TW205095B - - Google Patents

Description

205095 Printed by the Beigong Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economy V. Description of the invention (1) The invention is generally related to immunoassay methods using chemiluminescent compounds, and particularly to heterogeneous chemiluminescent immunoassay methods, in which The chemiluminescence signal generated by the immunochemical reaction through the cured product is expanded, which can result in a more sensitive analysis. The production of light due to the chemical reaction results is known in the art. For the total, see Schuster and Schmidt in " Chemi luminescence of Organic Compounds ", V. Gold and D. Bethel, eds ·, Advances in Physical Organ ic Chemistry 18 * 187-238 Academic Press, New York * (1982). Immunoassay methods using chemiluminescent markers as production compounds are known. The application of the generation and detection of chemiluminescence immunoassay is by W · R · Seitz General Manager, Immunoassay Labels Based on Chemi luminescence and Bioluminescence ", Clinical Biochemistry 17: 120-126 (1984). For example, the device and method for performing this analysis can be obtained from Ciba-Corning Diagnostics Magic-Lite 7 ^ system, in which chemiluminescent markers and magnetizable microparticles are used. Because brown microparticles optically interfere with the chemiluminescence signal, very low amounts of particles are used. This results in a very slow response. For example, an analysis of thyroid stimulating hormone (TSH) has been reported to have a three-hour incubation period. In addition, the many steps involved make it difficult to automate the configuration of this analysis. An enhanced chemiluminescence reaction, which is carried out on a white titration plate, and then read the ## Φ -in the notes before seeing the luminometer with a movable mask and a photomultiplier tube. 1 The binding line is too ttifcR娨 讧 ffl ΨΒ® A 4 Slip grid (210x297 public goods) 3 ώ 0509α Α6 Β6 Printed by the Beigong Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. Invention description (2) Read the signal generated by Lisenbee et al. , European Patent Case No. 194, 102 described, and incorporated into AMERLITE "W are sold by Amers-ham company. The latter reaction is pseudoenzymatic and is limited by the ELISA analysis of the coated plate, and the low diffusion rate of the reactant to the capture phase. In addition, the AMERLITE 7 ^ line applies a device separate from the luminometer, where the marker is triggered. A method for performing chemiluminescence analysis involves directly exciting and detecting chemiluminescence signals emitted by immune complexes, which are solidified in solid or porous elements, which serve as separation tools in heterogeneous immunoassays The device for this detection is described in the common US Patent Nos. 07/425, 643 and 07/206, 645, which share common ownership and are incorporated into this case by reference. The use of acridine compounds as markers in immunoassays and the subsequent generation of short-term chemiluminescence signals from these markers was described by I. Weeks et al., 'Acridinium Esters as Highly Specific Activity Labels in Immunoassays > '' Clin. Chemistry 19: 1474-1478 (1984). The use of stabilized azetamine sulfonamide is described in the common and co-owned patent case, PG Mattingly et al., U.S. Patent Case No. 92 1, 97 1, which is listed as a reference in this case and published in the European Patent Case No. 0273 1 1 5 The generation of long-lasting chemiluminescence signals has been described in the technology, and is produced by the action of enzymes or nucleophilic agents on dioxane compounds (containing an adamantane structure). Published European Patent Case No. 025405 1, (A. P-Schaap); Published PCT Patent Case W0 Read the back side first-the precautions before filling out this page binding line-4-Printed by Beigong Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs 20509ο Α6 _Β_6_ V. Description of the invention (3) 8 9 Ο 6 6 5 Ο 5 Ι · Bronstein et al. &Quot; 1.2 ~ Dioxeta-nes »Novel Chemiluminescent Substrates. App1i ca t i-on s to Immunoassays. &Quot; J . Bioluminescence and Chem-iluminescence 4:99 (1988) and the 5th International Conference on Bioluminescence and Chemiluminescence. F1orence-Bologna, Italy. Sept. 25-29 (1988). Use of signal enhancer, such as avidin-biotin Its use is also known. For example, U.S. Patent No. 4, 228, 237 described by Hev-ey et al. In the method uses biotin-labeled specific binding substrates for ligands, in which enzymes labeled with avidin are also used. The use of biotin-antibiotin is described in U.S. Patent No. 608, 849, published on May 10, 1 984, and it enjoys joint ownership and is hereby incorporated by reference (with European Patent Case No. 1 60 , Announcement No. 900 on November 13, 1985). Methods for enhancing and expanding chemiluminescent signals generated in immunoassays are known in the art. Therefore, U.S. Patent No. 4,927,769 describes the addition of surfactants to enhance the chemiluminescence signal generated in conjugates labeled with acridine monoester. At the same time, U.S. Patent No. 4,959,182 describes the addition of a surfactant and the adhesion of a fluorescent compound to expand the chemiluminescence signal produced by 1,2-dioxane catalyzed by alkaline basal enzymes. These known methods suffer from lack of specificity. Therefore, the signals generated by combining the chemiluminescent sassafras and the signals generated by the unwashed free state markers are expanded. The increase of the signal caused by this is equivalent to the desire (please read the back of the first, please fill out the notes before filling out this page) 装-线-太 蚯 张 尺 庶 1Λ 用 中 00 K 玄 itiJtiCNS) A 4 exploration grid (210X297 public potential) -5-Printed 20509b A6 __B_6 by Beigong Consumer Cooperative of Central Bureau of Standards, Ministry of Economic Affairs V. Description of invention (4) Response and background. Therefore, although the signal is expanded, it does not benefit from the sensitivity of the analysis. The present invention overcomes the shortcomings of the known art, that is, it proposes a chemiluminescent signal expansion method that uses the '' 's heterogeneous combination to the variability contained in the interaction. Therefore, the desired response signal is expanded to a greater extent than the background signal, thus improving the sensitivity of the analysis. The present invention further proposes a common heterogeneous expansion agent that can be used in various analyses. The invention proposes a method for determining the presence of an analyte in a test sample, which specifically expands the chemiluminescence signal produced in heterogeneous immunoassay. The method includes: a) the test sample containing the analyte and the analyte 1 The specific binding members are co-located to form a first mixture; (b) The first mixture is co-located for a period of time and the conditions are where the analyte / analyte specific binding member pair complex is formed; (c) analysis The analyte / analyte hetero-binding member complex is contacted with a probe containing an enhancer compound adhered to the analyte hetero-binding pair to form a second mixture; (d) co-locating the second mixture for a period of time The conditions are sufficient to form an analyte / analyte heterogeneous binding member pair / probe complex; (e) an analyte / analyte specific binding member pair / probe complex is in contact with the conjugate, which includes chemiluminescence The compound of the signal adheres to the binding member of the enhancer to form a third mixture; (f) The third mixture is co-located for a period of time, and the conditions are sufficient to form an analysis. / Analyte pending binding member pair / probe complex; and (g) Detect the detectable signal to determine the sample to be tested (please read the precautions on the back before filling out this page)-装 · 太 皈 薳 疳 ΐΛ Use Φ a Κ 定 itmiCNS) V4 short chess (210x297 Gongzhi) -6-^ 0δ09〇A 6 B6 Printed by the Beigong Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. The existence of analytes in the description of invention (5). The enhancer compound may be selected from haptens, fluorescent compounds and dinitrobenzene. The preferred enhancer compound is biotin. Chemiluminescent compounds can be selected from the following groups: azepine ester, azepine sulfonamide, 1,2-dioxane and luminol. The preferred chemiluminescent compound is BY-sulfonamide. Analyte-specific binding pair members can adhere to solid pairs. A set for expanding chemiluminescence analysis is also proposed. Detailed Description of the Invention The chemiluminescent properties of acridine compounds and their use in immunoassays have been described. Immunochemical tracers with acridinium esters or acridinium sulfonamide labels can be started by an alkaline peroxide solution to generate a chemiluminescent signal, which peaks after about 2 seconds. After about 10 seconds, the light exits completely. Acridine sulfonamide labeling chemistry can be applied in accordance with the present invention to split into stable tracers with high quantum yields. This method is described in the US Patent No. 371, 763 to which it belongs, and it has common ownership and is listed as a reference for this case. The long-lasting chemiluminescence of 1,2-dioxane in chemical catalysis can be produced in various ways. Therefore, ΕΡ 〇2 5 4 051 (shown above) describes a substitution with a silicon gas group; dioxane, which is 4- (6—third, butyldimethylsilyl group—2-pentyl)- 4 A gas spiro [1, 2-dipyrrolidine 3, 2 adamantane], which initiates a tetrabutyl ammonium gasification solution to produce a chemiluminescent signal that can last 20 minutes. At the same time, enzymes such as aryl esterase and alkaline phosphatase can react with aryl dimorpholine derivatives stabilized with adamantane structure to produce similar long-form chemiluminescent signals (please read the back side first. Note Please fill out this page again). Binding-Thread-Taizhao Dai Η / ίΐΛ Itinerary itit (CNS) A 4 New (210 to 297 cm) -7-Printed by Beigong Consumer Cooperatives, Central Bureau of Standards, Ministry of Economic Affairs Manufacturing 20509ο Α6 __Β6 V. Description of the invention (6) Ο Meanwhile, WO 881 00694 (WO 8906650, shown above) describes 3- (2 / -spiroadamantane) -4 monomethoxy-4 catalyzed by alkaline phosphatase A (3 〃 phosphoramidoxy) -phenyl-1,2-dioxane (AMPPD) reaction, and a similar / 3-galactosidase substrate-anti-2ϋ-continuous emission. The use of these compounds in immunoassays is also described. Therefore, labeling techniques for alkaline phosphatase are known, and the catalytic chemiluminescence of dioxane can be used to generate long-lasting signals. The present invention proposes an immunoassay that utilizes heterogeneous binding members. As used herein, a specific binding member " is a member of a specific binding pair, that is, one of two different molecules, one of which is specifically chemically or physically bound to the second molecule. Therefore, in addition to the specific binding pairs of antigens and antibodies in general immunoassays, other heterogeneous binding pairs include: biotin and avidin, water compounds and lectins, complementary nucleotide sequences, effectors and receptors Body molecules, cofactors and enzymes, enzyme inhibitors and enzymes, etc. Furthermore, specific binding pairs may include analogs of the original specific binding members, such as analyte analogs. Immunoreactive specific binding members include antigens, antigen fragments, antibodies and anti-skull fragments, all of which can be single strains or multiple strains, and complexes thereof, including those formed by recombinant DNA molecules. The term "hapten" here pseudo-refers to partial antigens or non-protein binding members, which can bind to antibodies, but cannot induce antibodies to form unless coupled to carrier proteins. '' Analyte " used here is the substance to be detected, which can be present in the test sample. The analyte can be any substance, and there is a natural presence for this (please read the precautions on the back before filling in this page). Packing and threading. 太 疳 i * m + BH ^ «miCNS) V4 Erbium (210x297 public potential)- 8-A6 B6 Printed by the Beigong Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs 1; ^ Si Mingshen § 4. Jia Gong (eg, antibodies), or specific binding members can be prepared. Therefore, an analyte is a substance that can bind to more than one specific binding member in the analysis. * Analytes 〃 also include any antigenic substances, semi-antigens, antibodies and mixtures thereof. As for specific binding members, analytes can be detected using naturally-occurring specific binding pairs. For example, using intrinsic factor proteins as members of specific binding pairs to determine vitamin Bih or using exogenous lectins as members of specific binding pairs, use To determine the water compound. Analytes may include proteins, peptides, amino acids, hormones, steroids, steroids, drugs (including those administered for therapeutic and illegal purposes), bacteria, viruses, and metabolites or antibodies to any of the foregoing. The preparation and use of this antibody as a specific binding member is well known to elites. As used herein, " capture reagents " refer to unlabeled binding members that are specific to analytes in sandwich analysis, indicator reagents or analytes in competitive analysis, or auxiliary specific binding members , And itself is specific to the analyte, as indirect analysis. The capture reagent can be directly or indirectly bound to the solid phase substance, and then the analysis or simultaneous analysis can be performed, so that the cured complex can be separated from the test sample. "The test sample" may be a biological fluid sample, such as whole blood or whole blood components, including red blood cells, white blood cells, platelets, serum and plasma; ascites, urine, cerebrospinal fluid; and other body components, which may contain important The analyte. Arbitrarily, the test sample can be obtained from water, soil, and plants. The probe " probe " used herein means adhered to the pending binding member of > enhancer compound ". The enhancer compound " can be any compound used in the analysis, which can enhance the signal generated by the chemiluminescent compound. (Please read the _Notes on the back before filling this page)

-9-Printed by the Employees ’Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs 20509b A6 ___B6 41¾ Compounds include haptens such as biotin, as well as melittin and dinitrophenol, etc. ** a chemiluminescent compound 〃 means all compounds that can generate chemiluminescent signals, such as azepine ester, azepine sulfonamide, 1,2-dioxane, luminol, or catalytic chemiluminescent substrate enzyme Wait. '' Conjugate " is used here to mean a chemiluminescent compound to which a compound having an opposite property to the enhancer compound (a specific binding member of the enhancer) is adhered. For example, if the enhancer compound used is biotin, avidin or avidin-specific compounds may be used. A solid phase can be used according to the method of the invention. "Solid phase" here refers to any substance that is insoluble or can be rendered insoluble by the entanglement reaction. The solid phase can be selected by its inherent ability to attract and solidify the capture reagent. In addition, the solid phase can retain additional receptors, It has the ability to attract and solidify the capture reagent. The additional receptor may include a negatively charged substance, which may have an opposite charge relative to the capture reagent itself, or relative to a charged substance conjugated to the capture reagent. The acceptor molecule can be any binding member to be differentiated, which is cured to the solid phase, and it has the ability to cure the capture reagent through a specific binding reaction. The acceptor molecule can allow the capture reagent to bind to the solid phase substance indirectly, Jie Jie Before or during analysis. The analysis device of the present invention can have many configurations, many of which depend on the solid phase selected. For example, the solid phase can include any suitable porous substance. '' Porous '' means The substance can easily pass the test sample, and includes absorbent and non-absorbent solid phase substances. In the present invention, the solid phase can include fiber glass, cellophane, or Nylon disc, used to have more than one layer and contain a kind of too earthworm; ???? Φ® S3 Zhai Xuanjin (CMS) A 4 sister grid (210x297 cm) -10 ~ (please read the precautions on the back before filling in This page) is installed. 'Order, line < 20509ο A 6 Β6 Printed by the employee consumer cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs # ± ^ 明 # 氍 & 1 pour and flow through the analysis and analysis device; used for dip and read analysis Liquid measuring rod; used for core strip (such as paper) or thin strip chromatography or capillary action (such as nitrocellulose) technology test strip; or other porous or open materials, known in the art (such as polyethylene Sheet material). However, the solid phase is not limited to porous materials. The solid phase can also include polymeric or glass beads, microparticles, tubes, H, plates, flakes, holes, patches, test tubes, etc., or other intrinsic charges or may Any substance that retains a charged substance. Natural, synthetic, or synthetically modified naturally occurring substances that can be used as a solid phase include polysaccharides, such as cellulose substances such as paper and cellulose derivatives, such as cellulose acetate and nitrocellulose ; Silicic acid salt; Inorganic substances such as deactivated Bauxite, diatomaceous earth, Mg S〇4, or other inorganic finely divided materials are evenly dispersed in the porous polymer matrix, polymers such as vinyl chloride, vinyl gas-propylene copolymer, and vinyl atmosphere-ethylene acetate copolymer ; Cloth, naturally occurring (such as cotton) and synthetic (such as nylon); porous gels such as silicone, agarose, dextran, and gelatin; polymeric films such as polypropylene amide; etc. The solids are reasonable The strength, or strength, can be provided by a solid phase, and it should not be resistant to the production of detectable signals. Preferred solid phase materials used in flow-through analysis devices include filter paper, such as porous fiberglass materials, or Other matrix materials. The thickness of this material is not critical, and the choice depends largely on the sample or analyte to be analyzed, such as the fluidity of the test sample. To change or strengthen the internal charge of the solid phase , Then the charged substance can be directly coated on the substance, or retained on the solid particles by the solid support. In addition, the microparticles can be used as a solid phase, which is retained in the column or suspended in the soluble reagent and (please read the precautions on the back before filling in this page)-Pack. Order ·-Line · Tai R 燎 iA 中 中 Η H «itmiCNS) V4 Siege (210x297 public power) -11-Printed by the Beigong Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 205095 A 6 __B_6 V. Description of the invention (1 ()) In the test sample mixture or the particles themselves can The solid support material is held and solidified. And "retention and solidification" means that the particles on or in the carrier material can substantially move to other locations on the skull-bearing material. The particles can be selected by any person skilled in the art from any suitable type of particulate matter, and include compounds such as polystyrene, polymethacrylate, polypropylene, latex, polytetrafluoroethylene, polypropylene eye, polyacrylate, or Similar substances. The size of the particles is not critical, but the average diameter of the better particles is smaller than the average pore size of the carrier material used. According to a preferred embodiment of the present invention, the test sample containing the analyte to be tested is in contact with the solid phase, where the binding pair members that are to be anisotropic to the analyte are adhered, thus forming a mixture. This mixture is incubated for a period of time, and the conditions are sufficient to allow analyte / analyte-specific binding pair member complexes to form. Afterwards, these complexes come into contact with the probe, which contains enhancer substances and adheres to members of the binding pair that are specific for the analyte, and is incubated again to form a second mixture. This second mixture is incubated for a period of time, and its conditions are sufficient to allow analyte / analyte-specific binding pair member / probe complex formation. The analyte / analyte-specific binding pair member / probe complex is then contacted with the conjugate, which contains the chemiluminescent signal-generating compound and is conjugated to the enhancer compound binding member, thus forming a third mixture. This third mixture is incubated for a period of time and under conditions sufficient to form an analyte / analyte specific binding pair member / probe / conjugate complex. The presence of the analyte in the test sample can be determined by detecting the signal generated by the chemiluminescent compound. It is also desirable to perform sandwich analysis, where soluble capture reagents may include analyte-specific binding members, which have been bound to charged substances such as yintai zfiA + A 4 gauge. Grid (210x297 public potential) ~ 12 ~ (please first Read the precautions on the back and then fill out this page) Binding. Order-205095 Attachment 2a: Amendment page to the specification of the patent application No. 8〇1〇9188-_____B6 Amended in August 81- Republic of China

V. Description of the invention (Sub-substance. The present invention can also be used for competitive analysis. In competitive configurations, soluble capture reagents also include specific binding members, which have adhered to charged substances such as anionic polymers, which can be bound to Specific binding pairing. The present invention will be illustrated by examples, which are used to illustrate that it does not limit the scope and meaning of the present invention. Examples printed by the Consumer Cooperative of the Bureau of Standards of the Ministry of Economic Affairs Example 1 Polystyrene latex with HTLV-1 antigen Preparation of particles Carboxylated polystyrene latex particles are purified by stirring with an equal w / w amount of mixed bed ion exchange resin for 3 hours at ambient temperature. The particles are separated and then 0.02M MES (2-[N- Phosphono] ethyl sulfonic acid, pH 6. ◦) diluted to ◦. 5% solid particles, and then into a suspension, then add 1 — ethyl one 3_ w_w ratio of 3: 1 (EDAC: latex) (3 -Dimethylaminopropyl) _Master diimine. This mixture was stirred at ambient temperature for 20 minutes, and then purified HTLV-1 antigen lysate was added to a final concentration of 60 μg / ml. After that, it was suspended The liquid was stirred at ambient temperature overnight The coated particles were separated by centrifugation and purified by re-suspending-centrifuging three cycles in a solution containing 0.1% Tween-2 0 ® at pH 7.2 0.01M phosphate and 0.15M NaCj ?. In the centrifugation step, the solid particles are diluted in the buffer solution to a concentration of 0.196. Example 2 ........: ...... t… (Please read the precautions on the back before C Uben Page) Binding-Binding · Thread i The paper size is printed in China National Standard (CNS) Grade 4 (210x297 mm) -13-81. 5. 20.000 (H) Printed by Beigong Consumer Cooperative, Bureau of Standards, Ministry of Economic Affairs dmn τίν-1 antigen was prepared from 0.1 Μ Boric acid buffer solution (ΡΗ8.5) containing 0.196 Triton-X-1 ΟΟΟ ® purified ΗTLV-1 antigen, to 5 mg / ml biotinamide hexanoic acid N-hydroxysuccinylamide DMF solution, treated with a ratio ranging from 0.3 to .6: 1 (antigen: ester). Allow the reaction mixture to stir at ambient temperature 3 After 4 hours, dialyze in a solution containing 0.01 M Ti * is, 0.15 M Na C ί containing 0.1 ^ Triton-X-1 00 ® at pH 7.5. Example 3 Luciferin Antigen preparation · Toxins and recombinant viral proteins are labeled with fluorescein isothiocyanate using the method of Samuel et al., J. Immunol. Methods, 107: 217-224 (1988). Example 4 Avidin labeled with acridine or Preparation of anti-luciferin 1 mg of 10-methyl-9- (N-toluenesulfonaldehyde, N- (2-carboxyethyl) acridinecarboxamide) dissolved in 100 μl of DMF, followed by 50 μl Part 5. 75 mg / ml N-hydroxysuccinimide DMF solution and 50 μl 9. 75 mg / ml 1-ethyl mono-3- (3-dimethylaminopropyl) mastered diimine DMF solution deal with. The solution was stirred at ambient temperature overnight. Coupling the activated acridine derivative to Taizhang Zhang R 疳 iA ffl China H V4 Fool (210x297 public power) -14-(Please read the precautions on the back before filling this page)

^ 0509¾ Printed by Beigong Consumer Cooperative of Central Bureau of Standards, Ministry of Economic Affairs A6 ___B6_ V. Description of the invention (13) The anti-biotin (or anti-fluorescein) is as follows. Anti-skeleton was dialyzed against 0.1M phosphate (pH 8.0) containing ◦. 15M NaC) 2, and then the protein was adjusted to a concentration of 1 mg / ml in the same buffer solution. Afterwards, the activated acridine derivatives in excess of 5 to 10 mils are added to the antibody solution at room temperature. After 10 minutes, the reaction mixture was centrifuged (12,000 rpm for 2 minutes) to remove aggregates, and the supernatant was refilled into a TSK-250 gel filtration column, which had previously been used with 0.01 M sodium phosphate, pH 6 . 3, containing 0.15M NaCi? Balance. Collect 1 ml fractions and track the absorbance at 280 millimetres and 369 millimetres. Collect the fractions containing the I g G peak and estimate the degree of incorporation of acridine as follows: The protein concentration is determined by the absorbance at 280 nm and corrected by the acridine sharing at this wavelength (corrected absorbance = A2S ° — [A3es x〇. 247]). 1% Using the molar extinction coefficient of Mohr concentration, respectively (4, 650 and 2 2 0, 0 0 0 M_; CMd to estimate acridine and IgG. Example 5 HTLV-1 analysis of 50 microliters of polystyrene latex particles 0.1% Solid suspension (as previously described in Example 1 has been coated with HTLV-1 antigen) at pH 7.5 10% sucrose (w / w%), 0.1% bovine serum albumin (BSA), 0.1 % Tween — 20®, 0.1M phosphate phosphate and 0.1% sodium azide solution, added to the 100 liter sample in the reaction hole. The suspension was then placed under 40 · 〇 2 0 points, then turn too 瀳 玥 + 03 ««; hang around (〇 < 5) Ping 4 Tanger (2〗 〇 佂 297 public power) -15-(Please read the precautions on the back before filling in this Page) Printed by the Beigong Consumer Cooperative of the Bureau of Standards, Ministry of Economic Affairs V. Invention Instructions (14) Moved to the capture membrane, where pH 7. 2, 0. 01M phosphate base, 0.1 5 M NaCi, containing 0. 1% sodium dinitrate in 300 liters of washing liquid was washed twice. The washed suspension was allowed to stand at 40 ° C for about 10 minutes, and then 30 μl of biotinylated HTLV-1 antigen ( Prepared as in Example 2) at ◦. 1M Tris (pH 8.5), 50% bovine serum And 0.1M NaC $ containing ◦. 1% sodium nitrate 167nm / ml solution treatment. The capture membrane is allowed to rest for another 20 minutes, then washed three times with 100μl each of pH 8.5 washing solution, washing The solution contains 0.1M Tris, 0.15 M NaCi, and 0.1% Triton ® contains 0.1% sodium azide. The washed capture membrane is then treated with 30 microliters of 167 milligrams / ml anti-biotic The solution was treated in 0.01M MES (pH 6.3), 0.15M NaC5, 2% BSA, and 0.5% Tritonr® containing 0.1% sodium azide, which has chemiluminescent acridine The ammonium sulfonamide is partially attached (see example 4). After 10 minutes of 401C co-location, the capture membrane was washed three times with 100 μl of pH 5.5 solution each, including 0.1M MES, 0.15M NaC Wen and 0.1% sodium krypton. The washed capture membrane was placed at 40¾ for 10 minutes, then treated with alkaline peroxide solution (◦. 25N NaOH containing 0.3% peroxide). Chemiluminescent total Read for 6 seconds, and decide whether to resist the existence of a Η TLV-1.. Example 6 Taicheng + A 4 group (210x297 public power) ~ 16 ~ A 6 B6 (Please read the notes on the back before filling this page )-Installed.

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ΛΒ Printed by the Employee Consumer Cooperative of the Bureau of Standards, Ministry of Economic Affairs V. Description of invention (Preparation of polystyrene latex particles coated with HCV (Hepatitis C virus) antigen. 20 micrograms of HCV antigen and 100 mg 2.66 Rice diameter polystyrene latex particles were mixed in 20 ml of pH 7. ◦. 1 M phosphate phosphate buffer solution and allowed to stir at room temperature overnight. Then centrifuged (17, 000 rpm, 25 Cent) Separate the solid particles by centrifugation, and then purify by resuspending and centrifuging for three cycles in 2◦mL pH 7.0 at 0.1M phosphate salt buffer solution (containing 0.005% Tween-20®). 7 Preparation of biotinylated HCV antigen 1 mg of biotinylamido hexanoic acid N-hydroxysuccinamide, added to 2.5 ml ◦. 05% pH8. 5 boric acid salt (containing 0.1% Tween 1 20®) of 50 mcg of HCV antigen. After stirring at room temperature for 2 hours, 50 mg of BSA was added, and the solution was subjected to two 500 ml portions of 0.02M Tris buffer solution solution at a ring temperature, pH 8 . 5 (containing 0. 0 0 2 M dithioisocyanurin and 0.1% Tween_20®) overnight dialysis. Example 8 HCV analysis 50 microliters at 0.1M ρ 7. 0 phosphate salt buffer solution (containing 0.005% Tween-20® (previously coated with HCV antigen, as shown in Example 6)) of 0.5% polystyrene latex particle solid (please read first The notes on the back will be written on this page). Packed. This paper size is used in the SB family standard (CNS) A 4 specifications (210X297 public goods) -17-81. 5. 20.000 (H) Ministry of Economic Affairs Central Standards Bureau Printed by the industrial and consumer cooperatives V. Description of invention (19

The suspension was added to 50 μl of pH 8.5. 02M borate buffer solution, which contained 1% Tween-20®, 0.025% cel-lquat, 01% hexadecyl pyridine Ingot, 0.05M EDTA, 0.03M NaC5 and 0.1% sodium azide, and then added to the reaction cavity 1 ◦ 0 microliter sample. After that, the suspension was placed at 40 * 0 for about 20 minutes, and then transferred to the reaction capture membrane, and washed twice with 300 microliters of washing liquid, and the washing liquid contained pH 7.5, 0.01 M phosphate buffer solution, 0.15M NaC and 0.1% sodium azide. The washed suspension was allowed to stand at 40 * 0 for about 10 minutes, and then treated with 30 liters of 660 nm / ml biotinylated recombinant HCV antigen solution (prepared as in Example 7) _ , Which is at 0. 02M Boron i

Acid salt (pH8.5), 5% BSA, and 5% Tee X-100® contain 0.1% sodium azide. The capture membrane was allowed to stand for another 20 minutes, and then washed with 3 parts of 100 liters of pH 8.0 wash solution containing 0.1M boric acid salt, 0.15M NaCJ2, and 0.05% lithium dodecyl sulfate (LDS ) And containing 0.1% sodium azide. After washing the capture membrane, apply 30 μl of 165 ng / ml of anti-biotin to the ◦. 01M phosphate salt (ρΗ6 · 0), 0 · 15M

NaC5, 5% bovine serum and 0.1% three-way @ solution containing 0.1% azide pin were treated, to which the chemiluminescent azepine sulfonamide part was adhered (prepared as in Example 4). After a further 10 minutes of co-location at 40¾, the capture membrane was washed three times with 00 microliters of PH8.5 solution, which contained 0.1 Μ boric acid salt, 15M NaCi? And 0.20% SDS, (please (Read the precautions on the back before filling the nest page) -Installation. -Line < This paper standard is common to China National Standards (CNS) A 4 specifications (210x297 g *) -18-Ordnance Industry Consumer Cooperative of Central Bureau of Standards, Ministry of Economic Affairs Printed Α6 ___ Β6__ 5. Description of the invention (1) 0.1% sodium nitride. The washed capture membrane was placed at 401C for 10 minutes, and then treated with an alkaline peroxide solution (0.S5N NaOH containing 0.3% pergasification). Chemiluminescence reads for 6 seconds and determines the presence or absence of anti-HCV. Example 9 Preparation of polystyrene latex particles coated with H IV antigen in 34. 2 8 ml of HIV antigen (10 mg) in 5 M borate buffer solution (ρ H 8. 5), and 10 ml The 0.5% polystyrene latex particle solid particle suspension is mixed, and then 55.72 ml of deionized water is added. Then the suspension was stirred at room temperature overnight. Afterwards, the solid particles were separated by centrifugation (17,000 rpm for 30 minutes), and then resuspended in 0.1 mM phosphate buffer solution (p Η 7.0) containing 0.1% Tween r ® and centrifuged for 3%. Purified by purification. The coated particles were resuspended and allowed to slowly search and mix at 561C for 1 day, and then stored at room temperature for use. Example 1 0 Preparation of biotinylated H IV antigen in 2.278 ml of 0.1 M borate buffer solution (pH 8.5) containing 250 mM NaCi? And 0.1% sodium kryptonide in HIV antigen ( 1. 9 mg) with 10% Tee® 0.125 ml for 30 minutes. After that, add 97 μl of 5 mg / ml biotin amide hexamide N-hydroxysuccinamide ester dissolved in DMF (please read the precautions on the back before filling out this page) * Package--Line · This For paper size, use China National Standards (CNS) Grade 4 (210x297 mm) -19-A 6 B 6 ^ 05093 5. Description of the invention (id. Let the reaction mixture be stirred at room temperature for about 2 hours. . 1 Μ boric acid buffer solution (pH 8.5, containing 250mM Na C ^; Ο. 1% SDS, and Ο. 1% sodium azide) full dialysis. Example 1 1 Η IV analysis 50 microliters about 0.2% polystyrene latex particles (previously coated with HIV antigen as described in Example 9) at pH 7.0 11% sucrose (w / w%), 0.01MEGTA, 0.1% CHAPS®, Ο. 1M phosphate salt and Ο. 1% β sodium nitride solution, added to the reaction hole 1 Ο Ο microliter sample. The suspension was then placed at 401C for about 20 minutes, and then transferred to the capture membrane, to 300μl of PH8.5 wash solution was washed three times, which contained PH8.5, 0.1 Μ borate buffer solution, 0.15M NaC)? And 0.01% lithium dodecyl sulfate (LDS) and contained 0 . 1% sodium azide. The washed suspension was allowed to stand at 40t! For about 10 minutes, and then 30 μl of 7 500 ng / ml was biotinylated H IV antigen (prepared according to Example 10) at 0.1 Μ Boric acid buffer solution (ρ Η 8.5) 1% E. coli lysate, 500 μg / ml CKS, 12.5% bovine serum, and 1.0% cholic acid (containing 0.1% sodium azide) Of solution. After that, the capture membrane was placed for a further 20 minutes, and then washed with 100 microliters of pH8.5 washing solution three times, containing 0.1M boric acid salt, 0.15M NaC5 and 0.03% LDS (inclusive (please read ^ Notes on the face and fill in the nest page) Install &Order; Line-Printed by the Beigong Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs. The paper standard is in the general Η B family sample standard (CNS) A 4 specifications (210x297 public «: ) -20-^ Οΰΰδδ A 6 Β6 V. Description of the invention (19

〇.1% day sodium nitride). The washed capture membrane was treated with 30 liters of 167 mM [m / ml of an anti-biotin solution, in which the chemiluminescent ammonium sulfonamide portion (prepared as in Example 4) had been adhered, and it was 0. 0 1 Μ phosphate wall (pH6.3), 0.15M Ν a C, and 5 96 BSA, and 1.0% three-way © containing 0.1% sodium azide. After 10 minutes at 40C, the capture membrane was washed with 5 parts of a pH 8.5 solution of 100 liters, which contained ◦. 1M boric acid tincture, 0.15M

NaCi? And 0.01% LDS, containing 0.1% sodium dinitrate. The washed capture membrane was placed at 40C for 10 minutes, and then treated with alkaline peroxidase (0.25N NaOH containing 0.3% peroxide). Chemiluminescence reads for 6 seconds and determines the presence or absence of anti-HIV.-Other modifications and changes to the specific specific examples of the invention shown here should be obvious to those skilled in the art. Therefore, the present invention is limited based on the scope of the attached patent application. (Please read the & notices before filling out this page) Pack ·-Order--Line · Printed by the Ministry of Economic Affairs, Central Standards Bureau, Beigong Consumer Cooperative Co., Ltd. This paper standard is used in a B family standard (CNS) A 4 specifications (210X297 Gong «) -21-

Claims (1)

A7 A7 6. Applicant's Patent Application Annex 1a: Amendment of the Chinese Patent Application No. 80109188 in the Chinese Patent Application Amendment in December 1981. 1. A specific expansion of the chemiluminescence signal generated from heterogeneous immunoassay to determine the antibody's acceptance. A method that exists in the test sample. This method includes: a. Contacting the test sample suspected of containing the antibody with the antigen bound to a solid phase to form a first mixture; b. Co-locating the first mixture for a period of time and The conditions are sufficient to form an antigen / antibody complex; c. The antigen / antibody complex is brought into contact with a probe containing a biotin-enhanced compound and adhered to the antigen to form a second mixture; d. The second mixture is co-located for a period of time and under conditions sufficient to form an antigen / antibody / probe complex; e. Make the antigen / antibody / probe complex and the chemiluminescent acridine sulfonamide compound The conjugate attached to the enhancer-to-be-unlike binding member contacts to form a third mixture; f. The third mixture is co-located for a period of time and the conditions are sufficient To antigen / antibody / probe / conjugate complexes;. G enabling the sample with a solid phase to separate; and h by chemiluminescence detection signal is determined in the presence of the antibody in the test sample. 2. According to the first method in the scope of patent application, wherein the enhancer compound is biotin and the enhancer is specific and the binding member is anti-biotin tensile. The scale of China National Standard (CNS) A 4 (210 X 297 mm) _ 1 ~ 81.9.10,000 A, please read the old note on the back and then write this page) • Installed. Ordered · Printed by the Ministry of Economic Affairs Central Sample Housing Xinggong Consumer Cooperative Society rkt B7 C7 D7 VI. Patent application Fan Yinhuang or avidin protein. 3. The method according to item 1 of the application scope, wherein the antibody is pseudo-specific to HCV antigen, HTLV antigen or HIV antigen. 4. The method according to item 1 of the patent application scope, in which the solid phase is a bead, particle removal, tube, flake, flat plate, glass plate, well, film or test tube. 5. A kit for expanded chemiluminescence analysis, including individual containers with the following substances: a solid phase with an antigen bound to it; a probe containing an enhancer compound adhered to an antigen Reagents; and a conjugate containing a chemiluminescent signal-generating compound adhered to an enhancer-to-iso binding member. 6. The kit according to item 5 of the patent application scope, in which the probe includes an antigen which is bound to an enhancer compound, biotin. 7. The kit according to item 5 of the patent application scope, wherein the conjugate includes an enhancer-specific binding member, which is bound to a chemiluminescent production compound, azetamide. 、 Please read the precautions first and then write the binding line. The Central Ministry of Economics, Central Ministry of Economic Affairs, β-Consumer Cooperative Printing Co., Ltd. Printed paper. The size of the paper is applicable to the Chinese National Standard (CNS) 4 (210 X 297 mm) _ 2 81.9.10,000 i Annex IIIa: Supplementary Example of Chinese Patent Application No. 80109188 in the Chinese Patent Case presented in August 1981 in the automatic analysis of detection of antibodies against HIV-1 and HIV-2 5, the improved H The sensitivity of IV seroconversion. Peterson, Bryan; Kite, A., La in iο, S., Wiese, J., Phelps, B., PIich, J .; and Stramer, SL Abbott Laboratori es, Abbott Park, 1160064 i〇 Foreword Abbott ’s wish Prism It is designed to be a porous tank device that can be used for immunoassay at the same time when testing, which is convenient for processing a large amount of blood ^ night screening test. Prism is a completely automatic device that can perform all of the analysis functions, including sample extraction, reagent addition force α, co-located reaction, reaction rate and data reduction. P "Analysis of virology 1 developed by ism ^ to include 1 includes test 1 antibodies against HIV ~ 10 1 and HIV-2 (combination analysis), HTLV-1 and HTLV-H (combination analysis) , HCV, and the core antigen of hepatitis B virus. The detection method for detecting tidal hepatitis B surface antigen has been established. The immunoassay method uses microparticle solid phase and chemiluminescence for signal determination. The analysis of the M method is based on the agglutination technique. This article describes the Prism immunoassay used to determine the sensitivity of seroconversion of antibodies against H IV-1 and H IV-2. Prism HIV_1 / HIV-2 assay Conjugates containing a biotin-labeled antigen and an avidin protein were used to determine " IgG " (Figure i). Serum conversion of the immunoassay T4 (210X297 male 5.) —I — Sensitivity data are also shown in anti-human conjugates (rDNA and assays based on virus lysate antigens) and other direct sandwich assays using H IV-1 and H IV-2 "D ΝΑ Α antigens Figure Ί Prism analysis format co-located with samples of HIV-1 and HIV-2 rDNA antigen coated microparticles 20 minutes. The final reaction mixture is transferred to the capture membrane in a non-contact manner, which prevents any sample from remaining. Wash the unbound antibody to allow it to flow into the adsorbent in the reaction tank. Then add the biotinylated antigen to Capture the membrane and co-locate it for another 2Q minutes. If an antibody against HIV antigen is bound to the particle, the probe antigen will be captured by the second antibody binding site. After co-location, unbound probe is washed away. Monoclonal antibody of biotin to a capture membrane, which is conjugated with a chemiluminescent acridine compound, and co-located for 10 minutes. After washing away unbound compounds of 卩 病 compound once, the chemiluminescent signal is sincere The urea-peroxidase solution is started and the excited photons are quantified by a photon amplification tube. A 4 (210X297 gong) 2.—