water and, after dissolution, is mixed with 100 ml of sample (short). The reaction is carried out at 340 (365) nmH photometrically.
    1367867
    the emulsion is frozen at and lyophilized.
    17.4 grams of mannitol, 4 grams of solid poly-ethnn glycol, 7 grams of urea, 7.47 grams of sodium deoxycholate, 0.82 grams of NaCl, 0.2 grams of sodium azide, 2.71 grams of Tris, and 0.4 grams Tris-HCl is crushed and intensively mixed with each other, to which is added a powdered dry emulsion and homogeneously mixed.
    90 mg of this powdered reagent are added to 2 ml of distilled water and, after dissolving, are mixed with 100 ml of sample (short). The reaction is carried out at 340 (365) nm Nl
    photometrically.
    Example5. 4.205 g polyethylene glycol with a molecular weight of 4000,
    4.205 g of cattle albumin, 1.22 g of sodium deoxycholate, 0.02 g of sodium azide and 0.034 g of colipase are dissolved in 100 ml of distilled water. While stirring, a solution of 3.5 g of triolenine and 7 ml of propanol is injected into this solution under pressure. The resulting emulsion is frozen at −40 ° C. and lyophilizate.
    17.4 g of mannitol, 4 g of polyethylene glycol with a molecular weight of 4000, 7 g of urea, 7.47 g of sodium deoxycholate, 0.82 g of NaCl, 0.2 g of sodium azide, 2.71 g of Tris, and 0.4 Tris-HCl is crushed 3g and intensively mixed with each other. To this, a powdered dry emulsion is added and mixed homogeneously,
    90 ml of this powdered reagent is added to 2 ml of distilled water and, after dissolving, is diluted with 100 ml of sample (serum). The reaction is carried out photometrically at 340 (365) nm N.
    PRI me R 6, the Formulation is given in 45 wt.%:
    Triglyceride
    Protective colloid
    Kaliev salt
    bile acid
    Colipase
    Alkaline Azide
    0.47 68.00
    50
    13.48 0.0066
    Example4. 8.41 g of alanine, 1.22 g of sodium deoxycholate, 0.02 g of sodium azide and 0.034 g of colipase are dissolved in 100 ml of distilled water, and a solution of 3.5 g of triolein in 7 ml of propanol is injected into this solution under pressure. . Received
    photometrically.
    Example5. 4.205 g polyethylene glycol with a molecular weight of 4000,
    0
    25

    thirty
    55
    4.205 g of cattle albumin, 1.22 g of sodium deoxycholate, 0.02 g of sodium azide and 0.034 g of colipase are dissolved in 100 ml of distilled water. While stirring, a solution of 3.5 g of triolenine and 7 ml of propanol is injected into this solution under pressure. The resulting emulsion is frozen at −40 ° C. and lyophilizate.
    17.4 g of mannitol, 4 g of polyethylene glycol with a molecular weight of 4000, 7 g of urea, 7.47 g of sodium deoxycholate, 0.82 g of NaCl, 0.2 g of sodium azide, 2.71 g of Tris, and 0.4 Tris-HCl is crushed 3g and intensively mixed with each other. To this, a powdered dry emulsion is added and mixed homogeneously,
    90 ml of this powdered reagent is added to 2 ml of distilled water and, after dissolving, is diluted with 100 ml of sample (serum). The reaction is carried out photometrically at 340 (365) nm N.
    PRI me R 6, the Formulation is given in 45 wt.%:
    Triglyceride
    Protective colloid
    Kaliev salt
    bile acid
    Colipase
    Alkaline Azide
    metal
    Urea
    Buffer substance
    Activator
    0.47 68.00
    50
    13.48 0.0066
    0.36 10.75 5.61 1.33
    PRI me R 7 Formulation given in May.%;
    Triglyceride 6.58 Protective Colloid, 56.05
    Kaliev salt
    bile acid 16,34
    Kolipaza0.06
    Alkaline Azide
    metal0.41
    Urea13,16
    Buffer substance5,85
    Activator, 1, 54
    The proposed reagent has a higher sensitivity in comparison with the known, and also has a higher reproducibility and stability of the results due to the stabilization of extinction during photometric measurement.
    权利要求:
    Claims (1)
    [1]
    Invention Formula
    The reagent for the turbidimetric determination of lipase containing pentose, hexose, sugar alcohol, polyethylene glycol or their mixture as a protective colloid, and liquid triglyceride, characterized in that, in order to determine the sensitivity of the reagent, the reagent contains liquid triglyceride.
    one
    367867 in
    eight
    ten
    15
    20
    , glycerin-tri-octanoin, glycerin tri-2-octanoin, glycerin-tri-tetradeloanine, glycerin-tri-2,4,6,8-tetradecanoin, glycerium-tri-eikozoan or glycerin tri-2,4,6,8,10,12,14,16-eicosanoin, additionally contains cattle albumin as a protective colloid, and in addition, the reagent additionally contains sodium azide, potassium or sodium salt of bile acid, colipase, boo-. ferric substance pH 6.0-10.0, selected from the group of: di- or tri-ethanolic buffer substances, activator, selected from the group: sodium chloride, calcium, magnesium, with the following ratio of components,% by weight:
    Liquid triglyceride 0,47-6,58
    Kaliev salt
    five
    bile acid
    Colipase
    Urea
    Alkaline azide
    metal
    Buffer substance
    Activator
    Protecting colloid
    13.5-34.4
    0,0065-0,05
    3.82-28.3
    0.36-0.87 4, "5-12,6 0,22-3,25
    Rest
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    法律状态:
    优先权:
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