• 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:
    Latex reagents for quantitative assay of a variety of steroid hormones or metabolites thereof which are contained in body fluid or excreted fluid of human beings comprise latex particles which are immunologically sensitized with a conjugate selected from a variety of steroid-serum albumin conjugates in which the steroid to be detected is bonded to serum albumin in a very small ratio ranging from 0.5 to 7 molecules per 1 molecule of the serum albumin used. Sensitization of latex particles with such a conjugate is performed using a limited amount dependent on the bonding ratio of steroid molecules per molecule of serum albumin in the conjugate and the particle size of the latex used. The latex reagents show very high sensitivity amounting to 0.2-0.04 nmole steroid equivalent/ml.
    公开号:SU1213975A3
    申请号:SU782629447
    申请日:1978-06-21
    公开日:1986-02-23
    发明作者:Огаса Кацухиро；Кубояма Морио；Саито Минору；Кудо Цутому；Харада Есицугу；Кавасири Акио；Такахаси Еидзи
    申请人:Моринага Милк Индастри Ко,Лтд (Фирма)；
    IPC主号:
    专利说明:

    This invention relates to medicine, in particular immunology.
    The purpose of the invention is to increase the sensitivity of the target product,
    Example 1. The following substances can be used as a steroid: follicular hormone (estrogen) J hormone of the corpus luteum (progesterone), male sex hormone (17-ketosterond) adrenocortical hormone (17-hydroxyxy-rticosteroid) and steroid, which can be a large number of metabolites listed steroi, valuable hormones.
    From syoritochink albumin, any purified short-lived albumin can be used - bovine gray-haired albumin (in SA), horse albumin (ES S) S sheep serum albumin (SS A), rabbit serum albumin (RSA) s human serum albumin (PZA).
    Among these substances, BSA and RSA are most preferred. Among the latex particles, polystyrene latex particles, polybutadiene latex particles, styrene-butadiene copolymer latex particles can be used. The particle size may lie in the Dnapaplane O, 234-0, 721 microns,
    10 mol steroid-glucuronide, hemuccinate steroid8. or steroid-10-carboxymethyl oxime per t mole of serum albumin is dissolved in dinetilformamnde. The same number of moles of tri-n-butylamine as a steroid hormone is added to this solution as an auxiliary activator5 and the mixture is rehumidified. To the solution obtained is the same number of moles of isobenate formate as sterotnogo hormone as activator, and the mixture is stirred about
    The resulting solution is designated as liquid A.
     Getting liquid B.
    Serum albumin is dissolved in deionized water. Adjust the pH with H.ijacTBopoM NaOH to SsO-IOjO. To the several solutions obtained, various amounts of dimethylformamide are added and 9 samples of liquid B are prepared that contain different amounts of RSA. The amount of dimethylformamide contained in each of the portions of fat. B, 139752
    set in such a way that the offensive amount of dimethylformamide contained in liquids A and B was equal to the amount of deionized water in liquid B. Preparation of the conjugate. Each of the liquids. A is added dropwise to each of the liquids B over one and a half hours with stirring.
    0 1 N NaOH solution is added to these solutions and the pH is adjusted to 8.0-10.0, after which each of the solutions is further stirred for 3 hours and as a result 9 samples of estriol-1b to -β-glucuronide-K8A- are obtained conjugate solutions
    Each of these solutions is dialyzed against distilled water, acetone in an amount of 2 hours, acetone per 1 part of the solution is added to the dialyzed solutions, and after stirring, 1N HCl solution is added to precipitate the conjugate. Precipitated conjugate in each
    The 2S from the solutions was separated by centrifugation for 5 minutes at a rotational speed of 10,000 rpm. Deionized water is added to each of the separated conjugates and, after setting, the pH is 8.0 with 1N. NaOH, each of these solutions is dialyzed against distilled water to remove acetone. The result is 9 kinds of conjugates,
    3 "; having different steroid binding numbers. The preparation of the conjugate is carried out at a low temperature (). i,
    Sensitization of latex particles. A suspension of latex particles is prepared by washing and / or diluting the latex particles with an appropriate buffer solution (pH 7e2-8.6) and the steroid-serum albumin conjugate is added at the desired concentration.
      into this suspension. The mixture was held at 37 ° C for 2 hours while stirring to obtain a suspension of sensitized latex particles. The resulting suspension is centrifuged.
    The 0 n precipitate is separated, then progressed with a buffer solution. Centrifugation and prosthesis are repeated several times. The pellet is re-suspended in a buffer solution to obtain
    55} w suspensions of latex particles sensitized by the conjugate. The amount of conjugate used to sensitize the latex particles.
    3
    is 1.8–7.2 mg per 100 mg of latex particles; the amount of conjugate used for sensitization of latex particles depends on the particle size (for the sensitization of particles of 0.234 μm and 0.721 μm in size, 72 mg and 18 mg per 1 part of latex particles).
    Antibody production.
    A steroid-serum albumin conjugate is obtained by a steroid reaction in the state it has in a liquid medium of a living organism or in an excreted liquid, or a steroid that has an immunological cross-reactivity with serum albumin, and then a semi-conjugate is administered, for example , by injection to a nutritional supplement, the serum of which differs from that used to prepare the conjugate. Get the serum.
    PRI mme R 2. Liquid A is prepared by dissolving 50 mg of estriol-16-oi-gl curonide in 10 ml of dimethylformamide, 26 ml of tri-n-butylamine and then 14 ml of isobutyl chloroformate are added with stirring.
    2.33 g of RSA is dissolved in 6 ml of deionized water containing 1.0 ml of NaOH, after which 50 ml of dimethylformamide is added. Get liquid B.
    Liquid A is added dropwise to the liquid and the mixture is stirred for 1 hour. Then 1.0 ml of 1N and NaOH solution is added and stirring is continued for another 3.5 hours. After the solution is dialyzed with deionized water for 1 hour. 2 parts of acetone and a homogeneous solution of HCl are added to precipitate the synthesized estriol-16 (x; -glucuronide-RSA conjugate. The resulting precipitate is separated by centrifugation at a rotation speed of 10,000 rpm for 5 minutes.
    Deionized water was added to the separated precipitate, the pH was adjusted to 8 with 1N. NaOH solution and dialyzed with deionized water to remove acetone. 1.78 g of conjugate is obtained (yield 75%). The process is carried out at 6 ° C. The binding number of estriol-1BL-glucuronide, measured by UV-Absorber39754
    tion and the method of dinitrophenylation are 2.2 and 1.9, respectively. 76 mg of the conjugate is dissolved in 400 ml of 40 mM veronal buffer
    5 (containing 150 mM NaCI, pH 7.8), 10 ml of the suspension (concentration 10%) of 0.234 µm polystyrene latex particles are added and the resulting mixture is held for
    to 2 hours at 37 ° C for sensitization. The sensitized latex suspension is centrifuged at a rotational speed of 4000 rpm for 20 minutes, the resulting sediment is suspended in 200 ml of veronal buffer and centrifuged twice in the same modes. The resulting precipitate is re-suspended in 50 ml of buffer and then sodium azide is added to a concentration of 0.1%. 50 ml of suspension (density 2%) of latex particles sensitized with estriol-16y-glucuronide-RSA are obtained.
    As a result of mixing in a pre-25 metute 0.1 ml of the obtained suspension of sensitized latex particles and O, 1 MP of a dilute solution of anti-estriol-16p |; -glucuronide-B5A, (diluted to obtain a titer equal to 0.04 nmol of estriol 16g-glucuronide-eq / ml), agglutination is observed for 1-2 minutes. However, in the case when the antibody is diluted to obtain a titer equal to 0.02 nmol of estriol-16 ° C-glucuronid.eq./ml, agglutination is not observed and the titer of the latex reagent obtained in this example is defined as 0.04 nmol of estriol -16cc-glucuronide-eq./ml.
     Example Using dehydro-epiandrosterone (DNEL) and BSA, 8 samples of conjugates are obtained, having different steroid binding numbers, ranging from 0.5-18.3.
     5 0.75 g of DNEA and 0.69 g of o-carboxymethylhydroxylamine hydrochloride are dissolved in 20 ml of ethyl alcohol. The mixture obtained is made alkaline by adding 2 ml of 6.4 M solution of suk50 sodium cinnate. The solution is heated under reflux for 1 hour and 5% is added to a final concentration.
    After washing the obtained
    55 solution of ether, the aqueous layer is acidified with concentrated HC1 and the precipitate is separated, which is recrystallized from ethanol. Get
    five
    51
    0.6 g DNEA-17- (0-carboxymethyl) - oxime (DNEA-SMO).
    To 1 ml of a 10% suspension of latex particles with a size of 0.721 microns was added 4 MP of 20 mM phosphate buffer solution pH 7.2, containing 150 mM NaCl. After thorough mixing, the solution is centrifuged at 4,000 rpm for 20 minutes. The resulting pellet is suspended in 5 ml of the same buffer, the suspension is centrifuged under the same conditions and the pellet is separated.
    Antiserum is obtained from rabbits immunized with a DNEA-CMO-ESA conjugate.
    Conjugate was prepared as follows.
    Liquid A is obtained by adding 0.5 g of DNEA-SMO in 100 MP of dimethylformamide, after which 0.33 mp of tri-n-butyl amine and 0.18 ml of isobutyl chloroformate are added with stirring. 18.0 g of BSA is added to 600 ml of deionized water, after which 500 ml of dimethylformamide is added, resulting in a liquid B,
    To this liquid, previously prepared liquid A is added dropwise and, after stirring for 1.5 hours, the pH of the mixture is set to 9.0 with 1N. NaOH solution and stirring is continued for 3.5 hours. The resulting solution is dialyzed with tap water and then for 1 hour, 2 hours of acetone are added to the solution. After that, the solution is added homogeneously mixed with 1N. HCl solution to precipitate the synthesized conjugate and the resulting solution is centrifuged at a speed of 5000 rpm for 20 minutes. Dionized water is added to the separated precipitate and, after adjusting the pH of the solution to 8 with 1N NaOH, the resulting solution is dialyzed with tap water to remove acetone. 13.0 g of DNEA-CMO-BSA conjugate are obtained (yield about 70%). The number of steroid binding (DNEA-SMO) of such a conjugate, measured by the method of dinitrophenylated, is 4.1.
    68 mg of the resulting conjugate is dissolved in 100 ml of 100 mM glycine buffer solution (containing 130 mM NaCl and 0.1% sodium azide, pH 8.2) and 10 ml of suspension is added.
    75-6
    (concentration 10%) polystyrene latex particles, then the resulting mixture is dialyzed with a buffer solution at room temperature for 5 days to sensitize. The sensitized latex suspension thus obtained is centrifuged at a rotational speed of 4000 rpm for 20 minutes and, after suspending the precipitate in 200 ml of the indicated buffer solution, this suspension is centrifuged and washed with the indicated buffer solution under the same conditions. The pellet is resuspended in 50 ml of the same buffer. 50 ml of suspension (concentration of 2%) of sensitized DNEA-CMO-BSA latex particles are obtained.
    As a result of mixing on a slide glass 0.05 ml and nmol / m DNEA with 0.05 ml of diluted antiserum and subsequent addition of O, 1 ml of the resulting suspension of sensitized latex particles, even after 10 minutes, no agglutination is observed. In the case when 0.1 ml of the suspension of sensitized latex particles is added to a mixture consisting of 0.05 ml of 0.02 nmol / ml of DNEA and 0.05 ml of the indicated diluted anti-short, agglutination is observed for 2-3 minutes. The latex reagent obtained in this example was taken as 0.05 nmol DNEA equiv. / ml.
    EXAMPLE 4 Liquid A was prepared by dissolving 1.1 g of testosterone-17-glucuronide in 200 ml of dimethylformamide, to the resulting solution, with stirring, 0.52 MP of tri-n-butylamine was successively added and then 0.28 ml isobutyl chloroformate.
    Liquid B is prepared by dissolving 46.6 g of RSA in 1.2 L of deionized water and after adjusting the pH to 9.0 with 1N. NaOH solution is added with 1.0 l of dimethylformamide. Analogously to Example 3, about 39.0 g of testosterone-17-glucuronide-RSACT-17-G-RSA are obtained. The yield is about 82%. The T-17-G steroid binding amount in this conjugate, measured by UV absorption and dinitrophenylation, is 2.3 and 2.1, respectively.
    Then to 100 ml of the suspension (concentration 10%) of polystyrene latex particles, having a particle size of 0.721 µm, add 400 NP of 20 mM phosphoric acid buffer solution containing 150 mM NaC1 (pH 7.2), after which the suspension is centrifuged at a rotational speed 4000 O & MIN for 20 min. The precipitate obtained in this way is suspended in 500 ml of an And-buffered solution and then, after centrifuging this suspension, the precipitate is separated. To the separated precipitate, 500 ml of a buffer solution containing 180 mg of the previously obtained conjugate is added. After the obtained suspension was centrifuged for 2 hours, then it was washed twice with buffer solution, and the centrifugation process was repeated. The resulting precipitate is re-suspended in 500 m of the buffer solution and sodium azide is added to a final concentration of 0.1%. As a result, 500 ml of suspension (concentration of 2%) of latex particles sensitized with T-17-G-RSA are obtained.
    Antibodies T-17-G-RSA are obtained and then the short is diluted with a buffer solution to obtain a titer of 0.05 nmol T-17-G eq / ml. On a glass slide, 0.1 ml of the diluted antiserum is mixed with O, 1 m of the resulting suspension of latex particles, and agglutination is observed for 1–2%. However, when antibodies are diluted to obtain a titer of 0.02 nmol of T-17-G-equiv / ml for 5 minutes, no agglutination is observed. Thus, the titer of the latex reagent obtained in this example is taken equal to O, 05 nmol T-17-G-eq. / Ml.
    Example 5. Liquid A is prepared by adding a number of moles of sodium hydrocortisone-21-hemisuccinate to the same number of moles of concentrated hydrochloric acid, after which the mixture obtained is concentrated and washed with water. Concentration and washing processes are repeated several times and then the substance is completely dried. 0.5 g of the obtained dry substance is dissolved in 100 ml of dimethylformamide, after which 0.26 ml of tri-n-butylamine and O, 14 ml of isobutyl chloro are successively added with stirring - formate.
    In the meantime, liquid B was prepared by dissolving 17.6 g of HSA in 600 ml of deionized water and the pH was adjusted to 9.0 with 1 and. NaOH solution and add 500 ml of dimethylformamide. Then in the same manner as in Example 3, 13.6 g of hydrocortisone-21-hemisuccinate-HSA (HC-21-HS-HSA) are obtained, yield about 75%.
    The number of steroid binding (HC-21-HS) in such a conjugate, as measured by dinitrophenylation, gives a value of 3.0.
    Then, 200 ml of a 40 mM veronal buffer solution containing 150 mM NaCI (pH 7.8) is added to a 50 MP suspension (concentration of 10% polystyrene latex particles), having a particle size of 0.721 µm, and the suspension thus obtained is centrifuged at rotational speed 4000 O6. / MIN for 20 minutes. The separated precipitate is suspended in 250 ml.
    buffer solution and then centrifuged under the indicated conditions and the resulting precipitate is collected. Then, 250 ml of a buffer solution containing 88 mg of the previously obtained conjugate and half
    The suspension is kept at 37 ° C for 2 hours, after which
    centrifuged to separate the precipitate. The precipitate is re-suspended in 250 ml of buffer solution and
    centrifuged, all these processes
    repeat once more. The resulting precipitate is re-suspended in 250 ml of the buffer solution and then sodium azide is added to a final concentration of 0.1%.
    250 ml of suspension (concentration of 2%) of latex particles sensitized with HC-21-HS-HSA are obtained.
    Then, 0.05 ml of 0.1 mol of hydrocortisone is mixed with 0.05 ml of diluted antibodies and diluted with a buffer solution to obtain a titer of 0.1 mol of HC-21-HS eq / mp on a glass slide with this solution. O, 1 ml of the previously prepared suspension of sensitized particles is mixed. Even after 10 minutes no agglutination is observed. However, when 0.05 ml of 0.05 nmol / ml of hydrocortisone and 0.05 ml of the indicated diluted antibody are mixed with each other and then 0 ml of the indicated suspension of sensibi is added to a glass slide 9121397510
    lysed latex particles agglyu- Sensitivity to detection
    Tinning occurs in the stream (over 2-3 min. of reagent containing estriol. The titer of the latex reagent in this at-16s. Cr-glucuronide is 0.8 measure taken equal to 0.05 nmol1.5 nmol / mp, the sensitivity from HC-21- HS - eq. / Ml.5 of the known reagent 2.5 nmol / ml.
    权利要求:
    Claims (1)
    [1]
    DIAGNOSTICUM FOR QUANTITATIVE DETERMINATION OF A STEROID HORMONE IN HUMAN ORGANISM BIOLOGICAL LIQUIDS, containing latex particles and a serum albumin conjugate with a hormone, characterized in that, in order to increase the sensitivity of the target product, it contains human albumin, albumin, ovine, , it contains estrogen, progesterone, androgen, 17-hydroxycorticosterone or their metabolites as a hormone, it contains polystyrene particles or a copolymer as latex particles styrene-butadiene size 0,234-0,721 microns with the following quantitative ratio of ingredients, β mg: S
    Latex particles 100
    The conjugate of serum albumin with a hormone of 1.8-7.2, while in the conjugate, 1 mol of serum albumin accounts for 0.57 mol of the hormone.
    SU .... 1213975
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    同族专利:
    公开号 | 公开日
    DE2827250A1|1979-01-04|
    AU3694578A|1979-12-13|
    AU522306B2|1982-05-27|
    JPS5819222B2|1983-04-16|
    NL7806681A|1978-12-27|
    JPS548715A|1979-01-23|
    GB2001992A|1979-02-14|
    SE7806961L|1978-12-21|
    FR2395507B1|1984-10-26|
    US4226847A|1980-10-07|
    DK151400B|1987-11-30|
    DK151400C|1988-05-16|
    DE2827250C2|1986-03-06|
    CA1107196A|1981-08-18|
    DK277178A|1978-12-22|
    GB2001992B|1982-03-24|
    FR2395507A1|1979-01-19|
    SE445149B|1986-06-02|
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    NL125235C|1965-04-15|
    JPS5226644B2|1972-05-30|1977-07-15|
    JPS5513306B2|1973-11-29|1980-04-08|
    JPS5434818B2|1974-03-14|1979-10-29|
    BE821053A|1974-08-01|1975-04-14|PRODUCT FOR DIAGNOSIS BY IMMUNOCHEMICAL DETERMINATION|
    JPS51112513A|1975-03-25|1976-10-05|Asahi Chem Ind Co Ltd|A method for determination of dha.sulfate|US4331657A|1980-02-06|1982-05-25|Commonwealth Scientific And Industrial Research Organization|Fecundity of domestic livestock|
    NZ196126A|1980-02-07|1985-05-31|Commw Scient Ind Res Org|Increasing ovulation rate in female cattle|
    US4340564A|1980-07-21|1982-07-20|Daryl Laboratories, Inc.|Immunoadsorptive surface coating for solid-phase immunosubstrate and solid-phase immunosubstrate|
    JPS589068A|1981-07-10|1983-01-19|Eiken Kagaku Kk|Immunochemical determination of estriol-16alpha- glucuronide|
    US4792527A|1981-07-17|1988-12-20|Toray Industries, Inc.|Method of assaying biologically active substances and labelling agents therefor|
    US4738932A|1985-12-03|1988-04-19|Advanced Polymer Systems, Inc.|Reaginic test for syphilis|
    ES2028124T3|1987-04-22|1992-07-01|Schiapparelli Biosystems, Inc.|METHOD FOR THE DETERMINATION OF ALBUMINE IN BIOLOGICAL FLUIDS.|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    JP52072775A|JPS5819222B2|1977-06-21|1977-06-21|
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