• 专利附录
  • 专利说明
  • 权利要求
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    • 专利摘要:
    A method and test means for determining a specific binding substance, such as an antigen or hapten, in a liquid sample wherein a solid, nonspecific adsorbent is contacted, preferably in sequence, with the sample and a labeled form of a binding partner for the substance to be determined, such as a labeled antibody. The amount of label which becomes associated with the adsorbent by binding of the labeled binding partner to the adsorbed substance to be determined, i.e., in a sandwich fashion, is a function of the amount of the substance to be determined in the liquid sample. Preferably, the adsorbent is an ion exchange material, and the assay steps are carried out under pH conditions between the isoelectric points(pI values) of the substance to be determined and the labeled binding partner. The method and test means are particularly suited for the detection of hepatitis B surface (HBs) antigen, especially where the label is a radioactive material and a column format is employed. The resultant radioimmunoadsorbent(RI-AdTM) assay and test means for HBS antigen offers significant manufacturing and user advantages over the prior art methods and test means.
    公开号:SU735153A3
    申请号:SU782608149
    申请日:1978-04-10
    公开日:1980-05-15
    发明作者:Алан Шик Ллойд；Кеннет Карпентр Стефен
    申请人:Майлз Лабораториз, Инк (Фирма)；
    IPC主号:
    专利说明:

    (54) METHOD FOR DETECTING SURFACE ANTIGEN-HEPATITIS B 1. The invention relates to the field of medicine, namely to the diagnosis of diseases of hepatitis B. There is a known method for detecting the surface antigen of hepatitis B in an assayed serum or plasma by determining the difference in the radioactivity of serum containing specific labeled antibodies, dO and after contact with a fixed on an inert carrier with hepatitis B antigen. However, the known method is not sufficiently sensitive. The aim of the invention is to increase the sensitivity of the method. This goal is achieved by passing the test sample through a column filled with an anion exchange material in the form of particles that are in equilibrium in a liquid at pH 5-8, washing the column at pH 5-8, then passing the labeled radioactive isotope specific antibodies with a predetermined level of radioactivity, incubated for 0.5-18 hours at 4-50 ° C, then the column is washed at pH 5-8 and the radioactivity of the anion exchange material or eluate from the previous stage is determined. The proposed method is used to detect hepatitis B surface antigen (HB) in liquid samples, such as serum or plasma samples. Various hydrophobic materials are used as adsorbents for the hepatitis antigen, including some alkyd-substituted polymers, as well as to -hadinoalkyl-substituted agars, but it is preferable to use ion-exchange materials. PH value for HB antigen. is in the range of 3.5-5, and for anti-HB 8, preferably pH is 6-8. As an adsorbent, anionic substances such as diethylaminoethyl-substituted polymers, in particular those containing polysaccharides, for example cellulose or dextran, are used as the main chains. DEAE-cellulose can be used as an adsorbent for HB antigen. A cation-exchange material, such as carboxyl methylcellulose, can be used as an adsorbent if the pH value of the labeled anti-HB is changed to a value that is lower than the same indicator for the HB antigen, for detecting HB antigen in serum or a plasma, the sample is introduced into a column containing an anion exchange substance in the form of particles, which are in equilibrium in a buffer liquid with a pH of 3 - in, preferably 6.5-7.5. Then, a buffer liquid with a pH of 5-8, preferably B, 5-7.5 is passed through the column in order to completely remove the HBg antigen, which is not adsorbed by the anion-exchange substance. . . After that, a predetermined volume of a solution labeled with a radioisotope anti-YBC having a certain level of radioactivity is introduced into the anion-exchange material. The solution is labeled with an anti-HB-labeled α-radioisotope in contact with the dione-exchange material 30 mich 18 h, preferably 1-3 h with, preferably 40-50 C. A buffer liquid with a pH of 5-8, preferably b, 5- is passed through the column 7, 5, to remove almost the entire anti-HB radioisotope that is not absorbed by the anion-exchange substance; time for; a link to adsorbed HB5 antigen. The radioactivity of the anion or material is measured or. the eluate of the preceding stage; and comparing Pa: Dio-activity measured at the preceding Stage; Radio radioactivity measured using a liquid sample that does not contain a significant amount of antibio gen HBj, instead of sykorit or kLaegl,:,; As buffer liquids, phosphate buffer, barbital buffer, trio (bxmethyl) amilomatome, new buffer, 4- (2-hydroxyethyl -1-pipera, zinethanol sulfonic acid (HEPES) and 1,4-piperazin-bis-zanesulfonic acid ( PIPES). Example 1. Preparation of an Analytical Column. The koyonks contain 0.5 MP of anion-exchange resin, DEAE-cellulose type DE-52, which is loaded into a 9.5 x 75 mm column made of polyethylene between two porous polyethylene discs. The column is: but it is plugged with the upper cap and the bottom cap. how it is filled with a volume of 0.02 M solution of a mixture of monobasic sodium phosphate — two ocHOBHOfd sodium phosphate, which functions as a buffer at pJ 6.8. The buffer solution must be sufficient to: saturate the resin and, moreover, to have a layer in the reservoir 1. above the resin level. Analysis method .. Remove the top cap and the column are fixed on the grate with the drain. In the buffer located in the tank above the DEAE-cellulose layer, D0 add 200 µl of sample to be analyzed serum or plasma or negative control sample. Next, the contents of the column are stirred to transfer the sample; and the buffer, and then the mixture flows down when the lower cap is removed. The resin bed was washed with two successive additions of 4 ml. 0.02 M phosphate buffer solution (pH 6.8). When the liquid from the column is completely drained, add on top of the resin layer. 200 µl of anti-HBs (specific activity of labeled anti-HB - 14–20 µCi / µg total activity of approximately 50,000 pulses / 1 1 in) are given. The radioactive preparation impregnates Slola, after which the lower hood is closed (anti-HB, used in conjunction with the anti-HB, cleaned in accordance with the Ling and Overby method and labeled according to the Chloramine T method). Then the column is incubated for 2 h at 45 ° C (assay 2 h 45 C) or overnight at room temperature 22 ° C. Then the bottom cover is removed and the resin layer is washed with two successive additions of 4 ml of 0.02 M phosphate buffer solution (pH 6.8). When the liquid is completely removed from the column, the bottom cover is put in place and the column is placed in the g socket of a gamma-radiation counter, such as a dammacord counter, and the radioactivity of the resin layer is determined. . A sample is considered positive if it gives a level of radioactivity that comes from a resin layer that exceeds (or gives a level of radioactivity from the flushing fluid less than) the limit value calculated as the average number of pulses / min of gamma radiation from the resin layer. The following is an example of the calculation of the boundary gamma radiation (pulse / min) for. a layer of resin. Seven similar negative control samples are separately subjected to an ASMU and the amount (counts / min) of gamma radiation, the source of which is a CMO-layer, is determined. ly (threshold pulse / min) for each sample. Sample Threshold, imp / min 10206 General 1 4 58 Average. Standard + 54. . deviation 1458 + (4 X Boundary X 54) 1678 value
    Therefore, if the sample being analyzed gives a threshold value greater than 1678 pulses / min, the sample is considered positive for the HBg antigen.
    Example 2. Radioimmunosorption analysis of the Hepatitis B surface antigen in the form of its immune complex with an antibody.
    A procedure is described for detecting HBc antigen present in a sample in a complex in anti-HB, i.e., in the form of its immune complex. In accordance with the proposed method, a serum sample can be analyzed for HBe antigen containing anti-HB, as well as produced HBg antigen analysis in the form of its immune complex in the corresponding samples.
    Columns prepare for analyzes in accordance with Example 1.
    Method of analysis. The bottom cover is closed, and the top is removed: phosphate buffer solution is poured from the tank, and 1 mp bm is replaced in its place
    solution of arbamide in O, O2M phosphate buffer solution (pH 6.8).
    200 µl of a serum or plasma sample or 5 negative control samples are added to a tank containing a mixture of carbamide buffer. The contents of the column are stirred to mix the sample and the buffer-carbamide mixture, and then allowed to stand for 10 minutes before draining, which is carried out.
    0 when removing the bottom cover.,
    After the solution is drained, the steps described in Example 1 are repeated.
    Detection of HBj antigen in serum 5 (endogenous anti-HBg). Negative control serum and oblique commercial serum containing low concentrations of the antigen HB and anti-HBc are analyzed using a modified method with 100: incubation for 2 hours and 45 ° C, methods
    Qusria, which was decommissioned in the previous example, and the Ousab methods of Abbott Laboratories, North Chicago. 5 The results are shown in the table.
    .4.0 3.0 2.0 1.0 0.4 0.2. 0.0
    The threshold value (imp / min) for the sample minus the limit value. The proposed example shows that it is possible to detect the HB antigen in the presence of endogenous anti-HB, .. In order to confirm the presence of the HB antigen, is carried out in serum. following in the centrifuge. A serum sample equilibrated with glycine buffer pact (pH 2.0) is treated with a sucrose gradient of 5–20% (w / w). The mixture is subjected to centrifuge separation at a flow rate of 3 r.p. rpm 3 H. The fractions are removed from the bottom of the tube. .in the Qusria j procedure for antigen VB were identified as positive. This confirms the presence of HB antigen in serum and. Clearly, it proves that HBs antigen, after separation from anti-HB, is detected in the centrifuge using the Qusria -j technique while it was not detected using the same technique when combined with anti-HB. Detection of antigen HBc, in the form of its immune complex. A series of samples are prepared for analysis by adding increasing amounts of positive serum YV antigen to a constant volume of high titer anti-HBv serum. The final volumes are equilibrated with normal serum, negative for both antigen HB and anti-fra. After incubation for 20 hours, the precipitate formed is removed in a centrifuge and the resulting upper layer is used as an anesthetized sample. Each sample obtained is analyzed by the RI-Ad method, and incubation is carried out for 2 hours at 45 ° C, by the method of Qusria-j-jr & also according to the Ousab method. The result; you are listed in the table. Hell / Av means the ratio of the volume of antigen YBV positive, serum, added to the positive anti-HB syringer syndrome,. . According to the modified RI-Ad method, the YV antigen o6Hapy5) (: fl (feaeTCf ErtIl5 cyTCT shows anti-HBg at the lowest T & C concentration of the added antigen HB (Ad / Av ratio - 0.2). In samples with anti-HB in samples is present in excess (Ad / Av 0.4) The method of Ousr.ia33 does not prevent the presence of HB antigen. On the other cfqpoHB, the presence of anti-HB is confirmed in samples that have LD / AV ratios less than or equal to 1.0 according to the Qusab method of Bnac tio myotda dyab can not detect anti-HBe, in the presence of an excess of HBV antigen (Hell / Av 2, a). Example 3. Investigation of adsorption properties various anionic metabolism anions for HB antigen. In the pH range 5-8, the ability to adsorb the antigen HB of the following anion-exchange substances was tested: D5AE-cellulose type DE-52, DEAE-Zerbadex,; QAS-Sephadex and DEAE-V1od eC, Volume 200 ml H-HB antigen (25 mg / mp) is added to each of several multi-columns, various concentrations of anion-exchange npvi of various known pH values in the contents of each column. Then each column is washed with two volumes of Mts of 4 ml of phosphate buffer with CoC) pH values corresponding to 1m. Then pn), the radioactivity of the columns is determined; gamma radiation meter counter. G - /.,.:, For each of the resins studied: optimal adsorption was observed for x 6.0-6.5. DEAE-cellulose DEAE-Sephadex and QAE-Sephadex IHSTs are very similar in maximum adherence to antigen-HB, while DEAE-Biogeft A exhibits a slightly lower ability to adsorb a tigena. In a more extensive study, it was found that DEAE-cellulose is the preferred resin for use in columns, because it does not create a WPR-Pr fertilizer for the flow through the column and has a minimal tendency to adsorb -anti-rVd. Example 4, Radioimmunosorption analysis (RI-Ad) for hepatitis B surface antigen. The RI-Ad method for detecting HB antigen, characterized in that the step between sample loading and the step of adding labeled anti-HBg is excluded. Each sample is analyzed by the method PH-Ad -2 h using a different series of J-anti-HBe ,. Example 5. The use of hydrophobic adsorbents in radioimmunosorption (RI-Ad) analysis of hepatitis B surface antigen. A series of t-amin.oalkyl-substituted agarose is investigated as adsorbents in accordance with the PH-Ad method. The preparation of the columns and the method for performing the analysis are described in Example G, except that the DEAE-cellulose adsorbent is replaced by different 6i-aminoalkyl substituted agars, and the adsorbent 1mg is washed with two successive additions of 2 MP ciiiecH tris-hydroxymethylaminomete-hydrochloride buffer, a buffer, 3-MP buffer, tris-hydroxymethyl buffer, tant-hydrochloride buffer, a buffer overgrown buffer, tris-hydroxymethylamine, and a hydrochloride buffer. sodium (pH 8.2). Two serum samples, one negative and one proliferative for the HBC antigen, are analyzed using a number of columns, each containing one of the following substances having a general formula. arai) OB- (CH2) f, -NHg ,, where n is 4-10 the number of carbon atoms in the alkyl side chain. L-AyinRalTs1igl-substituted agaroses having alkyl side chains containing more than four carbon atoms are more suitable for detecting ... HB antigen. Example 6. Columns are prepared for analysis in accordance with the method of example li. The top cap is removed and the column is placed on the drainage grate. To the buffer solution, located in the rezvévare above the DEAE-cellulose layer,. 200 ml of positive serum for antigen-HBc are added. The contents of the column are mixed to mix the sample and buffer, then the contents of the column are drained when the bottom cover is removed ..-. TlocJie. As the contents of the cream column, the Resin layer was washed in two successive portions of 4 ml of 0.02 M phosphate buffer solution (pH 6.8). Next, a layer of 200 ml of the sample to be analyzed or a negative sample of the control sample is applied to the upper disk and allowed to be absorbed into the resin layer. The bottom of the spout is set in place and the column is incubated for 2 hours at 45 ° C. The bottom cover is removed again and on top. a layer of 2000 ml is applied on the resin LAYER
    / E-anti-explosive and allowed to soak into the resin bed. Nizhnyun kryoik again for tearing down and incubate the column for another 2 h at 45 ° C.
    The bottom cover is removed again and the resin layer is washed with two successive batches of 4 ml of a 0.02 M phosphate buffer solution (pj 6.8),
    When the contents of the column are completely drained, the lower flap is closed again and the radioactivity of the column is measured during dry-cooling with T4ik g-radiation. ; The proposed method is more sensitive and makes it possible to quickly and accurately detect the hepatitis B surface antigen in the test sample.
    权利要求:
    Claims (1)
    [1]
    1. US patent 3,938,853, cl. 308-4, pub. 1976.
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