• 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:
    DIAGNOSTICUM FOR THE DETECTION OF HEPATITIS B ANTIGEN, containing 0.2-0.23 µm polystyrene particles with specific antibodies sorbed on them at a concentration of the last 0.05% and a saline solution, characterized in that, in order to increase the sensitivity of the diagnosticum, it additionally contains the normal output of guinea pigs, as a salt solution, it contains a physiological solution in the following proportion of components, wt%: Polystyrene particles of 0.2-0.23 µm in size with specific sorption on them with antibodies at a concentration of the last 0.05% 0.5-0.8 Normal serum of guinea pigs2-5 Physiological O) solutionOstal
    公开号:SU1091844A3
    申请号:SU2322065
    申请日:1976-02-13
    公开日:1984-05-07
    发明作者:Эдвард Лоук Джордж；Золтан Хорват Бела
    申请人:Варнер-Ламберт Компани (Фирма)；
    IPC主号:
    专利说明:

    QD
    00 4 The invention relates to medicine and relates to a diagnostic formulation for detecting hepatitis B. A diagnosticum is known for detecting hepatitis B antigen containing polystyrene particles with sorption -4 and specific antibodies on them and with a left solution 1 1. However, the known diagnosticum is not sufficiently high sensitivity. The aim of the invention is to increase the sensitivity of diagnosticum. This goal is achieved by the fact that a diagnostic tool for detecting the hepatitis B antigen containing polystyrene particles with a size of 052-0.23 μm and sor (specific antibodies on them at a concentration of the last 0.05% and salt the solution additionally contains normal sye.oruku sealskin pigs as a salt solution, it contains a physiological solution in the following quantitative ratio of components, wt,%: Polystyrene particles of 0.2–0523 µm in size, with with specific antibodies on them at a concentration of the last 0.05% 0.5-0.8 Normal shortening of guinea pigs2-5 Physiological solution Remaining Diagnosticum for detecting antihep on hepatitis B contains an aqueous suspension of finely crushed particles of the synthetic resin coated with purified antibody specific for the hepatitis B surface antigen. These particles are spherical polystyrene particles having a diameter of about 0.23 µm, but particles of polystyrene or other synthetic resins are also suitable, for example, acrylic o, having the same size 0.05-25O microns. Antibodies specific for hepatitis B surface antigen are preferably obtained from mammals, such as humans, sheep, goats, rabbits, or porpoises-HoKj or from avian sources, such as chickens or turkeys, which contain antibodies, or have been immunized in a highly purified manner. -i superficial; Hepatitis B antigen. derived from human blood. Guinea pigs or other animals: such as rabbits, goats, are immunized with the hepatitis B surface antigen according to a known method. Primary immunization with antigen in a full Freund excipient in the paw pads is accompanied by repeated intraperitoneal intramuscular, subcutaneous or intravenous antigen inoculations with or without the use of an auxiliary medicinal substance. After mixing the sera collected from immunization; in animals, the residual antibody for human serum proteins, if present, is removed by contacting the anti-Short with the human short. The gamma globulin can then be isolated by precipitation with 14% BonHbM sodium sulfate, dialyzed against phosphate-buffered aqueous physiological saline, and preferably up to 56 ° C for about 30 minutes to destroy complement. Next, the antibodies are purified by common known methods. Hepatitis B specific surface antibodies are purified from absorbed LShW sera (preferably guinea pig sera) by mixing these sera.:, In optimal proportions with human sera, with hepatitis B surface antigen, incubating at 37 ° C for an hour, and then overnight at i C, the precipitated (precipitate) of the antigen-antibody thus formed is washed twice in cold phosphate-buffered physiological saline solution to remove most of the residual nonspecific proteins. This amythenic-anthropyl precipitate is then dissolved in a cold 0.2 M acetic acid (2% sucrose: S pH 2.3) or various tons of chaotropic ions, such as 2.0 M sodium thiocyanate, pH 6.6. Other suitable buffer mixtures may be used to dissociate antigen-antibody complexes. These dissociated antigens and antibodies are separated by centrifugation at 25,000-28,000 rpm for 12-15 hours or at 30,000 rpm at 31 for 4 hours on a continuous sucrose gradient of about 10-40% in a Beckman type T1 rotor -15. The rotor is once loaded by pumping 50% sucrose into it. To mix the gradient, 20 m fractions are checked for ultraviolet absorption in a 1 cm well at 280t (L- ,. the peak fractions are mixed and a purified fraction of a specific antibody and a purified antigen fraction are obtained. Then both fractions are neutralized using 0.1N NaOH. The antibody fraction is concentrated to the volume of the original serum and dialyzed overnight against glycine-buffered saline sodium chloride. The purified antibody is then titrated and standardized. 0.1% sodium azide can be added as an anticoagulant for use in the preparation of a diagnostic reagent. Antibodies are diluted by preparing small dosages of diagnostic reagent with various antibody dilutions and testing them with a number of human sera containing hepatitis B surface antigen. Dilution giving better results than this When it covers resin particles, it is selected to prepare a diagnostic reagent. Diluted antibodies are mixed with 0.2 µm latex polystyrene grains. After mixing for one or two hours, these grains are washed three times with buffered glycine physiological saline solution. The aqueous suspension contains about 0.5% by weight / volume of resin particles, but may also contain 0.1-3% by weight / volume of resin particles. This suspension preferably contains about 0.05 wt.% / / Volume of antibody absorbed on the resin particles, but the composition may vary according to the concentration of the particles. The percentage of antibody should be sufficient to prevent auto-aggregation of rubber particles. For spherical polystyrene particles of the order of 0.23 microns in diameter, this is about one tenth of the particle weight. The slurry preferably contains a dispersion stabilizer (anticoagulant 44 liters) added after the antibody is absorbed on the particles, for example an inert protein, such as serum albumin at a concentration of, for example, 0.1 Bes. The diagnosticum also contains normal serum (that is, serum containing neither antibody specific for the hepatitis B surface antigen nor the antigen itself) obtained from the same animal species as the one from which serum containing antibodies was obtained. The presence of this normal serum significantly reduces the percentage of false-positive results when using diagnostic to detect the hepatitis B surface antigen in blood samples. Preferably, about one part of normal serum is contained in 20-50 parts (by volume) of an aqueous suspension of resin-coated particles of an antibody. If normal serum from the same animal species as the one from which the antibody-containing serum was obtained is not in the diagnostic system, about 10% of human sera that do not contain the hepatitis B surface antigen will agglutinate the resin particles. This is due to the presence of such substances in human sera, which react with other materials derived from the antisera of animals, from which the hepatitis B surface antigen is obtained. However, if the animal serum contains normal serum, only about 1-3% of human sera will agglutinate particles resin in the absence of hepatitis B surface antigen in these sera. This may occur if there is an animal in normal serum that can react with these substances in Human yvorotkah and thus prevent them from induce agglutination resin particles. Control of normal human serum is obtained, which is obtained from undiluted normal human serum; it is negative for hepatitis B surface antigen when tested with a latex reagent and a radioactive isotope method. Positive control serum should be prepared from the known known positively on the surface of the first human hepatitis B antigen gene. This serum is subjected to heat treatment at 60 ° C for 10 hours; and then diluted twice in succession in normal human serum. Dilution S used for this product 5 is a dilution giving a specific reaction with a latex reagent. Diagnosticum consists of the following, their components. AO Screening Test System 1, Hepatitis Antibody Absorbed on Latex Seeds (Beads, Balls), 2, Positive Control Serum. 3, Serum negative control. The system confirmatory test. 1. Hepatitis B antibodies (human), 2 ' Control of normal human serum (negative for hepatitis B surface antigen), 3, Rheumatoid factor uptake reagent, 4 o Thinner confirmatory test. Hepatitis-associated antibodies (human) are obtained from human plasma, which contains antibodies for the hepatitis B surface antigen. This plasma is taken from various plasmaphoresis (plasma preparation) centers, its gel is titrated by diffusion and converted into serum by adding calcium chloride and bovine thrombin . Then, gamma-globules are prepared from this serum by precipitation with 16% sodium sulfate. After washing the precipitate (precipitate) in 14% sodium sulfate, the composition is dialyzed for the whole time against the low molarity of phosphate buffer and passed through DEAE cellulose. balanced by the same buffer. Purified gamma globulin is then standardized in concentration and dialyzed against phosphate buffered saline sodium chloride. The reagent for rheumatoid factor absorbent consists of latex grains (beads, balls) that are coated with normal human gamma globulin. It is formed by preparing a sterile solution of purified gamma globulin in buffered | 1; 1, saline 110} 1 salt of salt with the addition of suspension of 0.2 µm polystyrene grains and by heating at 57 ° C for 5 minutes. This suspension is then diluted in glycine-buffered physiological saline diluent. A confirmatory test diluent is prepared by diluting a solution of 30% bovine serum albumin with phosphate-buffered physiological saline solution of parapt-EiHOK salt to 5% protein: The tests are carried out as follows. Blood samples are prepared. Blood sampling is the preferred form of a blood sample. If the blood test is whole blood, it must first be processed to dissolve the cells in it and prevent coagulation. For this purpose, it is treated with an old diluent 5 containing a non-ionic surfactant tic polyoxyethylated alkylphenol naphtamyl isooctylphenoxy polyoxyethyltanol and tartaric acid if you are testing a sample of blood, it must contain an anticoagulant extract and an extract) Preferably, it is also treated with the same cat4bJM aqueous diluent containing an ion-free surfactant as for whole blood; in order to dissolve any residual cells, add 3 in a b. The specificity of the reagent is determined by comparing it with a target of a series of positive and negative sera, supplied by the Bureau of Biological Sciences, as well as with a series of 100 negative serums collected from normal to volunteers. Re; g.ktsion; gu1o capacity is determined by suspending this reagent: vigorously, 3 drops of serum are poured into one of the test wells on the plastic laboratory alastins
    positive control. 3 drops of serum of the negative control are poured into the second adjacent depression
    One drop of reagent is added to each sample. Mix thoroughly using a separate mixer for each well. Rotate the plate on a mechanical rotator for 10 minutes at a speed of rotation of 150 revolutions per minute. The mixtures are checked through a viewing instrument illuminated by reflected or scattered light.
    Clearly positive, weak (1) the reaction should be observed with a positive control serum sample. No reaction should be observed with a negative control serum sample.
    The reactivity of the serum positive control is tested with a latex reagent to ensure reactivity on the order of 1,
    Serum negative controls are tested with a latex reagent to ensure a negative reaction.
    Control normal human serum.
    The specificity test of the wire is made to ensure that it does not react with the latex reagent and that it does not suppress positive serum against the hepatitis B surface antigen,
    Hepatitis B antibodies (human).
    Reactivity was tested by gel diffusion titration using standard hepatitis B surface antigen.
    Specificity tests are carried out to determine if this serum will specifically inhibit positive samples of the hepatitis B surface antigen,
    Absorption reagent.
    The reactivity is determined by testing the reagent with serums containing the rheumatoid factor to ensure its reaction with this factor and its partial absorption from the sera.
    Confirmatory Test Thinner.
    Specificity tests are carried out using this dilution.
    bodies to ensure that it does not react with the latex reagent and that it does not suppress the positive serum of the hepatitis B surface antigen.
    The test of serum samples (screening test) is carried out by separating 120 | O-E serum into a recess on a plastic plate.
    Add one drop of latex regent.
    Fully mix using a separate in-use mixer for each sample.
    Rotate the plate on the mechanical rotor for 10 minutes at a speed of 150 revolutions per minute.
    The mixtures are checked using a viewing instrument that is combined by reflected or scattered light.
    Reactions are calculated by points on a scale from 1 to 4, corresponding to an increase in the intensity of agglutination.
    Confirming neutralization procedure.
    Label one test tube with a volume of 10 X 75 mm with an identification number of the sample and index A; Mark the second tube with an identification number with a sample identification number and index B.
    Add two drops (approximately) of the associated with. hepatitis antibody (human) in a test tube equipped with an index A.
    Two drops of normal human serum of the negative control are added to a tube equipped with idex B,
    240jU.6 test samples are added to each of the tubes, labeled A and B,
    Mix the contents of these tubes and incubate for 30 minutes at 37 ° C in a water bath. - Test 120 | M- from tubes A and b using screening reagents and protsvdury. For most relatively few titrated positive samples, differentiation between specific and non-specific positive results can be without further testing, because sample A is unresponsive, while sample B remains positive, showing specific suppression by human antibody. For those samples that are specific but more heavily titrated, as well as those that are non-specific, further treatment is required. For each tube that requires further testing, an additional six tubes are labeled with dilutions from 1: 4 to. : 128, as well as the sample identification number and A or B indexes. Add 20014 diluent confirmatory test to each tube, including tubes that contain the patient's sample and human antiserum or normal human shortening, i.e. The initial tubes A and B, diluting them to about 1: 2, Dvage, diluted them with transfer 200 using separate micropipettes for the test tube and mixed well before removing the samples. Test from the test tube using screening agents and procedures. The titer obtained in row B (sample and normal human serum) must be four times (two tubes) higher than row A (sample and neutralizing hepatitis bound antibody (human) in order to treat the sample (sample ) as a specific positive surface hepatitis B antigen. In the case where both titer A and titer are higher than 1: 128, dilution should be done further until the end point is determined. Since a whole series of samples containing rheumatoid factor reacts with latex agent, it is necessary to differentiate the probyz content as a hepatitis B surface antigen and rheumatoid factor. plastic slide and spin for five minutes. If the sample contains rheumatoid factorJ it can be absorbed by adding 720 samples to a 10 X 75 tube and adding six Pel absorbing agent into this tube. Mix well and incubate at :: ambient temperature D for five minutes. Perform a confirmatory neutralization procedure on the absorbed sample. The preparation of purified antibody to hepatitis B surface antigen. Guinea pigs are immunized with hepatitis B surface antigen, purified in a known manner. The primary immunization with antigen in the Freund full complementary drug in the paw pads of guinea pigs is accompanied by two intraperitoneal inoculations of this antigen without the auxiliary drug substance. After combining the seraJ collected from the immunized animals, the residual protein, if necessary, is removed by contacting the antisera with normal human serum. The gamma globulin is then purified by precipitation with 14% w / v aqueous sodium sulfate, dialyzed against physiological saline solution buffered with aqueous phosphate, and preferably heated at 56 ° C. for about 30 minutes to dilute the complement. The purified serum is mixed with a human crank containing hepatitis B surface antigen, incubated at 4 ° C. The antigen-antibody primitive obtained in this way is then washed twice in cold phosphate-buffered saline saline to remove most of the residual non-specific proteins. Then this antigen-antibody precipitate is dissolved in cold 0.2 M acetic acid: hydrochloric acid (2% sucrose, pH 2.3 or chaotropic ions). These dissociated antigens and antibodies are separated by centrifugation at 25,000–28,000 rpm for 12–15 Celsius or at CrOOO revolutions per minute for 4 hours on a continuous 10–40% sucrose gradient in the Beckman type T1-15 rotor, The rotor is unloaded by injecting 50% sucrose into it to mix the gradient, and 20 ml fractions are checked for ultraviolet absorption in 1 cm of ggry cell 280 ml, and the peak fractions are combined to give purified. a specific fraction of the antibody, which is then neutralized with 0.1 n. NaOH. The antibody fraction is concentrated to the initial serum volume and dialyzed overnight against glycine-buffered physiological saline. Then purified antibodies are titrated and standardized. These antibodies are optimally diluted by preparing small dosages of diagnosticum with different dilutions of antibodies and testing them against a whole range of human sera, which are known to contain the hepatitis B surface antigen. Dilution giving the best indication against Resins, selected for the preparation of diagnosticum. The diagnosticum is prepared as follows. Example 1. 30 wt.% / Volume of an aqueous suspension of 0.23 µm polystyrene beads is diluted to 8 wt.% / / Volume buffered with glycine physiological saline solution and dialyzed against glycine-buffered saline saline throughout the night. It is then immediately added to 9 volumes of purified guinea pig antisera containing the antibody to the hepatitis B surface antigen (diluted to 0.05% w / v) and continuously mixed for 30 minutes. Thereafter, one volume of bovine serum albumin (1% w / v) and then sodium azide are added so that the concentration is in the order of 0.1% w / v in the final product. Example 2. 30% by weight / volume of a suspension of polystyrene beads (0.05-2.0 µm) is diluted to 8% by weight / volume with buffered glycine with physiological saline solution and sterilized by the addition of a hypochlorite solution. Then dialyzed against glycine-buffered saline to remove the hypochlorite. This formulation is then added to 9 volumes with an appropriately diluted, cleaned guinea pig serum — hepatitis B surface antigen antibodies. A12 The polystyrene suspension is added slowly with stirring, which continues for one hour. Polystyrene beads are centrifuged, washed twice in a centrifuge with buffered physiological saline solution, and again suspended in their original volume in buffered saline saline. Bovine serum albumin is added with stirring for 30 minutes. One tenth of a volume of diluted normal serum from non-immunized guinea pigs (diluted 1: 3.5 with buffered glycine physiological saline solution) is added to this formulation and the mixture is mixed again for 30 minutes, and finally the mixture is shaken vigorously to disperse any agglomeration of particles. All the solutions that were used were sterilized by filtration and contained 0.1% by weight / volume of sodium azide as a dispersion stabilizer (anticoagulant). Example 3. A positive control serum is prepared from a variety of human sera known as positive for the hepatitis B surface antigen. This serum is heat treated at 60 ° C for about 10 hours, and then twice in succession, diluted in normal human serum. Used dilution in-. It is such that it gives (1-4) a reaction with the latex reagent of examples 1 and 2. Then this composition is sterile filtered and placed in sterile containers. Example 4. The negative control serum was obtained from undiluted human serum, which was negative with respect to the hepatitis B surface antigen using the latex reagent of example 1 or example 2 using the radioactive isotope method. The composition is sterile filtered and placed in sterile containers. Example 5. Human plasma, which contains antibodies to the hepatitis B surface antigen, is titrated by gel diffusion and converted to serum by the addition of calcium chloride and bovine thrombin. Gamma-globulin is then prepared from this serum by precipitation with 16 wt.% / Volume sodium sulfate. After washing this precipitate at 14% w / v sodium sulphate, it is dialyzed against a low molar phosphate buffer and passed through a DELE type cellulose, which was previously equilibrated with the same buffer. Purified gamma globulin is then standardized and dialyzed against phosphate buffered saline. Then this composition is sterile filtered and placed in sterile containers. Example 6. A standard solution of purified human gamma globulin in glycine-buffered saline saline is added to a suspension of 0.2 µm polystyrene latex and heated at 15 minutes. The suspension used to absorb the rheumatoid factor in the test sera is diluted in a buffered salt with glycine saline and cooked in sterile containers. Example 7. A confirmatory test diluent was prepared by diluting a solution of 30% w / w bovine serum albumin solution in phosphate-buffered physiological saline to 5% protein concentration. This solution is then sterile diluted and placed in sterile containers. Example 8, Regarding the concentration of purified guinea pig antibodies; The volume of polystyrene suspension is mixed with such a volume of purified guinea pig antisera that the resulting suspension will have an antibody concentration of 0.05 percent i.v., i.e. about; 0.8 percent V / V of polystyrene spheres and about 0.05% V / V antibodies to hepatitis B surface antigen. Example 9. Similar to Example 1, the product contains 0.8 percent of polystyrene spheres. In addition, it contains one tenth of the volume (10 percent w / w) diluted in the ratio of 1: 3.5 normal serum of guinea pigs. A serum diluted in a ratio of 1: 3.5 corresponds to a solution, which contains approximately 3D percent IV. Accordingly, the final product contains about 3 percent IV normal serum of guinea pigs. The proposed diagnostics has a high sensitivity for detecting the hepatitis B antigen due to the fact that BbiCOKo uses purified antibodies and normal serum of the same animal species from which specific antibodies were obtained, thus reducing the number of false positives,
    权利要求:
    Claims (1)
    [1]
    DIAGNOSTICUM FOR IDENTIFICATION OF HEPATITIS B ANTIGEN, containing polystyrene particles 0.2-0.23 microns in size with specific antibodies adsorbed on them at a concentration of the last 0.05% and saline solution, characterized in that, in order to increase the sensitivity of the diagnosticum, it additionally contains normal serum of guinea pigs, as saline, it contains physiological saline in the following quantitative ratio of components, wt.%:
    Polystyrene particles 0.2-0.23 microns in size with specific antibodies adsorbed on them at a concentration of the last 0.05% Normal guinea pig serum Saline
    0.5-0.8 2 ~ 5 g
    The rest is sy _ <„> 1091844
    109'1844
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    同族专利:
    公开号 | 公开日
    FI760326A|1976-08-20|
    DK144395C|1982-08-02|
    ZA76721B|1977-01-26|
    FI63493B|1983-02-28|
    CS188129B2|1979-02-28|
    US3992517A|1976-11-16|
    FR2301825A1|1976-09-17|
    ATA91776A|1979-02-15|
    GB1529891A|1978-10-25|
    JPS51106723A|1976-09-21|
    DK144395B|1982-03-01|
    SE429481B|1983-09-05|
    DD124128A5|1977-02-02|
    SE7600797L|1976-08-20|
    DK60876A|1976-08-20|
    IT1066096B|1985-03-04|
    EG12696A|1979-06-30|
    IL48927A|1978-12-17|
    LU74348A1|1976-12-31|
    DE2604844C3|1978-09-21|
    NZ179988A|1978-06-02|
    BR7600906A|1976-10-12|
    MX4962E|1983-01-20|
    IL48927D0|1976-03-31|
    AR210753A1|1977-09-15|
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    BE838474A|1976-08-12|
    CH613048A5|1979-08-31|
    DE2604844A1|1976-09-02|
    AT352290B|1979-09-10|
    引用文献:
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    US4542103A|1982-09-13|1985-09-17|Miles Laboratories Inc.|Latex agglutination determination of IgG in neonatals and in colostrum|
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    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    US05/550,949|US3992517A|1975-02-19|1975-02-19|Detection of hepatitis B surface antigen by latex agglutination|
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